@article {3106, title = {Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.}, journal = {ISME J}, volume = {10}, year = {2016}, month = {2016 Feb}, pages = {310-20}, abstract = {

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Cytochrome c Group, Electron Transport, Ferric Compounds, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, Proteomics, Sequence Alignment, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2015.113}, author = {Yun, Jiae and Malvankar, Nikhil S and Ueki, Toshiyuki and Lovley, Derek R} } @article {1743, title = {Methanocaldococcus bathoardescens sp. nov., a hyperthermophilic methanogen isolated from a volcanically active deep-sea hydrothermal vent.}, journal = {Int J Syst Evol Microbiol}, volume = {65}, year = {2015}, month = {2015 Apr}, pages = {1280-3}, abstract = {

A hyperthermophilic methanogen, strain JH146(T), was isolated from 26 {\textdegree}C hydrothermal vent fluid emanating from a crack in basaltic rock at Marker 113 vent, Axial Seamount in the northeastern Pacific Ocean. It was identified as an obligate anaerobe that uses only H2 and CO2 for growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain is more than 97\% similar to other species of the genus Methanocaldococcus . Therefore, overall genome relatedness index analyses were performed to establish that strain JH146(T) represents a novel species. For each analysis, strain JH146(T) was most similar to Methanocaldococcus sp. FS406-22, which can fix N2 and also comes from Marker 113 vent. However, strain JH146(T) differs from strain FS406-22 in that it cannot fix N2. The average nucleotide identity score for strain JH146(T) was 87\%, the genome-to-genome direct comparison score was 33-55\% and the species identification score was 93\%. For each analysis, strain JH146(T) was below the species delineation cut-off. Full-genome gene synteny analysis showed that strain JH146(T) and strain FS406-22 have 97\% genome synteny, but strain JH146(T) was missing the operons necessary for N2 fixation and assimilatory nitrate reduction that are present in strain FS406-22. Based on its whole genome sequence, strain JH146(T) is suggested to represent a novel species of the genus Methanocaldococcus for which the name Methanocaldococcus bathoardescens is proposed. The type strain is JH146(T) ( = DSM 27223(T) = KACC 18232(T)).

}, keywords = {DNA, Bacterial, Hydrothermal Vents, Methanocaldococcus, Molecular Sequence Data, Pacific Ocean, Phylogeny, RNA, Ribosomal, 16S, Seawater, Sequence Analysis, DNA}, issn = {1466-5034}, doi = {10.1099/ijs.0.000097}, author = {Stewart, Lucy C and Jung, Jong-Hyun and Kim, You-Tae and Kwon, Soon-Wo and Park, Cheon-Seok and Holden, James F} } @article {3131, title = {A Geobacter sulfurreducens strain expressing pseudomonas aeruginosa type IV pili localizes OmcS on pili but is deficient in Fe(III) oxide reduction and current production.}, journal = {Appl Environ Microbiol}, volume = {80}, year = {2014}, month = {2014 Feb}, pages = {1219-24}, abstract = {

The conductive pili of Geobacter species play an important role in electron transfer to Fe(III) oxides, in long-range electron transport through current-producing biofilms, and in direct interspecies electron transfer. Although multiple lines of evidence have indicated that the pili of Geobacter sulfurreducens have a metal-like conductivity, independent of the presence of c-type cytochromes, this claim is still controversial. In order to further investigate this phenomenon, a strain of G. sulfurreducens, designated strain PA, was constructed in which the gene for the native PilA, the structural pilin protein, was replaced with the PilA gene of Pseudomonas aeruginosa PAO1. Strain PA expressed and properly assembled P. aeruginosa PilA subunits into pili and exhibited a profile of outer surface c-type cytochromes similar to that of a control strain expressing the G. sulfurreducens PilA. Surprisingly, the strain PA pili were decorated with the c-type cytochrome OmcS in a manner similar to the control strain. However, the strain PA pili were 14-fold less conductive than the pili of the control strain, and strain PA was severely impaired in Fe(III) oxide reduction and current production. These results demonstrate that the presence of OmcS on pili is not sufficient to confer conductivity to pili and suggest that there are unique structural features of the G. sulfurreducens PilA that are necessary for conductivity.

}, keywords = {Amino Acid Sequence, Cytochromes c, Electricity, Ferric Compounds, Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Methanosarcinaceae, Molecular Sequence Data, Oxidation-Reduction, Pseudomonas aeruginosa, Sequence Alignment}, issn = {1098-5336}, doi = {10.1128/AEM.02938-13}, author = {Liu, Xing and Tremblay, Pier-Luc and Malvankar, Nikhil S and Nevin, Kelly P and Lovley, Derek R and Vargas, Madeline} } @article {1420, title = {Response of free-living nitrogen-fixing microorganisms to land use change in the Amazon rainforest.}, journal = {Appl Environ Microbiol}, volume = {80}, year = {2014}, month = {2014 Jan}, pages = {281-8}, abstract = {

The Amazon rainforest, the largest equatorial forest in the world, is being cleared for pasture and agricultural use at alarming rates. Tropical deforestation is known to cause alterations in microbial communities at taxonomic and phylogenetic levels, but it is unclear whether microbial functional groups are altered. We asked whether free-living nitrogen-fixing microorganisms (diazotrophs) respond to deforestation in the Amazon rainforest, using analysis of the marker gene nifH. Clone libraries were generated from soil samples collected from a primary forest, a 5-year-old pasture originally converted from primary forest, and a secondary forest established after pasture abandonment. Although diazotroph richness did not significantly change among the three plots, diazotroph community composition was altered with forest-to-pasture conversion, and phylogenetic similarity was higher among pasture communities than among those in forests. There was also 10-fold increase in nifH gene abundance following conversion from primary forest to pasture. Three environmental factors were associated with the observed changes: soil acidity, total N concentration, and C/N ratio. Our results suggest a partial restoration to initial levels of abundance and community structure of diazotrophs following pasture abandonment, with primary and secondary forests sharing similar communities. We postulate that the response of diazotrophs to land use change is a direct consequence of changes in plant communities, particularly the higher N demand of pasture plant communities for supporting aboveground plant growth.

}, keywords = {Agriculture, Bacteria, Biota, Carbon, Cluster Analysis, Conservation of Natural Resources, Human Activities, Hydrogen-Ion Concentration, Molecular Sequence Data, Nitrogen, Nitrogen Fixation, Oxidoreductases, Phylogeny, Sequence Analysis, DNA, Soil, Soil Microbiology, South America, Trees}, issn = {1098-5336}, doi = {10.1128/AEM.02362-13}, author = {Mirza, Babur S and Potisap, Chotima and N{\"u}sslein, Klaus and Bohannan, Brendan J M and Rodrigues, Jorge L M} } @article {3135, title = {Sulfur oxidation to sulfate coupled with electron transfer to electrodes by Desulfuromonas strain TZ1.}, journal = {Microbiology (Reading)}, volume = {160}, year = {2014}, month = {2014 Jan}, pages = {123-129}, abstract = {

Microbial oxidation of elemental sulfur with an electrode serving as the electron acceptor is of interest because this may play an important role in the recovery of electrons from sulfidic wastes and for current production in marine benthic microbial fuel cells. Enrichments initiated with a marine sediment inoculum, with elemental sulfur as the electron donor and a positively poised (+300 mV versus Ag/AgCl) anode as the electron acceptor, yielded an anode biofilm with a diversity of micro-organisms, including Thiobacillus, Sulfurimonas, Pseudomonas, Clostridium and Desulfuromonas species. Further enrichment of the anode biofilm inoculum in medium with elemental sulfur as the electron donor and Fe(III) oxide as the electron acceptor, followed by isolation in solidified sulfur/Fe(III) medium yielded a strain of Desulfuromonas, designated strain TZ1. Strain TZ1 effectively oxidized elemental sulfur to sulfate with an anode serving as the sole electron acceptor, at rates faster than Desulfobulbus propionicus, the only other organism in pure culture previously shown to oxidize S{\textdegree} with current production. The abundance of Desulfuromonas species enriched on the anodes of marine benthic fuel cells has previously been interpreted as acetate oxidation driving current production, but the results presented here suggest that sulfur-driven current production is a likely alternative.

}, keywords = {Bioelectric Energy Sources, Desulfuromonas, DNA, Bacterial, Electricity, Electrodes, Geologic Sediments, Molecular Sequence Data, Oxidation-Reduction, Sequence Analysis, DNA, Sulfates, Sulfur}, issn = {1465-2080}, doi = {10.1099/mic.0.069930-0}, author = {Zhang, Tian and Bain, Timothy S and Barlett, Melissa A and Dar, Shabir A and Snoeyenbos-West, Oona L and Nevin, Kelly P and Lovley, Derek R} } @article {1410, title = {Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. of hyperthermophilic heterotrophs from deep-sea hydrothermal vents.}, journal = {Int J Syst Evol Microbiol}, volume = {64}, year = {2014}, month = {2014 Nov}, pages = {3655-9}, abstract = {

Two heterotrophic hyperthermophilic strains, ES1(T) and CL1(T), were isolated from Paralvinella sp. polychaete worms collected from active hydrothermal vent chimneys in the north-eastern Pacific Ocean. Both were obligately anaerobic and produced H2S in the presence of elemental sulfur and H2. Complete genome sequences are available for both strains. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strains are more than 97\% similar to most other species of the genus Thermococcus. Therefore, overall genome relatedness index analyses were performed to establish that these strains are novel species. For each analysis, strain ES1(T) was determined to be most similar to Thermococcus barophilus MP(T), while strain CL1(T) was determined to be most similar to Thermococcus sp. 4557. The average nucleotide identity scores for these strains were 84\% for strain ES1(T) and 81\% for strain CL1(T), genome-to-genome direct comparison scores were 23\% for strain ES1(T) and 47\% for strain CL1(T), and the species identification scores were 89\% for strain ES1(T) and 88\% for strain CL1(T). For each analysis, strains ES1(T) and CL1(T) were below the species delineation cut-off. Therefore, based on their whole genome sequences, strains ES1(T) and CL1(T) are suggested to represent novel species of the genus Thermococcus for which the names Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. are proposed, respectively. The type strains are ES1(T) ( =DSM 27261(T) =KACC 17923(T)) and CL1(T) ( =DSM 27260(T) =KACC 17922(T)).

}, keywords = {Animals, DNA, Archaeal, Hydrothermal Vents, Molecular Sequence Data, Pacific Ocean, Phylogeny, Polychaeta, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Thermococcus}, issn = {1466-5034}, doi = {10.1099/ijs.0.066100-0}, author = {Hensley, Sarah A and Jung, Jong-Hyun and Park, Cheon-Seok and Holden, James F} } @article {3145, title = {Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater.}, journal = {ISME J}, volume = {7}, year = {2013}, month = {2013 Jul}, pages = {1286-98}, abstract = {

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80\% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100\% of the sequences recovered during this phase of the bioremediation. These results suggest a prey-predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.

}, keywords = {Acetates, Biodegradation, Environmental, Eukaryota, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, RNA, Ribosomal, 18S, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2013.20}, author = {Holmes, Dawn E and Giloteaux, Ludovic and Williams, Kenneth H and Wrighton, Kelly C and Wilkins, Michael J and Thompson, Courtney A and Roper, Thomas J and Long, Philip E and Lovley, Derek R} } @article {3147, title = {Molecular analysis of the in situ growth rates of subsurface Geobacter species.}, journal = {Appl Environ Microbiol}, volume = {79}, year = {2013}, month = {2013 Mar}, pages = {1646-53}, abstract = {

Molecular tools that can provide an estimate of the in situ growth rate of Geobacter species could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate of Geobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins, rpsC and rplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance of rpsC correlated best (r(2) = 0.90) with specific growth rates. Therefore, expression patterns of rpsC were used to estimate specific growth rates of Geobacter species during an in situ uranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression of Geobacter rpsC, and the increase in the number of Geobacter cells estimated with fluorescent in situ hybridization compared well with specific growth rates estimated from levels of in situ rpsC expression. However, in later phases, cell number increases were substantially lower than predicted from rpsC transcript abundance. This change coincided with a bloom of protozoa and increased attachment of Geobacter species to solid phases. These results suggest that monitoring rpsC expression may better reflect the actual rate that Geobacter species are metabolizing and growing during in situ uranium bioremediation than changes in cell abundance.

}, keywords = {Acetates, Biodegradation, Environmental, DNA, Bacterial, Gene Expression Profiling, Geobacter, Groundwater, In Situ Hybridization, Fluorescence, Molecular Sequence Data, Ribosomal Proteins, Sequence Analysis, DNA, Uranium}, issn = {1098-5336}, doi = {10.1128/AEM.03263-12}, author = {Holmes, Dawn E and Giloteaux, Ludovic and Barlett, Melissa and Chavan, Milind A and Smith, Jessica A and Williams, Kenneth H and Wilkins, Michael and Long, Philip and Lovley, Derek R} } @article {3155, title = {Anaerobic benzene oxidation by Geobacter species.}, journal = {Appl Environ Microbiol}, volume = {78}, year = {2012}, month = {2012 Dec}, pages = {8304-10}, abstract = {

The abundance of Geobacter species in contaminated aquifers in which benzene is anaerobically degraded has led to the suggestion that some Geobacter species might be capable of anaerobic benzene degradation, but this has never been documented. A strain of Geobacter, designated strain Ben, was isolated from sediments from the Fe(III)-reducing zone of a petroleum-contaminated aquifer in which there was significant capacity for anaerobic benzene oxidation. Strain Ben grew in a medium with benzene as the sole electron donor and Fe(III) oxide as the sole electron acceptor. Furthermore, additional evaluation of Geobacter metallireducens demonstrated that it could also grow in benzene-Fe(III) medium. In both strain Ben and G. metallireducens the stoichiometry of benzene metabolism and Fe(III) reduction was consistent with the oxidation of benzene to carbon dioxide with Fe(III) serving as the sole electron acceptor. With benzene as the electron donor, and Fe(III) oxide (strain Ben) or Fe(III) citrate (G. metallireducens) as the electron acceptor, the cell yields of strain Ben and G. metallireducens were 3.2 {\texttimes} 10(9) and 8.4 {\texttimes} 10(9) cells/mmol of Fe(III) reduced, respectively. Strain Ben also oxidized benzene with anthraquinone-2,6-disulfonate (AQDS) as the sole electron acceptor with cell yields of 5.9 {\texttimes} 10(9) cells/mmol of AQDS reduced. Strain Ben serves as model organism for the study of anaerobic benzene metabolism in petroleum-contaminated aquifers, and G. metallireducens is the first anaerobic benzene-degrading organism that can be genetically manipulated.

}, keywords = {Anaerobiosis, Benzene, Carbon Dioxide, Cluster Analysis, Culture Media, DNA, Bacterial, DNA, Ribosomal, Ferric Compounds, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1098-5336}, doi = {10.1128/AEM.02469-12}, author = {Zhang, Tian and Bain, Timothy S and Nevin, Kelly P and Barlett, Melissa A and Lovley, Derek R} } @article {356, title = {Analysis of the genome of the sexually transmitted insect virus Helicoverpa zea nudivirus 2.}, journal = {Viruses}, volume = {4}, year = {2012}, month = {2012 Jan}, pages = {28-61}, abstract = {The sexually transmitted insect virus Helicoverpa zea nudivirus 2 (HzNV-2) was determined to have a circular double-stranded DNA genome of 231,621 bp coding for an estimated 113 open reading frames (ORFs). HzNV-2 is most closely related to the nudiviruses, a sister group of the insect baculoviruses. Several putative ORFs that share homology with the baculovirus core genes were identified in the viral genome. However, HzNV-2 lacks several key genetic features of baculoviruses including the late transcriptional regulation factor, LEF-1 and the palindromic hrs, which serve as origins of replication. The HzNV-2 genome was found to code for three ORFs that had significant sequence homology to cellular genes which are not generally found in viral genomes. These included a presumed juvenile hormone esterase gene, a gene coding for a putative zinc-dependent matrix metalloprotease, and a major facilitator superfamily protein gene; all of which are believed to play a role in the cellular proliferation and the tissue hypertrophy observed in the malformation of reproductive organs observed in HzNV-2 infected corn earworm moths, Helicoverpa zea.}, keywords = {Amino Acid Sequence, Animals, Baculoviridae, Base Sequence, Biological Evolution, Consensus Sequence, DNA Viruses, Female, Genome, Viral, Insect Viruses, Molecular Sequence Data, Moths, Open Reading Frames, Phylogeny, Sequence Alignment, Sequence Homology, Species Specificity, Viral Proteins}, issn = {1999-4915}, doi = {10.3390/v4010028}, author = {Burand, John P and Kim, Woojin and Afonso, Claudio L and Tulman, Edan R and Kutish, Gerald F and Lu, Zhiqiang and Rock, Daniel L} } @article {1207, title = {Complete genome sequence of the hyperthermophilic archaeon Pyrococcus sp. strain ST04, isolated from a deep-sea hydrothermal sulfide chimney on the Juan de Fuca Ridge.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Aug}, pages = {4434-5}, abstract = {

Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na(+) gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential.

}, keywords = {Adenosine Triphosphate, Anaerobiosis, DNA, Archaeal, Genome, Archaeal, Heterotrophic Processes, Hydrothermal Vents, Molecular Sequence Data, Pacific Ocean, Polysaccharides, Pyrococcus, Seawater, Sequence Analysis, DNA, Sodium Chloride, Sulfides}, issn = {1098-5530}, doi = {10.1128/JB.00824-12}, author = {Jung, Jong-Hyun and Lee, Ju-Hoon and Holden, James F and Seo, Dong-Ho and Shin, Hakdong and Kim, Hae-Yeong and Kim, Wooki and Ryu, Sangryeol and Park, Cheon-Seok} } @article {1205, title = {Complete genome sequence of the hyperthermophilic archaeon Thermococcus sp. strain CL1, isolated from a Paralvinella sp. polychaete worm collected from a hydrothermal vent.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Sep}, pages = {4769-70}, abstract = {

Thermococcus sp. strain CL1 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a Paralvinella sp. polychaete worm living on an active deep-sea hydrothermal sulfide chimney on the Cleft Segment of the Juan de Fuca Ridge. To further understand the distinct characteristics of this archaeon at the genome level, its genome was completely sequenced and analyzed. Here, we announce the complete genome sequence (1,950,313 bp) of Thermococcus sp. strain CL1, with a focus on H(2)- and energy-producing capabilities and its amino acid biosynthesis and acquisition in an extreme habitat.

}, keywords = {Animals, Base Sequence, Chromosome Mapping, DNA, Archaeal, DNA, Ribosomal, Genome, Bacterial, Hydrothermal Vents, Molecular Sequence Data, Phylogeny, Polychaeta, Sequence Analysis, DNA, Thermococcus}, issn = {1098-5530}, doi = {10.1128/JB.01016-12}, author = {Jung, Jong-Hyun and Holden, James F and Seo, Dong-Ho and Park, Kwan-Hwa and Shin, Hakdong and Ryu, Sangryeol and Lee, Ju-Hoon and Park, Cheon-Seok} } @article {3149, title = {The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features.}, journal = {BMC Genomics}, volume = {13}, year = {2012}, month = {2012 Dec 10}, pages = {690}, abstract = {

BACKGROUND: The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species.

RESULTS: Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate:ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires.

CONCLUSIONS: Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.

}, keywords = {Base Pairing, Base Sequence, Butylene Glycols, Choline, Deltaproteobacteria, Ethanolamine, Ethylene Glycol, Genome, Bacterial, Glycerol, Metabolic Networks and Pathways, Molecular Sequence Annotation, Molecular Sequence Data, Mutation, Oxidation-Reduction, Oxidoreductases, Propylene Glycols, RNA, Transfer, Asn, Sequence Analysis, DNA}, issn = {1471-2164}, doi = {10.1186/1471-2164-13-690}, author = {Aklujkar, Muktak and Haveman, Shelley A and DiDonato, Raymond and Chertkov, Olga and Han, Cliff S and Land, Miriam L and Brown, Peter and Lovley, Derek R} } @article {1206, title = {Hydrogen-limited growth of hyperthermophilic methanogens at deep-sea hydrothermal vents.}, journal = {Proc Natl Acad Sci U S A}, volume = {109}, year = {2012}, month = {2012 Aug 21}, pages = {13674-9}, abstract = {

Microbial productivity at hydrothermal vents is among the highest found anywhere in the deep ocean, but constraints on microbial growth and metabolism at vents are lacking. We used a combination of cultivation, molecular, and geochemical tools to verify pure culture H(2) threshold measurements for hyperthermophilic methanogenesis in low-temperature hydrothermal fluids from Axial Volcano and Endeavour Segment in the northeastern Pacific Ocean. Two Methanocaldococcus strains from Axial and Methanocaldococcus jannaschii showed similar Monod growth kinetics when grown in a bioreactor at varying H(2) concentrations. Their H(2) half-saturation value was 66 μM, and growth ceased below 17-23 μM H(2), 10-fold lower than previously predicted. By comparison, measured H(2) and CH(4) concentrations in fluids suggest that there was generally sufficient H(2) for Methanocaldococcus growth at Axial but not at Endeavour. Fluids from one vent at Axial (Marker 113) had anomalously high CH(4) concentrations and contained various thermal classes of methanogens based on cultivation and mcrA/mrtA analyses. At Endeavour, methanogens were largely undetectable in fluid samples based on cultivation and molecular screens, although abundances of hyperthermophilic heterotrophs were relatively high. Where present, Methanocaldococcus genes were the predominant mcrA/mrtA sequences recovered and comprised \~{}0.2-6\% of the total archaeal community. Field and coculture data suggest that H(2) limitation may be partly ameliorated by H(2) syntrophy with hyperthermophilic heterotrophs. These data support our estimated H(2) threshold for hyperthermophilic methanogenesis at vents and highlight the need for coupled laboratory and field measurements to constrain microbial distribution and biogeochemical impacts in the deep sea.

}, keywords = {Archaea, Biodiversity, Coculture Techniques, DNA, Ribosomal, Ecosystem, Gases, Geography, Hydrogen, Hydrothermal Vents, Kinetics, Methane, Molecular Sequence Data, Temperature, Time Factors, Water Microbiology}, issn = {1091-6490}, doi = {10.1073/pnas.1206632109}, author = {Ver Eecke, Helene C and Butterfield, David A and Huber, Julie A and Lilley, Marvin D and Olson, Eric J and Roe, Kevin K and Evans, Leigh J and Merkel, Alexandr Y and Cantin, Holly V and Holden, James F} } @article {728, title = {Performance of a pilot-scale packed bed reactor for perchlorate reduction using a sulfur oxidizing bacterial consortium.}, journal = {Biotechnol Bioeng}, volume = {109}, year = {2012}, month = {2012 Mar}, pages = {637-46}, abstract = {A novel sulfur-utilizing perchlorate reducing bacterial consortium successfully treated perchlorate (ClO$_{4}$$^{-}$) in prior batch and bench-scale packed bed reactor (PBR) studies. This study examined the scale up of this process for treatment of water from a ClO $_{4}$$^{-}$ and RDX contaminated aquifer in Cape Cod Massachusetts. A pilot-scale upflow PBR (\~{}250-L) was constructed with elemental sulfur and crushed oyster shell packing media. The reactor was inoculated with sulfur oxidizing ClO$_{4}$$^{-}$ reducing cultures enriched from a wastewater seed. Sodium sulfite provided a good method of dissolved oxygen removal in batch cultures, but was found to promote the growth of bacteria that carry out sulfur disproportionation and sulfate reduction, which inhibited ClO$_{4}$$^{-}$ reduction in the pilot system. After terminating sulfite addition, the PBR successfully removed 96\% of the influent ClO$_{4}$$^{-}$ in the groundwater at an empty bed contact time (EBCT) of 12 h (effluent ClO$_{4}$$^{-}$ of 4.2 {\textmu}g L(-1)). Simultaneous ClO$_{4}$$^{-}$ and NO$_{3}$$^{-}$ reduction was observed in the lower half of the reactor before reactions shifted to sulfur disproportionation and sulfate reduction. Analyses of water quality profiles were supported by molecular analysis, which showed distinct groupings of ClO$_{4}$$^{-}$ and NO$_{3}$$^{-}$ degrading organisms at the inlet of the PBR, while sulfur disproportionation was the primary biological process occurring in the top potion of the reactor.}, keywords = {Bioreactors, Cluster Analysis, DNA, Bacterial, DNA, Ribosomal, Massachusetts, Microbial Consortia, Molecular Sequence Data, Oxidation-Reduction, Perchloric Acid, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Sulfites, Sulfur, Water Microbiology, Water Pollutants, Chemical, Water Purification}, issn = {1097-0290}, doi = {10.1002/bit.24354}, author = {Boles, Amber R and Conneely, Teresa and McKeever, Robert and Nixon, Paul and N{\"u}sslein, Klaus R and Ergas, Sarina J} } @article {804, title = {Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 May}, pages = {2551-63}, abstract = {Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein. The short PilA isoform is found predominantly in an intracellular fraction. It seems to stabilize the long isoform and to influence the secretion of several outer-surface c-type cytochromes. The long PilA isoform is required for secretion of PilA to the outer cell surface, a process that requires coexpression of pilA with nine downstream genes. The long isoform was determined to be essential for biofilm formation on certain surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxides.}, keywords = {Amino Acid Sequence, Bacterial Adhesion, Base Sequence, Biofilms, Cytochromes, Electron Transport, Fimbriae Proteins, Fimbriae, Bacterial, Gene Expression Regulation, Bacterial, Geobacter, Molecular Sequence Data, Protein Isoforms, Protein Transport, Reverse Transcriptase Polymerase Chain Reaction, Surface Properties}, issn = {1098-5530}, doi = {10.1128/JB.06366-11}, author = {Richter, Lubna V and Sandler, Steven J and Weis, Robert M} } @article {426, title = {Development of a biomarker for Geobacter activity and strain composition; proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI).}, journal = {Microb Biotechnol}, volume = {4}, year = {2011}, month = {2011 Jan}, pages = {55-63}, abstract = {Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy{\textquoteright}s Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample-specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)-reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Biological Markers, Citrate (si)-Synthase, Geobacter, Groundwater, Molecular Sequence Data, Phylogeny, Proteomics, Sequence Alignment, Uranium}, issn = {1751-7915}, doi = {10.1111/j.1751-7915.2010.00194.x}, author = {Wilkins, Michael J and Callister, Stephen J and Miletto, Marzia and Williams, Kenneth H and Nicora, Carrie D and Lovley, Derek R and Long, Philip E and Lipton, Mary S} } @article {374, title = {Effects of selected root exudate components on soil bacterial communities.}, journal = {FEMS Microbiol Ecol}, volume = {77}, year = {2011}, month = {2011 Sep}, pages = {600-10}, abstract = {Low-molecular-weight organic compounds in root exudates play a key role in plant-microorganism interactions by influencing the structure and function of soil microbial communities. Model exudate solutions, based on organic acids (OAs) (quinic, lactic, maleic acids) and sugars (glucose, sucrose, fructose), previously identified in the rhizosphere of Pinus radiata, were applied to soil microcosms. Root exudate compound solutions stimulated soil dehydrogenase activity and the addition of OAs increased soil pH. The structure of active bacterial communities, based on reverse-transcribed 16S rRNA gene PCR, was assessed by denaturing gradient gel electrophoresis and PhyloChip microarrays. Bacterial taxon richness was greater in all treatments than that in control soil, with a wide range of taxa (88-1043) responding positively to exudate solutions and fewer (<24) responding negatively. OAs caused significantly greater increases than sugars in the detectable richness of the soil bacterial community and larger shifts of dominant taxa. The greater response of bacteria to OAs may be due to the higher amounts of added carbon, solubilization of soil organic matter or shifts in soil pH. Our results indicate that OAs play a significant role in shaping soil bacterial communities and this may therefore have a significant impact on plant growth.}, keywords = {Bacteria, DNA, Bacterial, Molecular Sequence Data, Organic Chemicals, Phylogeny, Pinus, Plant Exudates, Plant Roots, Rhizosphere, RNA, Ribosomal, 16S, Soil, Soil Microbiology}, issn = {1574-6941}, doi = {10.1111/j.1574-6941.2011.01150.x}, author = {Shi, Shengjing and Richardson, Alan E and O{\textquoteright}Callaghan, Maureen and Deangelis, Kristen M and Jones, Eirian E and Stewart, Alison and Firestone, Mary K and Condron, Leo M} } @article {1569, title = {Field application of nitrogen and phenylacetylene to mitigate greenhouse gas emissions from landfill cover soils: effects on microbial community structure.}, journal = {Appl Microbiol Biotechnol}, volume = {89}, year = {2011}, month = {2011 Jan}, pages = {189-200}, abstract = {

Landfills are large sources of CH(4), but a considerable amount of CH(4) can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N(2)O production. To examine the effects of nitrogen and a selective inhibitor on CH(4) oxidation and N(2)O production in situ, 0.5 M of NH(4)Cl and 0.25 M of KNO(3), with and without 0.01\% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N(2)O production but had no effect on CH(4) oxidation. The addition of phenylacetylene stimulated CH(4) oxidation while reducing N(2)O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N(2)O production by selectively inhibiting AOAs and/or type II methanotrophs.

}, keywords = {Acetylene, Archaea, Archaeal Proteins, Bacteria, Bacterial Proteins, Gases, Greenhouse Effect, Methane, Molecular Sequence Data, Nitrogen, Refuse Disposal, Soil, Soil Microbiology}, issn = {1432-0614}, doi = {10.1007/s00253-010-2811-0}, author = {Im, Jeongdae and Lee, Sung-Woo and Bodrossy, Levente and Barcelona, Michael J and Semrau, Jeremy D} } @article {357, title = {Identification of a nucleopolyhedrovirus in winter moth populations from Massachusetts.}, journal = {J Invertebr Pathol}, volume = {108}, year = {2011}, month = {2011 Nov}, pages = {217-9}, abstract = {Winter moth, Operophtera brumata, originally from Europe, has invaded eastern Massachusetts causing widespread defoliation and damage to many deciduous tree species and a variety of crop plants in the infested area. We identified O. brumata nucleopolyhedrovirus (OpbuNPV) in winter moth larvae collected from field sites in Massachusetts by using PCR to amplify a 482 bp region of the baculovirus polyhedrin gene. Viral sequences were also detected in winter moth pupae that failed to emerge, suggesting that these insects may have died as a result of viral infection. This represents the first report of OpbuNPV in winter moth populations in the US.}, keywords = {Animals, Base Sequence, Environmental Monitoring, Larva, Massachusetts, Molecular Sequence Data, Moths, Nucleopolyhedrovirus, Viral Proteins, Virus Diseases}, issn = {1096-0805}, doi = {10.1016/j.jip.2011.08.005}, author = {Burand, John P and Kim, Woojin and Welch, Anna and Elkinton, Joseph S} } @article {420, title = {Molecular analysis of the metabolic rates of discrete subsurface populations of sulfate reducers.}, journal = {Appl Environ Microbiol}, volume = {77}, year = {2011}, month = {2011 Sep}, pages = {6502-9}, abstract = {Elucidating the in situ metabolic activity of phylogenetically diverse populations of sulfate-reducing microorganisms that populate anoxic sedimentary environments is key to understanding subsurface ecology. Previous pure culture studies have demonstrated that the transcript abundance of dissimilatory (bi)sulfite reductase genes is correlated with the sulfate-reducing activity of individual cells. To evaluate whether expression of these genes was diagnostic for subsurface communities, dissimilatory (bi)sulfite reductase gene transcript abundance in phylogenetically distinct sulfate-reducing populations was quantified during a field experiment in which acetate was added to uranium-contaminated groundwater. Analysis of dsrAB sequences prior to the addition of acetate indicated that Desulfobacteraceae, Desulfobulbaceae, and Syntrophaceae-related sulfate reducers were the most abundant. Quantifying dsrB transcripts of the individual populations suggested that Desulfobacteraceae initially had higher dsrB transcripts per cell than Desulfobulbaceae or Syntrophaceae populations and that the activity of Desulfobacteraceae increased further when the metabolism of dissimilatory metal reducers competing for the added acetate declined. In contrast, dsrB transcript abundance in Desulfobulbaceae and Syntrophaceae remained relatively constant, suggesting a lack of stimulation by added acetate. The indication of higher sulfate-reducing activity in the Desulfobacteraceae was consistent with the finding that Desulfobacteraceae became the predominant component of the sulfate-reducing community. Discontinuing acetate additions resulted in a decline in dsrB transcript abundance in the Desulfobacteraceae. These results suggest that monitoring transcripts of dissimilatory (bi)sulfite reductase genes in distinct populations of sulfate reducers can provide insight into the relative rates of metabolism of different components of the sulfate-reducing community and their ability to respond to environmental perturbations.}, keywords = {Biodiversity, Deltaproteobacteria, DNA, Bacterial, Gene Expression Profiling, Hydrogensulfite Reductase, Molecular Sequence Data, Oxidation-Reduction, Sequence Analysis, DNA, Soil Microbiology, Sulfates, Water Microbiology}, issn = {1098-5336}, doi = {10.1128/AEM.00576-11}, author = {Miletto, M and Williams, K H and N{\textquoteright}Guessan, A L and Lovley, D R} } @article {805, title = {Structure of the SSB-DNA polymerase III interface and its role in DNA replication.}, journal = {EMBO J}, volume = {30}, year = {2011}, month = {2011 Oct 19}, pages = {4236-47}, abstract = {Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB{\textquoteright}s association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, DNA Polymerase III, DNA Replication, DNA, Single-Stranded, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Holoenzymes, Molecular Sequence Data}, issn = {1460-2075}, doi = {10.1038/emboj.2011.305}, author = {Marceau, Aimee H and Bahng, Soon and Massoni, Shawn C and George, Nicholas P and Sandler, Steven J and Marians, Kenneth J and Keck, James L} } @article {452, title = {Genome-wide gene regulation of biosynthesis and energy generation by a novel transcriptional repressor in Geobacter species.}, journal = {Nucleic Acids Res}, volume = {38}, year = {2010}, month = {2010 Jan}, pages = {810-21}, abstract = {Geobacter species play important roles in bioremediation of contaminated environments and in electricity production from waste organic matter in microbial fuel cells. To better understand physiology of Geobacter species, expression and function of citrate synthase, a key enzyme in the TCA cycle that is important for organic acid oxidation in Geobacter species, was investigated. Geobacter sulfurreducens did not require citrate synthase for growth with hydrogen as the electron donor and fumarate as the electron acceptor. Expression of the citrate synthase gene, gltA, was repressed by a transcription factor under this growth condition. Functional and comparative genomics approaches, coupled with genetic and biochemical assays, identified a novel transcription factor termed HgtR that acts as a repressor for gltA. Further analysis revealed that HgtR is a global regulator for genes involved in biosynthesis and energy generation in Geobacter species. The hgtR gene was essential for growth with hydrogen, during which hgtR expression was induced. These findings provide important new insights into the mechanisms by which Geobacter species regulate their central metabolism under different environmental conditions.}, keywords = {Bacterial Proteins, Base Sequence, Citrate (si)-Synthase, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Molecular Sequence Data, Promoter Regions, Genetic, Repressor Proteins, Transcription, Genetic}, issn = {1362-4962}, doi = {10.1093/nar/gkp1085}, author = {Ueki, Toshiyuki and Lovley, Derek R} } @article {436, title = {Genome-wide survey for PilR recognition sites of the metal-reducing prokaryote Geobacter sulfurreducens.}, journal = {Gene}, volume = {469}, year = {2010}, month = {2010 Dec 1}, pages = {31-44}, abstract = {Geobacter sulfurreducens is a species from the bacterial family Geobacteraceae, members of which participate in bioenergy production and in environmental bioremediation. G. sulfurreducens pili are electrically conductive and are required for Fe(III) oxide reduction and for optimal current production in microbial fuel cells. PilR is an enhancer binding protein, which is an activator acting together with the alternative sigma factor, RpoN, in transcriptional regulation. Both RpoN and PilR are involved in regulation of expression of the pilA gene, whose product is pilin, a structural component of a pilus. Using bioinformatic approaches, we predicted G. sulfurreducens sequence elements that are likely to be regulated by PilR. The functional importance of the genome region containing a PilR binding site predicted upstream of the pilA gene was experimentally validated. The predicted G. sulfurreducens PilR binding sites are similar to PilR binding sites of Pseudomonas and Moraxella. While the number of predicted PilR-regulated sites did not deviate from that expected by chance, multiple sites were predicted upstream of genes with roles in biosynthesis and function of pili and flagella, in secretory pathways, and in cell wall biogenesis, suggesting the possible involvement of G. sulfurreducens PilR in regulation of production and assembly of pili and flagella.}, keywords = {Bacterial Proteins, Base Sequence, Binding Sites, Conserved Sequence, Ferric Compounds, Fimbriae Proteins, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Molecular Sequence Data, Promoter Regions, Genetic, Transcription Factors, Transcription, Genetic}, issn = {1879-0038}, doi = {10.1016/j.gene.2010.08.005}, author = {Krushkal, Julia and Ju{\'a}rez, Katy and Barbe, Jose F and Qu, Yanhua and Andrade, Angel and Puljic, Marko and Adkins, Ronald M and Lovley, Derek R and Ueki, Toshiyuki} } @article {439, title = {Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus.}, journal = {BMC Evol Biol}, volume = {10}, year = {2010}, month = {2010}, pages = {230}, abstract = {BACKGROUND: Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. RESULTS: CRISPR spacer $\#$1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer $\#$1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. CONCLUSIONS: The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer $\#$1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of CRISPR on genome-scale evolution by interference with an essential gene.}, keywords = {Base Sequence, Comparative Genomic Hybridization, Computational Biology, Deltaproteobacteria, DNA, Bacterial, DNA, Intergenic, Evolution, Molecular, Genes, Bacterial, Genome, Bacterial, Geobacillus, Histidine-tRNA Ligase, Inverted Repeat Sequences, Molecular Sequence Data, Phylogeny, Sequence Alignment, Sequence Analysis, DNA}, issn = {1471-2148}, doi = {10.1186/1471-2148-10-230}, author = {Aklujkar, Muktak and Lovley, Derek R} } @article {730, title = {Phytoprotective influence of bacteria on growth and cadmium accumulation in the aquatic plant Lemna minor.}, journal = {Water Res}, volume = {44}, year = {2010}, month = {2010 Sep}, pages = {4970-9}, abstract = {Certain plants are known to accumulate heavy metals, and can be used in remediation of polluted soil or water. Plant-associated bacteria, especially those that are metal tolerant, may enhance the total amount of metal accumulated by the plant, but this process is still unclear. In this study, we investigated metal enhancement vs. exclusion by plants, and the phytoprotective role plant-associated bacteria might provide to plants exposed to heavy metal. We isolated cadmium-tolerant bacteria from the roots of the aquatic plant Lemna minor grown in heavy metal-polluted waters, and tested these isolates for tolerance to cadmium. The efficiency of plants to accumulate heavy metal from their surrounding environment was then tested by comparing L. minor plants grown with added metal tolerant bacteria to plants grown axenically to determine, whether bacteria associated with these plants increase metal accumulation in the plant. Unexpectedly, cadmium tolerance was not seen in all bacterial isolates that had been exposed to cadmium. Axenic plants accumulated slightly more cadmium than plants inoculated with bacterial isolates. Certain isolates promoted root growth, but overall, addition of bacterial strains did not enhance plant cadmium uptake, and in some cases, inhibited cadmium accumulation by plants. This suggests that bacteria serve a phytoprotective role in their relationship with Lemna minor, preventing toxic cadmium from entering plants.}, keywords = {Adaptation, Physiological, Araceae, Bacteria, Biodegradation, Environmental, Cadmium, Hydrogen-Ion Concentration, Molecular Sequence Data, Plant Roots, Siderophores, Water}, issn = {1879-2448}, doi = {10.1016/j.watres.2010.07.073}, author = {Stout, Lisa M and Dodova, Elena N and Tyson, Julian F and N{\"u}sslein, Klaus} } @article {445, title = {Purification and characterization of OmcZ, an outer-surface, octaheme c-type cytochrome essential for optimal current production by Geobacter sulfurreducens.}, journal = {Appl Environ Microbiol}, volume = {76}, year = {2010}, month = {2010 Jun}, pages = {3999-4007}, abstract = {Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZ(L); 50-kDa) and small (OmcZ(S); 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZ(S) was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZ(S) and molecular weight measurements indicated that OmcZ(S) is a cleaved product of OmcZ(L) retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX(14)CH. The purified OmcZ(S) was remarkably thermally stable (thermal-denaturing temperature, 94.2 degrees C). Redox titration analysis revealed that the midpoint reduction potential of OmcZ(S) is approximately -220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (-420 to -60 mV). OmcZ(S) transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens.}, keywords = {Binding Sites, Bioelectric Energy Sources, Cytochromes c, Electricity, Electron Transport, Geobacter, Heme, Hot Temperature, Molecular Sequence Data, Molecular Weight, Oxidation-Reduction, Protein Binding, Protein Stability, Sequence Alignment, Sequence Analysis, Protein}, issn = {1098-5336}, doi = {10.1128/AEM.00027-10}, author = {Inoue, Kengo and Qian, Xinlei and Morgado, Leonor and Kim, Byoung-Chan and Mester, T{\"u}nde and Izallalen, Mounir and Salgueiro, Carlos A and Lovley, Derek R} } @article {462, title = {Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells.}, journal = {PLoS One}, volume = {4}, year = {2009}, month = {2009}, pages = {e5628}, abstract = {The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 microm) biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Bioelectric Energy Sources, Biofilms, Cytochromes, Electrodes, Electron Transport, Fumarates, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genetic Complementation Test, Geobacter, Microscopy, Confocal, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, RNA, Messenger, Up-Regulation}, issn = {1932-6203}, doi = {10.1371/journal.pone.0005628}, author = {Nevin, Kelly P and Kim, Byoung-Chan and Glaven, Richard H and Johnson, Jessica P and Woodard, Trevor L and Meth{\'e}, Barbara A and Didonato, Raymond J and Covalla, Sean F and Franks, Ashley E and Liu, Anna and Lovley, Derek R} } @article {377, title = {Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip arrays and 16S rRNA gene clone library sequencing.}, journal = {Appl Environ Microbiol}, volume = {75}, year = {2009}, month = {2009 Mar}, pages = {1566-74}, abstract = {The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. "Candidatus Liberibacter asiaticus" was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of "Candidatus Liberibacter asiaticus" in symptomatic leaves. These data implicate "Candidatus Liberibacter asiaticus" as the pathogen responsible for HLB disease.}, keywords = {Bacteria, Biodiversity, Citrus, DNA, Bacterial, DNA, Ribosomal, Genes, rRNA, Microarray Analysis, Molecular Sequence Data, Phylogeny, Plant Diseases, Plant Leaves, Rhizobiaceae, RNA, Bacterial, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Sequence Homology, Nucleic Acid}, issn = {1098-5336}, doi = {10.1128/AEM.02404-08}, author = {Sagaram, Uma Shankar and Deangelis, Kristen M and Trivedi, Pankaj and Andersen, Gary L and Lu, Shi-En and Wang, Nian} } @article {736, title = {Hydrogenotrophic denitrification and perchlorate reduction in ion exchange brines using membrane biofilm reactors.}, journal = {Biotechnol Bioeng}, volume = {104}, year = {2009}, month = {2009 Oct 15}, pages = {483-91}, abstract = {Halophilic (salt loving), hydrogenotrophic (H(2) oxidizing) denitrifying bacteria were investigated for treatment of nitrate (NO3-) and perchlorate (ClO4-) contaminated groundwater and ion exchange (IX) brines. Hydrogenotrophic denitrifying bacteria were enriched from a denitrifying wastewater seed under both halophilc and non-halophilc conditions. The cultures were inoculated into bench-scale membrane biofilm reactors (MBfRs) with an "outside in" configuration, with contaminated water supplied to the lumen of the membranes and H(2) supplied to the shell. Abiotic mass transfer tests showed that H(2) mass transfer coefficients were lower in brines than in tap water at highest Reynolds number, possibly due to increased transport of salts and decreased H(2) solubility at the membrane/liquid interface. An average NO3- removal efficiency of 93\% was observed for the MBfR operated in continuous flow mode with synthetic contaminated groundwater. Removal efficiencies of 30\% for NO3- and 42\% for ClO4- were observed for the MBfR operated with synthetic IX brine in batch operating mode with a reaction time of 53 h. Phylogenetic analysis focused on the active microbial community and revealed that halotolerant, NO3- -reducing bacteria of the bacterial classes Gamma-Proteobacteria and Sphingobacteria were the metabolically dominant members within the stabilized biofilm. This study shows that, despite decreased H(2) transfer under high salt conditions, hydrogenotrophic biological reduction may be successfully used for the treatment of NO3- and ClO- in a MBfR.}, keywords = {Bacteroidetes, Biofilms, Cluster Analysis, DNA, Bacterial, DNA, Ribosomal, Gammaproteobacteria, Hydrogen, Ion Exchange, Membranes, Molecular Sequence Data, Nitrites, Oxidation-Reduction, Perchloric Acid, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Water Pollutants, Chemical, Water Purification}, issn = {1097-0290}, doi = {10.1002/bit.22414}, author = {Sahu, Ashish K and Conneely, Teresa and N{\"u}sslein, Klaus and Ergas, Sarina J} } @article {767, title = {The origin of malignant malaria.}, journal = {Proc Natl Acad Sci U S A}, volume = {106}, year = {2009}, month = {2009 Sep 1}, pages = {14902-7}, abstract = {Plasmodium falciparum, the causative agent of malignant malaria, is among the most severe human infectious diseases. The closest known relative of P. falciparum is a chimpanzee parasite, Plasmodium reichenowi, of which one single isolate was previously known. The co-speciation hypothesis suggests that both parasites evolved separately from a common ancestor over the last 5-7 million years, in parallel with the divergence of their hosts, the hominin and chimpanzee lineages. Genetic analysis of eight new isolates of P. reichenowi, from wild and wild-born captive chimpanzees in Cameroon and C{\^o}te d{\textquoteright}Ivoire, shows that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite. The genetic lineage comprising the totality of global P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. This finding is inconsistent with the co-speciation hypothesis. Phylogenetic analysis indicates that all extant P. falciparum populations originated from P. reichenowi, likely by a single host transfer, which may have occurred as early as 2-3 million years ago, or as recently as 10,000 years ago. The evolutionary history of this relationship may be explained by two critical genetic mutations. First, inactivation of the CMAH gene in the human lineage rendered human ancestors unable to generate the sialic acid Neu5Gc from its precursor Neu5Ac, and likely made humans resistant to P. reichenowi. More recently, mutations in the dominant invasion receptor EBA 175 in the P. falciparum lineage provided the parasite with preference for the overabundant Neu5Ac precursor, accounting for its extreme human pathogenicity.}, keywords = {Amino Acid Sequence, Animals, Glycoproteins, Humans, Malaria, Molecular Sequence Data, Mutation, N-Acetylneuraminic Acid, Pan troglodytes, Phylogeny, Plasmodium, Plasmodium falciparum, Protozoan Infections, Animal, Protozoan Proteins, Sequence Alignment}, issn = {1091-6490}, doi = {10.1073/pnas.0907740106}, author = {Rich, Stephen M and Leendertz, Fabian H and Xu, Guang and LeBreton, Matthew and Djoko, Cyrille F and Aminake, Makoah N and Takang, Eric E and Diffo, Joseph L D and Pike, Brian L and Rosenthal, Benjamin M and Formenty, Pierre and Boesch, Christophe and Ayala, Francisco J and Wolfe, Nathan D} } @article {457, title = {Proteogenomic monitoring of Geobacter physiology during stimulated uranium bioremediation.}, journal = {Appl Environ Microbiol}, volume = {75}, year = {2009}, month = {2009 Oct}, pages = {6591-9}, abstract = {Implementation of uranium bioremediation requires methods for monitoring the membership and activities of the subsurface microbial communities that are responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a proteomics-based approach for simultaneously documenting the strain membership and microbial physiology of the dominant Geobacter community members during in situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three planktonic Geobacter-dominated samples were obtained from two wells down-gradient of acetate addition. Over 2,500 proteins from each of these samples were identified by matching liquid chromatography-tandem mass spectrometry spectra to peptides predicted from seven isolate Geobacter genomes. Genome-specific peptides indicate early proliferation of multiple M21 and Geobacter bemidjiensis-like strains and later possible emergence of M21 and G. bemidjiensis-like strains more closely related to Geobacter lovleyi. Throughout biostimulation, the proteome is dominated by enzymes that convert acetate to acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides matching tricarboxylic acid cycle proteins and ATP synthase subunits were also detected, indicating the importance of energy generation during the period of rapid growth following the start of biostimulation. Evolving Geobacter strain composition may be linked to changes in protein abundance over the course of biostimulation and may reflect changes in metabolic functioning. Thus, metagenomics-independent community proteogenomics can be used to diagnose the status of the subsurface consortia upon which remediation biotechnology relies.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Genomics, Geobacter, Molecular Sequence Data, Oxidation-Reduction, Peptide Mapping, Plankton, Proteomics, Uranium, Water Microbiology, Water Pollutants, Radioactive}, issn = {1098-5336}, doi = {10.1128/AEM.01064-09}, author = {Wilkins, Michael J and VerBerkmoes, Nathan C and Williams, Kenneth H and Callister, Stephen J and Mouser, Paula J and Elifantz, Hila and N{\textquoteright}guessan, Lucie A and Thomas, Brian C and Nicora, Carrie D and Shah, Manesh B and Abraham, Paul and Lipton, Mary S and Lovley, Derek R and Hettich, Robert L and Long, Philip E and Banfield, Jillian F} } @article {471, title = {Transcriptome of Geobacter uraniireducens growing in uranium-contaminated subsurface sediments.}, journal = {ISME J}, volume = {3}, year = {2009}, month = {2009 Feb}, pages = {216-30}, abstract = {To learn more about the physiological state of Geobacter species living in subsurface sediments, heat-sterilized sediments from a uranium-contaminated aquifer in Rifle, Colorado, were inoculated with Geobacter uraniireducens, a pure culture representative of the Geobacter species that predominates during in situ uranium bioremediation at this site. Whole-genome microarray analysis comparing sediment-grown G. uraniireducens with cells grown in defined culture medium indicated that there were 1084 genes that had higher transcript levels during growth in sediments. Thirty-four c-type cytochrome genes were upregulated in the sediment-grown cells, including several genes that are homologous to cytochromes that are required for optimal Fe(III) and U(VI) reduction by G. sulfurreducens. Sediment-grown cells also had higher levels of transcripts, indicative of such physiological states as nitrogen limitation, phosphate limitation and heavy metal stress. Quantitative reverse transcription PCR showed that many of the metabolic indicator genes that appeared to be upregulated in sediment-grown G. uraniireducens also showed an increase in expression in the natural community of Geobacter species present during an in situ uranium bioremediation field experiment at the Rifle site. These results demonstrate that it is feasible to monitor gene expression of a microorganism growing in sediments on a genome scale and that analysis of the physiological status of a pure culture growing in subsurface sediments can provide insights into the factors controlling the physiology of natural subsurface communities.}, keywords = {Colorado, DNA, Bacterial, Environmental Microbiology, Gene Expression Profiling, Geobacter, Geologic Sediments, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Sequence Analysis, DNA, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2008.89}, author = {Holmes, Dawn E and O{\textquoteright}Neil, Regina A and Chavan, Milind A and N{\textquoteright}guessan, Lucie A and Vrionis, Helen A and Perpetua, Lorrie A and Larrahondo, M Juliana and DiDonato, Raymond and Liu, Anna and Lovley, Derek R} } @article {470, title = {Comparative genomics of Geobacter chemotaxis genes reveals diverse signaling function.}, journal = {BMC Genomics}, volume = {9}, year = {2008}, month = {2008}, pages = {471}, abstract = {BACKGROUND: Geobacter species are delta-Proteobacteria and are often the predominant species in a variety of sedimentary environments where Fe(III) reduction is important. Their ability to remediate contaminated environments and produce electricity makes them attractive for further study. Cell motility, biofilm formation, and type IV pili all appear important for the growth of Geobacter in changing environments and for electricity production. Recent studies in other bacteria have demonstrated that signaling pathways homologous to the paradigm established for Escherichia coli chemotaxis can regulate type IV pili-dependent motility, the synthesis of flagella and type IV pili, the production of extracellular matrix material, and biofilm formation. The classification of these pathways by comparative genomics improves the ability to understand how Geobacter thrives in natural environments and better their use in microbial fuel cells. RESULTS: The genomes of G. sulfurreducens, G. metallireducens, and G. uraniireducens contain multiple (approximately 70) homologs of chemotaxis genes arranged in several major clusters (six, seven, and seven, respectively). Unlike the single gene cluster of E. coli, the Geobacter clusters are not all located near the flagellar genes. The probable functions of some Geobacter clusters are assignable by homology to known pathways; others appear to be unique to the Geobacter sp. and contain genes of unknown function. We identified large numbers of methyl-accepting chemotaxis protein (MCP) homologs that have diverse sensing domain architectures and generate a potential for sensing a great variety of environmental signals. We discuss mechanisms for class-specific segregation of the MCPs in the cell membrane, which serve to maintain pathway specificity and diminish crosstalk. Finally, the regulation of gene expression in Geobacter differs from E. coli. The sequences of predicted promoter elements suggest that the alternative sigma factors sigma28 and sigma54 play a role in regulating the Geobacter chemotaxis gene expression. CONCLUSION: The numerous chemoreceptors and chemotaxis-like gene clusters of Geobacter appear to be responsible for a diverse set of signaling functions in addition to chemotaxis, including gene regulation and biofilm formation, through functionally and spatially distinct signaling pathways.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Chemotaxis, Escherichia coli, Gene Expression Regulation, Bacterial, Genome, Bacterial, Genomics, Geobacter, Membrane Proteins, Molecular Sequence Data, Multigene Family, Promoter Regions, Genetic, Sequence Alignment, Sequence Homology, Amino Acid}, issn = {1471-2164}, doi = {10.1186/1471-2164-9-471}, author = {Tran, Hoa T and Krushkal, Julia and Antommattei, Frances M and Lovley, Derek R and Weis, Robert M} } @article {489, title = {Computational and experimental analysis of redundancy in the central metabolism of Geobacter sulfurreducens.}, journal = {PLoS Comput Biol}, volume = {4}, year = {2008}, month = {2008 Feb}, pages = {e36}, abstract = {Previous model-based analysis of the metabolic network of Geobacter sulfurreducens suggested the existence of several redundant pathways. Here, we identified eight sets of redundant pathways that included redundancy for the assimilation of acetate, and for the conversion of pyruvate into acetyl-CoA. These equivalent pathways and two other sub-optimal pathways were studied using 5 single-gene deletion mutants in those pathways for the evaluation of the predictive capacity of the model. The growth phenotypes of these mutants were studied under 12 different conditions of electron donor and acceptor availability. The comparison of the model predictions with the resulting experimental phenotypes indicated that pyruvate ferredoxin oxidoreductase is the only activity able to convert pyruvate into acetyl-CoA. However, the results and the modeling showed that the two acetate activation pathways present are not only active, but needed due to the additional role of the acetyl-CoA transferase in the TCA cycle, probably reflecting the adaptation of these bacteria to acetate utilization. In other cases, the data reconciliation suggested additional capacity constraints that were confirmed with biochemical assays. The results demonstrate the need to experimentally verify the activity of key enzymes when developing in silico models of microbial physiology based on sequence-based reconstruction of metabolic networks.}, keywords = {Bacterial Proteins, Base Sequence, Computer Simulation, Gene Expression Regulation, Bacterial, Geobacter, Models, Biological, Molecular Sequence Data, Multienzyme Complexes, Signal Transduction}, issn = {1553-7358}, doi = {10.1371/journal.pcbi.0040036}, author = {Segura, Daniel and Mahadevan, Radhakrishnan and Ju{\'a}rez, Katy and Lovley, Derek R} } @article {483, title = {Genes for two multicopper proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes.}, journal = {Microbiology}, volume = {154}, year = {2008}, month = {2008 May}, pages = {1422-35}, abstract = {Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 \% similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.}, keywords = {Acetates, Amino Acid Sequence, Bacterial Outer Membrane Proteins, Electrodes, Ferric Compounds, Gene Deletion, Gene Expression Profiling, Geobacter, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, Sequence Alignment, Sequence Homology, Nucleic Acid, Soil Microbiology, Uranium}, issn = {1350-0872}, doi = {10.1099/mic.0.2007/014365-0}, author = {Holmes, Dawn E and Mester, T{\"u}nde and O{\textquoteright}Neil, Regina A and Perpetua, Lorrie A and Larrahondo, M Juliana and Glaven, Richard and Sharma, Manju L and Ward, Joy E and Nevin, Kelly P and Lovley, Derek R} } @article {484, title = {Geobacter uraniireducens sp. nov., isolated from subsurface sediment undergoing uranium bioremediation.}, journal = {Int J Syst Evol Microbiol}, volume = {58}, year = {2008}, month = {2008 May}, pages = {1075-8}, abstract = {A Gram-negative, rod-shaped, motile bacterium, strain Rf4T, which conserves energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation, was isolated from subsurface sediment undergoing uranium bioremediation. The 16S rRNA gene sequence of strain Rf4T matched sequences recovered in 16S rRNA gene clone libraries constructed from DNA extracted from groundwater sampled at the same time as the source sediment. Cells of strain Rf4T were regular, motile rods, 1.2-2.0 microm long and 0.5-0.6 microm in diameter, with rounded ends. Cells had one lateral flagellum. Growth was optimal at pH 6.5-7.0 and 32 degrees C. With acetate as the electron donor, strain Rf4T used Fe(III), Mn(IV), anthraquinone-2,6-disulfonate, malate and fumarate as electron acceptors and reduced U(VI) in cell suspensions. With poorly crystalline Fe(III) oxide as the electron acceptor, strain Rf4T oxidized the following electron donors: acetate, lactate, pyruvate and ethanol. Phylogenetic analysis of the 16S rRNA gene sequence of strain Rf4T placed it in the genus Geobacter. Strain Rf4T was most closely related to {\textquoteright}Geobacter humireducens{\textquoteright} JW3 (95.9 \% sequence similarity), Geobacter bremensis Dfr1T (95.4 \%) and Geobacter bemidjiensis BemT (95.1 \%). Based on phylogenetic analysis and phenotypic differences between strain Rf4T and closely related Geobacter species, this strain is described as a representative of a novel species, Geobacter uraniireducens sp. nov. The type strain is Rf4T (=ATCC BAA-1134T =JCM 13001T).}, keywords = {Bacterial Typing Techniques, DNA, Bacterial, Genes, rRNA, Genotype, Geobacter, Geologic Sediments, Molecular Sequence Data, Oxidation-Reduction, Phenotype, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Species Specificity, Uranium}, issn = {1466-5026}, doi = {10.1099/ijs.0.65377-0}, author = {Shelobolina, Evgenya S and Vrionis, Helen A and Findlay, Robert H and Lovley, Derek R} } @article {500, title = {Quantifying expression of a dissimilatory (bi)sulfite reductase gene in petroleum-contaminated marine harbor sediments.}, journal = {Microb Ecol}, volume = {55}, year = {2008}, month = {2008 Apr}, pages = {489-99}, abstract = {The possibility of quantifying in situ levels of transcripts for dissimilatory (bi)sulfite reductase (dsr) genes to track the activity of sulfate-reducing microorganisms in petroleum-contaminated marine harbor sediments was evaluated. Phylogenetic analysis of the cDNA generated from mRNA for a ca. 1.4 kbp portion of the contiguous dsrA and dsrB genes suggested that Desulfosarcina species, closely related to cultures known to anaerobically oxidize aromatic hydrocarbons, were active sulfate reducers in the sediments. The levels of dsrA transcripts (per mug total mRNA) were quantified in sediments incubated anaerobically at the in situ temperature as well as in sediments incubated at higher temperatures and/or with added acetate to increase the rate of sulfate reduction. Levels of dsrA transcripts were low when there was no sulfate reduction because the sediments were depleted of sulfate or if sulfate reduction was inhibited with added molybdate. There was a direct correlation between dsrA transcript levels and rates of sulfate reduction when sulfate was at ca. 10 mM in the various sediment treatments, but it was also apparent that within a given sediment, dsrA levels increased over time as long as sulfate was available, even when sulfate reduction rates did not increase. These results suggest that phylogenetic analysis of dsr transcript sequences may provide insight into the active sulfate reducers in marine sediments and that quantifying levels of dsrA transcripts can indicate whether sulfate reducers are active in particular sediment. Furthermore, it may only be possible to use dsrA transcript levels to compare the relative rates of sulfate reduction in sediments when sulfate concentrations, and possibly other environmental conditions, are comparable.}, keywords = {Anaerobiosis, Desulfitobacterium, DNA, Bacterial, DNA, Ribosomal, Gene Expression, Geologic Sediments, Hydrogensulfite Reductase, Molecular Sequence Data, Petroleum, Phylogeny, RNA, Bacterial, RNA, Messenger, RNA, Ribosomal, 16S, Temperature}, issn = {0095-3628}, doi = {10.1007/s00248-007-9294-2}, author = {Chin, Kuk-Jeong and Sharma, Manju L and Russell, Lyndsey A and O{\textquoteright}Neill, Kathleen R and Lovley, Derek R} } @article {746, title = {Comparative bacterial diversity in recent Hawaiian volcanic deposits of different ages.}, journal = {FEMS Microbiol Ecol}, volume = {60}, year = {2007}, month = {2007 Apr}, pages = {60-73}, abstract = {Volcanic activity creates new landforms that can change dramatically over time as a consequence of biotic succession. Nonetheless, volcanic deposits present severe constraints for microbial colonization and activity. We have characterized bacterial diversity on four recent deposits at Kilauea volcano, Hawaii (KVD). Much of the diversity was either closely related to uncultured organisms or distinct from any reported 16S rRNA gene sequences. Diversity indices suggested that diversity was highest in a moderately vegetated 210-year-old ash deposit (1790-KVD), and lowest for a 79-year-old lava flow (1921-KVD). Diversity for a 41-year-old tephra deposit (1959-KVD) and a 300-year-old rainforest (1700-KVD) reached intermediate values. The 1959-KVD and 1790-KVD communities were dominated by Acidobacteria, Alpha- and Gammaproteobacteria, Actinobacteria, Cyanobacteria, and many unclassified phylotypes. The 1921-KVD, an unvegetated low pH deposit, was dominated by unclassified phylotypes. In contrast, 1700-KVD was primarily populated by Alphaproteobacteria with very few unclassified phylotypes. Similar diversity indices and levels of trace gas flux were found for 1959-KVD and 1790-KVD; however, statistical analyses indicated significantly different communities. This study not only showed that microorganisms colonize recent volcanic deposits and are able to establish diverse communities, but also that their composition is governed by variations in local deposit parameters.}, keywords = {Bacteria, DNA, Bacterial, Ecosystem, Genes, rRNA, Genetic Variation, Hawaii, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Soil Microbiology, Time Factors, Volcanic Eruptions}, issn = {0168-6496}, doi = {10.1111/j.1574-6941.2006.00253.x}, author = {Gomez-Alvarez, Vicente and King, Gary M and N{\"u}sslein, Klaus} } @article {508, title = {Geobacter pickeringii sp. nov., Geobacter argillaceus sp. nov. and Pelosinus fermentans gen. nov., sp. nov., isolated from subsurface kaolin lenses.}, journal = {Int J Syst Evol Microbiol}, volume = {57}, year = {2007}, month = {2007 Jan}, pages = {126-35}, abstract = {The goal of this project was to isolate representative Fe(III)-reducing bacteria from kaolin clays that may influence iron mineralogy in kaolin. Two novel dissimilatory Fe(III)-reducing bacteria, strains G12(T) and G13(T), were isolated from sedimentary kaolin strata in Georgia (USA). Cells of strains G12(T) and G13(T) were motile, non-spore-forming regular rods, 1-2 mum long and 0.6 mum in diameter. Cells had one lateral flagellum. Phylogenetic analyses using the 16S rRNA gene sequence of the novel strains demonstrated their affiliation to the genus Geobacter. Strain G12(T) was most closely related to Geobacter pelophilus (94.7 \%) and Geobacter chapellei (94.1 \%). Strain G13(T) was most closely related to Geobacter grbiciae (95.3 \%) and Geobacter metallireducens (95.1 \%). Based on phylogenetic analyses and phenotypic differences between the novel isolates and other closely related species of the genus Geobacter, the isolates are proposed as representing two novel species, Geobacter argillaceus sp. nov. (type strain G12(T)=ATCC BAA-1139(T)=JCM 12999(T)) and Geobacter pickeringii sp. nov. (type strain G13(T)=ATCC BAA-1140(T)=DSM 17153(T)=JCM 13000(T)). Another isolate, strain R7(T), was derived from a primary kaolin deposit in Russia. The cells of strain R7(T) were motile, spore-forming, slightly curved rods, 0.6 x 2.0-6.0 microm in size and with up to six peritrichous flagella. Strain R7(T) was capable of reducing Fe(III) only in the presence of a fermentable substrate. 16S rRNA gene sequence analysis demonstrated that this isolate is unique, showing less than 92 \% similarity to bacteria of the Sporomusa-Pectinatus-Selenomomas phyletic group, including {\textquoteright}Anaerospora hongkongensis{\textquoteright} (90.2 \%), Acetonema longum (90.6 \%), Dendrosporobacter quercicolus (90.9 \%) and Anaerosinus glycerini (91.5 \%). On the basis of phylogenetic analysis and physiological tests, strain R7(T) is proposed to represent a novel genus and species, Pelosinus fermentans gen. nov., sp. nov. (type strain R7(T)=DSM 17108(T)=ATCC BAA-1133(T)), in the Sporomusa-Pectinatus-Selenomonas group.}, keywords = {Bacterial Typing Techniques, Base Composition, DNA, Bacterial, DNA, Ribosomal, Ferric Compounds, Genes, rRNA, Geobacter, Geologic Sediments, Georgia, Kaolin, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Russia, Sequence Analysis, DNA, Species Specificity}, issn = {1466-5026}, doi = {10.1099/ijs.0.64221-0}, author = {Shelobolina, Evgenya S and Nevin, Kelly P and Blakeney-Hayward, Jessie D and Johnsen, Claudia V and Plaia, Todd W and Krader, Paul and Woodard, Trevor and Holmes, Dawn E and Vanpraagh, Catherine Gaw and Lovley, Derek R} } @article {507, title = {Heat-shock sigma factor RpoH from Geobacter sulfurreducens.}, journal = {Microbiology}, volume = {153}, year = {2007}, month = {2007 Mar}, pages = {838-46}, abstract = {Recent studies with Myxococcus xanthus have suggested that homologues of the Escherichia coli heat-shock sigma factor, RpoH, may not be involved in the heat-shock response in this delta-proteobacterium. The genome of another delta-proteobacterium, Geobacter sulfurreducens, which is considered to be a representative of the Fe(III)-reducing Geobacteraceae that predominate in a diversity of subsurface environments, contains an rpoH homologue. Characterization of the G. sulfurreducens rpoH homologue revealed that it was induced by a temperature shift from 30 degrees C to 42 degrees C and that an rpoH-deficient mutant was unable to grow at 42 degrees C. The predicted heat-shock genes, hrcA, grpE, dnaK, groES and htpG, were heat-shock inducible in an rpoH-dependent manner, and comparison of promoter regions of these genes identified the consensus sequences for the -10 and -35 promoter elements. In addition, DNA elements identical to the CIRCE consensus sequence were found in promoters of rpoH, hrcA and groES, suggesting that these genes are regulated by a homologue of the repressor HrcA, which is known to bind the CIRCE element. These results suggest that the G. sulfurreducens RpoH homologue is the heat-shock sigma factor and that heat-shock response in G. sulfurreducens is regulated positively by RpoH as well as negatively by the HrcA/CIRCE system.}, keywords = {Adaptation, Physiological, Base Sequence, Binding Sites, Consensus Sequence, DNA, Bacterial, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Heat-Shock Proteins, Hot Temperature, Molecular Sequence Data, Promoter Regions, Genetic, RNA, Bacterial, RNA, Messenger, Sigma Factor, Transcription, Genetic}, issn = {1350-0872}, doi = {10.1099/mic.0.2006/000638-0}, author = {Ueki, Toshiyuki and Lovley, Derek R} } @article {681, title = {Pepitope: epitope mapping from affinity-selected peptides.}, journal = {Bioinformatics}, volume = {23}, year = {2007}, month = {2007 Dec 1}, pages = {3244-6}, abstract = {Identifying the epitope to which an antibody binds is central for many immunological applications such as drug design and vaccine development. The Pepitope server is a web-based tool that aims at predicting discontinuous epitopes based on a set of peptides that were affinity-selected against a monoclonal antibody of interest. The server implements three different algorithms for epitope mapping: PepSurf, Mapitope, and a combination of the two. The rationale behind these algorithms is that the set of peptides mimics the genuine epitope in terms of physicochemical properties and spatial organization. When the three-dimensional (3D) structure of the antigen is known, the information in these peptides can be used to computationally infer the corresponding epitope. A user-friendly web interface and a graphical tool that allows viewing the predicted epitopes were developed. Pepitope can also be applied for inferring other types of protein-protein interactions beyond the immunological context, and as a general tool for aligning linear sequences to a 3D structure. AVAILABILITY: http://pepitope.tau.ac.il/}, keywords = {Algorithms, Amino Acid Sequence, Binding Sites, Epitope Mapping, Molecular Sequence Data, Peptides, Protein Binding, Sequence Alignment, Sequence Analysis, Protein, Software}, issn = {1367-4811}, doi = {10.1093/bioinformatics/btm493}, author = {Mayrose, Itay and Penn, Osnat and Erez, Elana and Rubinstein, Nimrod D and Shlomi, Tomer and Freund, Natalia Tarnovitski and Bublil, Erez M and Ruppin, Eytan and Sharan, Roded and Gershoni, Jonathan M and Martz, Eric and Pupko, Tal} } @article {504, title = {Prolixibacter bellariivorans gen. nov., sp. nov., a sugar-fermenting, psychrotolerant anaerobe of the phylum Bacteroidetes, isolated from a marine-sediment fuel cell.}, journal = {Int J Syst Evol Microbiol}, volume = {57}, year = {2007}, month = {2007 Apr}, pages = {701-7}, abstract = {A Gram-negative, non-motile, filamentous, rod-shaped, non-spore-forming bacterium (strain F2(T)) was isolated from the surface of an electricity-harvesting electrode incubated in marine sediments. Strain F2(T) does not contain c-type cytochromes, flexirubin or carotenoids. It is a facultative anaerobe that can ferment sugars by using a mixed acid fermentation pathway and it can grow over a wide range of temperatures (4-42 degrees C). The DNA G+C (44.9 mol\%) content and chemotaxonomic characteristics (major fatty acids, a-15 : 0 and 15 : 0) were consistent with those of species within the phylum Bacteroidetes. Phylogenetic analysis of the 16S rRNA nucleotide and elongation factor G amino acid sequences indicated that strain F2(T) represents a unique phylogenetic cluster within the phylum Bacteroidetes. On the basis of 16S rRNA gene sequence phylogeny, the closest relative available in pure culture, Alkaliflexus imshenetskii, is only 87.5 \% similar to strain F2(T). Results from physiological, biochemical and phylogenetic analyses showed that strain F2(T) should be classified as a novel genus and species within the phylum Bacteroidetes, for which the name Prolixibacter bellariivorans gen. nov., sp. nov. is proposed. The type strain is F2(T) (=ATCC BAA-1284(T)=JCM 13498(T)).}, keywords = {Bacteroidetes, Carbohydrate Metabolism, Cold Temperature, DNA, Bacterial, DNA, Ribosomal, Energy-Generating Resources, Geologic Sediments, Molecular Sequence Data, Phylogeny, RNA, Ribosomal, 16S, Seawater}, issn = {1466-5026}, doi = {10.1099/ijs.0.64296-0}, author = {Holmes, Dawn E and Nevin, Kelly P and Woodard, Trevor L and Peacock, Aaron D and Lovley, Derek R} } @article {744, title = {Salinity constraints on subsurface archaeal diversity and methanogenesis in sedimentary rock rich in organic matter.}, journal = {Appl Environ Microbiol}, volume = {73}, year = {2007}, month = {2007 Jul}, pages = {4171-9}, abstract = {The diversity of microorganisms active within sedimentary rocks provides important controls on the geochemistry of many subsurface environments. In particular, biodegradation of organic matter in sedimentary rocks contributes to the biogeochemical cycling of carbon and other elements and strongly impacts the recovery and quality of fossil fuel resources. In this study, archaeal diversity was investigated along a salinity gradient spanning 8 to 3,490 mM Cl(-) in a subsurface shale rich in CH(4) derived from biodegradation of sedimentary hydrocarbons. Shale pore waters collected from wells in the main CH(4)-producing zone lacked electron acceptors such as O(2), NO(3)(-), Fe(3+), or SO(4)(2-). Acetate was detected only in high-salinity waters, suggesting that acetoclastic methanogenesis is inhibited at Cl(-) concentrations above approximately 1,000 mM. Most-probable-number series revealed differences in methanogen substrate utilization (acetate, trimethylamine, or H(2)/CO(2)) associated with chlorinity. The greatest methane production in enrichment cultures was observed for incubations with salinity at or close to the native pore water salinity of the inoculum. Restriction fragment length polymorphism analyses of archaeal 16S rRNA genes from seven wells indicated that there were links between archaeal communities and pore water salinity. Archaeal clone libraries constructed from sequences from 16S rRNA genes isolated from two wells revealed phylotypes similar to a halophilic methylotrophic Methanohalophilus species and a hydrogenotrophic Methanoplanus species at high salinity and a single phylotype closely related to Methanocorpusculum bavaricum at low salinity. These results show that several distinct communities of methanogens persist in this subsurface, CH(4)-producing environment and that each community is adapted to particular conditions of salinity and preferential substrate use and each community induces distinct geochemical signatures in shale formation waters.}, keywords = {Archaea, Base Sequence, Cloning, Molecular, DNA Primers, Genes, Archaeal, Genetic Variation, Geologic Sediments, Methane, Michigan, Molecular Sequence Data, Organic Chemicals, Phylogeny, RNA, Archaeal, RNA, Ribosomal, 16S, Sodium Chloride, Soil, Soil Microbiology, Water Microbiology}, issn = {0099-2240}, doi = {10.1128/AEM.02810-06}, author = {Waldron, Patricia J and Petsch, Steven T and Martini, Anna M and N{\"u}sslein, Klaus and N{\"u}slein, Klaus} } @article {682, title = {Selecton 2007: advanced models for detecting positive and purifying selection using a Bayesian inference approach.}, journal = {Nucleic Acids Res}, volume = {35}, year = {2007}, month = {2007 Jul}, pages = {W506-11}, abstract = {Biologically significant sites in a protein may be identified by contrasting the rates of synonymous (K(s)) and non-synonymous (K(a)) substitutions. This enables the inference of site-specific positive Darwinian selection and purifying selection. We present here Selecton version 2.2 (http://selecton.bioinfo.tau.ac.il), a web server which automatically calculates the ratio between K(a) and K(s) (omega) at each site of the protein. This ratio is graphically displayed on each site using a color-coding scheme, indicating either positive selection, purifying selection or lack of selection. Selecton implements an assembly of different evolutionary models, which allow for statistical testing of the hypothesis that a protein has undergone positive selection. Specifically, the recently developed mechanistic-empirical model is introduced, which takes into account the physicochemical properties of amino acids. Advanced options were introduced to allow maximal fine tuning of the server to the user{\textquoteright}s specific needs, including calculation of statistical support of the omega values, an advanced graphic display of the protein{\textquoteright}s 3-dimensional structure, use of different genetic codes and inputting of a pre-built phylogenetic tree. Selecton version 2.2 is an effective, user-friendly and freely available web server which implements up-to-date methods for computing site-specific selection forces, and the visualization of these forces on the protein{\textquoteright}s sequence and structure.}, keywords = {Algorithms, Amino Acid Sequence, Amino Acid Substitution, Amino Acids, Animals, Bayes Theorem, Computational Biology, Computer Simulation, Humans, Internet, Models, Molecular, Molecular Sequence Data, Phylogeny, Proteins, Sequence Analysis, Protein, Software}, issn = {1362-4962}, doi = {10.1093/nar/gkm382}, author = {Stern, Adi and Doron-Faigenboim, Adi and Erez, Elana and Martz, Eric and Bacharach, Eran and Pupko, Tal} } @article {493, title = {Subsurface clade of Geobacteraceae that predominates in a diversity of Fe(III)-reducing subsurface environments.}, journal = {ISME J}, volume = {1}, year = {2007}, month = {2007 Dec}, pages = {663-77}, abstract = {There are distinct differences in the physiology of Geobacter species available in pure culture. Therefore, to understand the ecology of Geobacter species in subsurface environments, it is important to know which species predominate. Clone libraries were assembled with 16S rRNA genes and transcripts amplified from three subsurface environments in which Geobacter species are known to be important members of the microbial community: (1) a uranium-contaminated aquifer located in Rifle, CO, USA undergoing in situ bioremediation; (2) an acetate-impacted aquifer that serves as an analog for the long-term acetate amendments proposed for in situ uranium bioremediation and (3) a petroleum-contaminated aquifer in which Geobacter species play a role in the oxidation of aromatic hydrocarbons coupled with the reduction of Fe(III). The majority of Geobacteraceae 16S rRNA sequences found in these environments clustered in a phylogenetically coherent subsurface clade, which also contains a number of Geobacter species isolated from subsurface environments. Concatamers constructed with 43 Geobacter genes amplified from these sites also clustered within this subsurface clade. 16S rRNA transcript and gene sequences in the sediments and groundwater at the Rifle site were highly similar, suggesting that sampling groundwater via monitoring wells can recover the most active Geobacter species. These results suggest that further study of Geobacter species in the subsurface clade is necessary to accurately model the behavior of Geobacter species during subsurface bioremediation of metal and organic contaminants.}, keywords = {Biodegradation, Environmental, Ecosystem, Ferric Compounds, Geobacter, Hydrocarbons, Aromatic, Molecular Sequence Data, Oxidation-Reduction, Petroleum, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Uranium}, issn = {1751-7362}, doi = {10.1038/ismej.2007.85}, author = {Holmes, Dawn E and O{\textquoteright}Neil, Regina A and Vrionis, Helen A and N{\textquoteright}guessan, Lucie A and Ortiz-Bernad, Irene and Larrahondo, Maria J and Adams, Lorrie A and Ward, Joy A and Nicoll, Julie S and Nevin, Kelly P and Chavan, Milind A and Johnson, Jessica P and Long, Philip E and Lovley, Derek R} } @article {817, title = {UvrD limits the number and intensities of RecA-green fluorescent protein structures in Escherichia coli K-12.}, journal = {J Bacteriol}, volume = {189}, year = {2007}, month = {2007 Apr}, pages = {2915-20}, abstract = {RecA is important for recombination, DNA repair, and SOS induction. In Escherichia coli, RecBCD, RecFOR, and RecJQ prepare DNA substrates onto which RecA binds. UvrD is a 3{\textquoteright}-to-5{\textquoteright} helicase that participates in methyl-directed mismatch repair and nucleotide excision repair. uvrD deletion mutants are sensitive to UV irradiation, hypermutable, and hyper-rec. In vitro, UvrD can dissociate RecA from single-stranded DNA. Other experiments suggest that UvrD removes RecA from DNA where it promotes unproductive reactions. To test if UvrD limits the number and/or the size of RecA-DNA structures in vivo, an uvrD mutation was combined with recA-gfp. This recA allele allows the number of RecA structures and the amount of RecA at these structures to be assayed in living cells. uvrD mutants show a threefold increase in the number of RecA-GFP foci, and these foci are, on average, nearly twofold higher in relative intensity. The increased number of RecA-green fluorescent protein foci in the uvrD mutant is dependent on recF, recO, recR, recJ, and recQ. The increase in average relative intensity is dependent on recO and recQ. These data support an in vivo role for UvrD in removing RecA from the DNA.}, keywords = {DNA Helicases, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli K12, Escherichia coli Proteins, Green Fluorescent Proteins, Molecular Sequence Data, Mutagenesis, Rec A Recombinases, Recombinant Fusion Proteins}, issn = {0021-9193}, doi = {10.1128/JB.01777-06}, author = {Centore, Richard C and Sandler, Steven J} } @article {38, title = {Analysis of Opi1p repressor mutants.}, journal = {Curr Genet}, volume = {49}, year = {2006}, month = {2006 Jan}, pages = {30-8}, abstract = {Opi1p is the only known repressor protein specific to the phospholipid biosynthetic pathway. Opi1p is required for repression in response to inositol and choline supplementation. However, the mechanism of Opi1p repression is not completely understood. In part, this is because previously identified opi1 mutants contained nonsense mutations and thus provided little insight into the mechanism of Opi1p function. We have recently reported isolating novel opi1 mutants (rum and dim mutants) that contain missense mutations. Here, we show that these opi1 mutants produce Opi1p product at levels comparable to a wild-type strain. However, these mutants mis-regulate expression of two target genes, INO2-HIS3 and INO1-lacZ, and are also defective in autoregulation. An opi1-S339F mutant is particularly interesting because it completely eliminated autoregulation, but only abated regulation of an INO1-lacZ reporter. Two of the mutations in OPI1 (V343Q and S339F) provide genetic evidence for an interaction between Opi1p and the Ino2p activator since they reside in a region of Opi1p recently shown to interact with Ino2p in vitro. A third mutation (L252F) resides in a region of Opi1p with no known function.}, keywords = {Amino Acid Sequence, Basic Helix-Loop-Helix Transcription Factors, Conserved Sequence, Molecular Sequence Data, Mutation, Missense, Myo-Inositol-1-Phosphate Synthase, Repressor Proteins, Saccharomyces cerevisiae Proteins, Transcription Factors}, issn = {0172-8083}, doi = {10.1007/s00294-005-0021-6}, author = {Kaadige, Mohan R and Lopes, John M} } @article {512, title = {Computational prediction of RpoS and RpoD regulatory sites in Geobacter sulfurreducens using sequence and gene expression information.}, journal = {Gene}, volume = {384}, year = {2006}, month = {2006 Dec 15}, pages = {73-95}, abstract = {RpoS, the sigma S subunit of RNA polymerase, is vital during the growth and survival of Geobacter sulfurreducens under conditions typically encountered in its native subsurface environments. We investigated the conservation of sites that may be important for RpoS function in G. sulfurreducens. We also employed sequence information and expression microarray data to predict G. sulfurreducens genome sites that may be related to RpoS regulation. Hierarchical clustering identified three clusters of significantly downregulated genes in the rpoS deletion mutant. The search for conserved overrepresented motifs in co-regulated operons identified likely -35 and -10 promoter elements upstream of a number of functionally important G. sulfurreducens operons that were downregulated in the rpoS deletion mutant. Putative -35/-10 promoter elements were also identified in the G. sulfurreducens genome using sequence similarity searches to matrices of -35/-10 promoter elements found in G. sulfurreducens and in Escherichia coli. Due to a sufficient degree of sequence similarity between -35/-10 promoter elements for RpoS, RpoD, and other sigma factors, both the sequence similarity searches and the search for conserved overrepresented motifs using microarray data may identify promoter elements for both RpoS and other sigma factors.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, Citrates, Computational Biology, Conserved Sequence, DNA-Directed RNA Polymerases, Escherichia coli, Escherichia coli Proteins, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genome, Bacterial, Geobacter, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Operon, Promoter Regions, Genetic, Sigma Factor, Transcription, Genetic}, issn = {0378-1119}, doi = {10.1016/j.gene.2006.06.025}, author = {Yan, Bin and N{\'u}{\~n}ez, Cinthia and Ueki, Toshiyuki and Esteve-N{\'u}{\~n}ez, Abraham and Puljic, Marko and Adkins, Ronald M and Meth{\'e}, Barbara A and Lovley, Derek R and Krushkal, Julia} } @article {525, title = {Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens.}, journal = {J Bacteriol}, volume = {188}, year = {2006}, month = {2006 Jan}, pages = {450-5}, abstract = {The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.}, keywords = {Culture Media, Dicarboxylic Acids, Fumarates, Geobacter, Molecular Sequence Data, Operon, Oxidation-Reduction, Recombinant Proteins, Substrate Specificity, Succinate Dehydrogenase, Succinic Acid}, issn = {0021-9193}, doi = {10.1128/JB.188.2.450-455.2006}, author = {Butler, Jessica E and Glaven, Richard H and Esteve-N{\'u}{\~n}ez, Abraham and N{\'u}{\~n}ez, Cinthia and Shelobolina, Evgenya S and Bond, Daniel R and Lovley, Derek R} } @article {692, title = {PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria.}, journal = {J Biol Chem}, volume = {281}, year = {2006}, month = {2006 Sep 1}, pages = {25143-55}, abstract = {Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.}, keywords = {Amino Acid Sequence, Cell Proliferation, Cell Wall, Cell-Free System, Genome, Bacterial, Humans, Mannose, Mannosides, Mannosyltransferases, Molecular Sequence Data, Mycobacterium, Mycobacterium smegmatis, Phosphates, Phosphatidylinositols, Sequence Homology, Amino Acid}, issn = {0021-9258}, doi = {10.1074/jbc.M604214200}, author = {Morita, Yasu S and Sena, Chubert B C and Waller, Ross F and Kurokawa, Ken and Sernee, M Fleur and Nakatani, Fumiki and Haites, Ruth E and Billman-Jacobe, Helen and McConville, Malcolm J and Maeda, Yusuke and Kinoshita, Taroh} } @article {533, title = {Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 Jul}, pages = {3858-65}, abstract = {Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.}, keywords = {Amino Acid Sequence, Citrate (si)-Synthase, Culture Media, Deltaproteobacteria, DNA, Bacterial, Eukaryotic Cells, Geobacter, Kinetics, Molecular Sequence Data, Phylogeny, Sequence Analysis, DNA}, issn = {0099-2240}, doi = {10.1128/AEM.71.7.3858-3865.2005}, author = {Bond, Daniel R and Mester, T{\"u}nde and Nesb{\o}, Camilla L and Izquierdo-Lopez, Andrea V and Collart, Frank L and Lovley, Derek R} } @article {532, title = {Geobacter bemidjiensis sp. nov. and Geobacter psychrophilus sp. nov., two novel Fe(III)-reducing subsurface isolates.}, journal = {Int J Syst Evol Microbiol}, volume = {55}, year = {2005}, month = {2005 Jul}, pages = {1667-74}, abstract = {Fe(III)-reducing isolates were recovered from two aquifers in which Fe(III) reduction is known to be important. Strain Bem(T) was enriched from subsurface sediments collected in Bemidji, MN, USA, near a site where Fe(III) reduction is important in aromatic hydrocarbon degradation. Strains P11, P35(T) and P39 were isolated from the groundwater of an aquifer in Plymouth, MA, USA, in which Fe(III) reduction is important because of long-term inputs of acetate as a highway de-icing agent to the subsurface. All four isolates were Gram-negative, slightly curved rods that grew best in freshwater media. Strains P11, P35(T) and P39 exhibited motility via means of monotrichous flagella. Analysis of the 16S rRNA and nifD genes indicated that all four strains are delta-proteobacteria and members of the Geobacter cluster of the Geobacteraceae. Differences in phenotypic and phylogenetic characteristics indicated that the four isolates represent two novel species within the genus Geobacter. All of the isolates coupled the oxidation of acetate to the reduction of Fe(III) [iron(III) citrate, amorphous iron(III) oxide, iron(III) pyrophosphate and iron(III) nitrilotriacetate]. All four strains utilized ethanol, lactate, malate, pyruvate and succinate as electron donors and malate and fumarate as electron acceptors. Strain Bem(T) grew fastest at 30 degrees C, whereas strains P11, P35(T) and P39 grew equally well at 17, 22 and 30 degrees C. In addition, strains P11, P35(T) and P39 were capable of growth at 4 degrees C. The names Geobacter bemidjiensis sp. nov. (type strain Bem(T)=ATCC BAA-1014(T)=DSM 16622(T)=JCM 12645(T)) and Geobacter psychrophilus sp. nov. (strains P11, P35(T) and P39; type strain P35(T)=ATCC BAA-1013(T)=DSM 16674(T)=JCM 12644(T)) are proposed.}, keywords = {Bacterial Proteins, Bacterial Typing Techniques, Cold Temperature, DNA, Bacterial, DNA, Ribosomal, Ferric Compounds, Fresh Water, Genes, rRNA, Geobacter, Geologic Sediments, Massachusetts, Minnesota, Molecular Sequence Data, Oxidation-Reduction, RNA, Ribosomal, 16S, Species Specificity, Water Supply}, issn = {1466-5026}, doi = {10.1099/ijs.0.63417-0}, author = {Nevin, Kelly P and Holmes, Dawn E and Woodard, Trevor L and Hinlein, Erich S and Ostendorf, David W and Lovley, Derek R} } @article {534, title = {A novel Geobacteraceae-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe(III) and Mn(IV) oxides in Geobacter sulfurreducens.}, journal = {BMC Microbiol}, volume = {5}, year = {2005}, month = {2005}, pages = {41}, abstract = {BACKGROUND: Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. RESULTS: When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe(III) and insoluble Mn(IV) oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50\% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal reduction. CONCLUSION: OmpJ is a putative porin found in the outer membrane of the model metal reducer G. sulfurreducens that is required for respiration of extracellular electron acceptors such as soluble and insoluble metals. The effect of OmpJ in extracellular electron transfer is indirect, as OmpJ is required to keep the integrity of the periplasmic space necessary for proper folding and functioning of periplasmic and outer membrane electron transport components. The exclusive presence of ompJ in members of the Geobacteraceae family as well as its role in metal reduction suggest that the ompJ sequence may be useful in tracking the growth or activity of Geobacteraceae in sedimentary environments.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Base Sequence, Biological Transport, Deltaproteobacteria, DNA Primers, Ferric Compounds, Gene Deletion, Genome, Bacterial, Geobacter, Manganese Compounds, Molecular Sequence Data, Oxides, Peptide Fragments, Phylogeny, Protein Structure, Secondary, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, issn = {1471-2180}, doi = {10.1186/1471-2180-5-41}, author = {Afkar, Eman and Reguera, Gemma and Schiffer, Marianne and Lovley, Derek R} } @article {536, title = {OmcF, a putative c-Type monoheme outer membrane cytochrome required for the expression of other outer membrane cytochromes in Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {187}, year = {2005}, month = {2005 Jul}, pages = {4505-13}, abstract = {Outer membrane cytochromes are often proposed as likely agents for electron transfer to extracellular electron acceptors, such as Fe(III). The omcF gene in the dissimilatory Fe(III)-reducing microorganism Geobacter sulfurreducens is predicted to code for a small outer membrane monoheme c-type cytochrome. An OmcF-deficient strain was constructed, and its ability to reduce and grow on Fe(III) citrate was found to be impaired. Following a prolonged lag phase (150 h), the OmcF-deficient strain developed the ability to grow in Fe(III) citrate medium with doubling times and yields that were ca. 145\% and 70\% of those of the wild type, respectively. Comparison of the c-type cytochrome contents of outer membrane-enriched fractions prepared from wild-type and OmcF-deficient cultures confirmed the outer membrane association of OmcF and revealed multiple changes in the cytochrome content of the OmcF-deficient strain. These changes included loss of expression of two previously characterized outer membrane cytochromes, OmcB and OmcC, and overexpression of a third previously characterized outer membrane cytochrome, OmcS, during growth on Fe(III) citrate. The omcB and omcC transcripts could not be detected in the OmcF-deficient mutant by either reverse transcriptase PCR or Northern blot analyses. Expression of the omcF gene in trans restored both the capacity of the OmcF-deficient mutant to reduce Fe(III) and wild-type levels of omcB and omcC mRNA and protein. Thus, elimination of OmcF may impair Fe(III) reduction by influencing expression of OmcB, which has previously been demonstrated to play a critical role in Fe(III) reduction.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Cytochromes c, Ferric Compounds, Gene Deletion, Gene Expression Regulation, Bacterial, Geobacter, Molecular Sequence Data, Oxidation-Reduction, Sequence Alignment}, issn = {0021-9193}, doi = {10.1128/JB.187.13.4505-4513.2005}, author = {Kim, Byoung-Chan and Leang, Ching and Ding, Yan-Huai R and Glaven, Richard H and Coppi, Maddalena V and Lovley, Derek R} } @article {527, title = {Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 Dec}, pages = {8634-41}, abstract = {The potential role of outer membrane proteins in electron transfer to insoluble Fe(III) oxides by Geobacter sulfurreducens was investigated because this organism is closely related to the Fe(III) oxide-reducing organisms that are predominant in many Fe(III)-reducing environments. Two of the most abundant proteins that were easily sheared from the outer surfaces of intact cells were c-type cytochromes. One, designated OmcS, has a molecular mass of ca. 50 kDa and is predicted to be an outer membrane hexaheme c-type cytochrome. Transcripts for omcS could be detected during growth on Fe(III) oxide, but not on soluble Fe(III) citrate. The omcS mRNA consisted primarily of a monocistronic transcript, and to a lesser extent, a longer transcript that also contained the downstream gene omcT, which is predicted to encode a second hexaheme outer membrane cytochrome with 62.6\% amino acid sequence identity to OmcS. The other abundant c-type cytochrome sheared from the outer surface of G. sulfurreducens, designated OmcE, has a molecular mass of ca. 30 kDa and is predicted to be an outer membrane tetraheme c-type cytochrome. When either omcS or omcE was deleted, G. sulfurreducens could no longer reduce Fe(III) oxide but could still reduce soluble electron acceptors, including Fe(III) citrate. The mutants could reduce Fe(III) in Fe(III) oxide medium only if the Fe(III) chelator, nitrilotriacetic acid, or the electron shuttle, anthraquinone 2,6-disulfonate, was added. Expressing omcS or omcE in trans restored the capacity for Fe(III) oxide reduction. OmcT was not detected among the sheared proteins, and genetic studies indicated that G. sulfurreducens could not reduce Fe(III) oxide when omcT was expressed but OmcS was absent. In contrast, Fe(III) oxide was reduced when omcS was expressed in the absence of OmcT. These results suggest that OmcS and OmcE are involved in electron transfer to Fe(III) oxides in G. sulfurreducens. They also emphasize the importance of evaluating mechanisms for Fe(III) reduction with environmentally relevant Fe(III) oxide, rather than the more commonly utilized Fe(III) citrate, because additional electron transfer components are required for Fe(III) oxide reduction that are not required for Fe(III) citrate reduction.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Bacterial Proteins, Base Sequence, Cytochromes c, DNA Primers, Ferric Compounds, Geobacter, Kinetics, Manganese Compounds, Molecular Sequence Data, Oxidation-Reduction, Oxides, Peptide Fragments}, issn = {0099-2240}, doi = {10.1128/AEM.71.12.8634-8641.2005}, author = {Mehta, T and Coppi, M V and Childers, S E and Lovley, D R} } @article {698, title = {PIG-V involved in transferring the second mannose in glycosylphosphatidylinositol.}, journal = {J Biol Chem}, volume = {280}, year = {2005}, month = {2005 Mar 11}, pages = {9489-97}, abstract = {Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors many proteins to the eukaryotic cell surface. The biosynthetic pathway of GPI is mediated by sequential additions of sugars and other components to phosphatidylinositol. Four mannoses in the GPI are transferred from dolichol-phosphate-mannose (Dol-P-Man) and are linked through different glycosidic linkages. Therefore, four Dol-P-Man-dependent mannosyltransferases, GPI-MT-I, -MT-II, -MT-III, and -MT-IV for the first, second, third, and fourth mannoses, respectively, are required for generation of GPI. GPI-MT-I (PIG-M), GPI-MT-III (PIG-B), and GPI-MT-IV (SMP3) were previously reported, but GPI-MT-II remains to be identified. Here we report the cloning of PIG-V involved in transferring the second mannose in the GPI anchor. Human PIG-V encodes a 493-amino acid, endoplasmic reticulum (ER) resident protein with eight putative transmembrane regions. Saccharomyces cerevisiae protein encoded in open reading frame YBR004c, which we termed GPI18, has 25\% amino acid identity to human PIG-V. Viability of the yeast gpi18 deletion mutant was restored by human PIG-V cDNA. PIG-V has two functionally important conserved regions facing the ER lumen. Taken together, we suggest that PIG-V is the second mannosyltransferase in GPI anchor biosynthesis.}, keywords = {Amino Acid Sequence, Animals, Cell Line, Tumor, CHO Cells, Cloning, Molecular, Conserved Sequence, Cricetinae, DNA Primers, Glioma, Glycosylphosphatidylinositols, Humans, Mannose, Mannosyltransferases, Molecular Sequence Data, Open Reading Frames, Rats, Restriction Mapping, Saccharomyces cerevisiae, Sequence Alignment, Sequence Homology, Amino Acid}, issn = {0021-9258}, doi = {10.1074/jbc.M413867200}, author = {Kang, Ji Young and Hong, Yeongjin and Ashida, Hisashi and Shishioh, Nobue and Murakami, Yoshiko and Morita, Yasu S and Maeda, Yusuke and Kinoshita, Taroh} } @article {537, title = {Regulation of two highly similar genes, omcB and omcC, in a 10 kb chromosomal duplication in Geobacter sulfurreducens.}, journal = {Microbiology}, volume = {151}, year = {2005}, month = {2005 Jun}, pages = {1761-7}, abstract = {The Fe(III)-reducing micro-organism Geobacter sulfurreducens requires an outer-membrane c-type cytochrome, OmcB, for Fe(III) reduction, but a related cytochrome, OmcC, which is 73 \% identical to OmcB, is not required. The omcB and omcC genes are part of a tandem chromosomal duplication consisting of two repeated clusters of four genes. The 2.7 kb sequences preceding omcB and omcC are identical with the exception of a single base pair change. Studies that combined genetic, Northern blotting and primer extension analyses demonstrated that both omcB and omcC are transcribed as monocistronic and polycistronic (orf1-orf2-omcB/omcC) transcripts. All of the promoters for the various transcripts were found to be located within the 2.7 kb identical region upstream of omcB and omcC. The sequences of the promoter regions for the two monocistronic transcripts are identical and equidistant from the omcB or omcC start codons. The promoters for the two polycistronic transcripts, in contrast, are distinct. One is specific for transcription of orf1-orf2-omcB and the other is associated with transcription of orf1-orf2-omcC. Studies with an RpoS-deficient mutant suggested that transcription from all four promoters is RpoS dependent under one or more growth conditions. Deletion of orfR, a gene immediately upstream of orf1-orf2-omcB that encodes a putative transcriptional regulator, significantly lowered the omcB transcription when Fe(III) was the electron acceptor and partially inhibited Fe(III) reduction. In contrast, levels of omcC transcripts were unaffected in the orfR mutant. These results indicate that omcB and omcC operons represent a rare instance in which duplicated operons, located in tandem on the chromosome, have different transcriptional regulation.}, keywords = {Bacterial Outer Membrane Proteins, Bacterial Proteins, Blotting, Northern, DNA, Bacterial, Gene Deletion, Gene Expression Regulation, Bacterial, Geobacter, Molecular Sequence Data, Operon, Promoter Regions, Genetic, RNA, Bacterial, RNA, Messenger, Sequence Analysis, DNA, Sigma Factor, Transcription, Genetic}, issn = {1350-0872}, doi = {10.1099/mic.0.27870-0}, author = {Leang, Ching and Lovley, Derek R} } @article {751, title = {Shifts in rhizoplane communities of aquatic plants after cadmium exposure.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 May}, pages = {2484-92}, abstract = {In this study we present the comparative molecular analysis of bacterial communities of the aquatic plant Lemna minor from a contaminated site (RCP) and from a laboratory culture (EPA), as well as each of these with the addition of cadmium. Plants were identified as L. minor by analysis of the rpl16 chloroplast region. Comparative bacterial community studies were based on the analyses of 16S rRNA clone libraries, each containing about 100 clones from the root surfaces of plants. Bacterial communities were compared at three phylogenetic levels of resolution. At the level of bacterial divisions, differences in diversity index scores between treatments, with and without cadmium within the same plant type (EPA or RCP), were small, indicating that cadmium had little effect. When we compared genera within the most dominant group, the beta-proteobacteria, differences between unamended and cadmium-amended libraries were much larger. Bacterial diversity increased upon cadmium addition for both EPA and RCP libraries. Analyses of diversity at the phylotype level showed parallel shifts to more even communities upon cadmium addition; that is, percentage changes in diversity indices due to cadmium addition were the same for either plant type, indicating that contamination history might be independent of disturbance-induced diversity shifts. At finer phylogenetic levels of resolution, the effects of cadmium addition on bacterial communities were very noticeable. This study is a first step in understanding the role of aquatic plant-associated microbial communities in phytoremediation of heavy metals.}, keywords = {Araceae, Bacteria, Base Sequence, Cadmium, Molecular Sequence Data, Phylogeny, Plant Roots, RNA, Ribosomal, 16S}, issn = {0099-2240}, doi = {10.1128/AEM.71.5.2484-2492.2005}, author = {Stout, Lisa M and N{\"u}sslein, Klaus} } @article {547, title = {Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov.}, journal = {Int J Syst Evol Microbiol}, volume = {54}, year = {2004}, month = {2004 Sep}, pages = {1591-9}, abstract = {The sequences of five conserved genes, in addition to the 16S rRNA gene, were investigated in 30 members of the Geobacteraceae fam. nov. All members of the Geobacteraceae examined contained nifD, suggesting that they are capable of nitrogen fixation, which may explain their ability to compete effectively in nitrogen-poor subsurface environments undergoing remediation for petroleum or metal contamination. The phylogenies predicted from rpoB, gyrB, fusA, recA and nifD were generally in agreement with the phylogeny predicted from 16S rRNA gene sequences. Furthermore, phylogenetic analysis of concatemers constructed from all five protein-coding genes corresponded closely with the 16S rRNA gene-based phylogeny. This study demonstrated that the Geobacteraceae is a phylogenetically coherent family within the delta-subclass of the Proteobacteria that is composed of three distinct phylogenetic clusters: Geobacter, Desulfuromonas and Desulfuromusa. The sequence data provided here will make it possible to discriminate better between physiologically distinct members of the Geobacteraceae, such as Pelobacter propionicus and Geobacter species, in geobacteraceae-dominated microbial communities and greatly expands the potential to identify geobacteraceae sequences in libraries of environmental genomic DNA.}, keywords = {Bacterial Proteins, Deltaproteobacteria, Desulfuromonas, DNA Gyrase, DNA, Bacterial, DNA, Ribosomal, DNA-Directed RNA Polymerases, Genes, Bacterial, Genes, rRNA, Geobacter, Molecular Sequence Data, Nitrogen Fixation, Peptide Elongation Factor G, Phylogeny, Rec A Recombinases, RNA, Bacterial, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1466-5026}, doi = {10.1099/ijs.0.02958-0}, author = {Holmes, Dawn E and Nevin, Kelly P and Lovley, Derek R} } @article {824, title = {A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12.}, journal = {Genetics}, volume = {167}, year = {2004}, month = {2004 Jun}, pages = {569-78}, abstract = {The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.}, keywords = {Base Sequence, Codon, DNA Primers, DNA Repair, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli K12, Escherichia coli Proteins, Molecular Sequence Data, Mutation, Plasmids, Polymerase Chain Reaction, Sequence Deletion}, issn = {0016-6731}, doi = {10.1534/genetics.103.025296}, author = {McCool, Jesse D and Ford, Christopher C and Sandler, Steven J} } @article {554, title = {Isolation, characterization, and U(VI)-reducing potential of a facultatively anaerobic, acid-resistant Bacterium from Low-pH, nitrate- and U(VI)-contaminated subsurface sediment and description of Salmonella subterranea sp. nov.}, journal = {Appl Environ Microbiol}, volume = {70}, year = {2004}, month = {2004 May}, pages = {2959-65}, abstract = {A facultatively anaerobic, acid-resistant bacterium, designated strain FRCl, was isolated from a low-pH, nitrate- and U(VI)-contaminated subsurface sediment at site FW-024 at the Natural and Accelerated Bioremediation Research Field Research Center in Oak Ridge, Tenn. Strain FRCl was enriched at pH 4.5 in minimal medium with nitrate as the electron acceptor, hydrogen as the electron donor, and acetate as the carbon source. Clones with 16S ribosomal DNA (rDNA) sequences identical to the sequence of strain FRCl were also detected in a U(VI)-reducing enrichment culture derived from the same sediment. Cells of strain FRCl were gram-negative motile regular rods 2.0 to 3.4 micro m long and 0.7 to 0.9 microm in diameter. Strain FRCl was positive for indole production, by the methyl red test, and for ornithine decarboxylase; it was negative by the Voges-Proskauer test (for acetylmethylcarbinol production), for urea hydrolysis, for arginine dihydrolase, for lysine decarboxylase, for phenylalanine deaminase, for H(2)S production, and for gelatin hydrolysis. Strain FRCl was capable of using O(2), NO(3)(-), S(2)O(3)(2-), fumarate, and malate as terminal electron acceptors and of reducing U(VI) in the cell suspension. Analysis of the 16S rDNA sequence of the isolate indicated that this strain was 96.4\% similar to Salmonella bongori and 96.3\% similar to Enterobacter cloacae. Physiological and phylogenetic analyses suggested that strain FRCl belongs to the genus Salmonella and represents a new species, Salmonella subterranea sp. nov.}, keywords = {Anaerobiosis, Culture Media, DNA, Ribosomal, Fresh Water, Geologic Sediments, Hydrogen-Ion Concentration, Molecular Sequence Data, Nitrates, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Salmonella, Sequence Analysis, DNA, Uranium, Water Pollution, Chemical}, issn = {0099-2240}, author = {Shelobolina, Evgenya S and Sullivan, Sara A and O{\textquoteright}Neill, Kathleen R and Nevin, Kelly P and Lovley, Derek R} } @article {551, title = {MacA, a diheme c-type cytochrome involved in Fe(III) reduction by Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {186}, year = {2004}, month = {2004 Jun}, pages = {4042-5}, abstract = {A 36-kDa diheme c-type cytochrome abundant in Fe(III)-respiring Geobacter sulfurreducens, designated MacA, was more highly expressed during growth with Fe(III) as the electron acceptor than with fumarate. Although MacA has homology to proteins with in vitro peroxidase activity, deletion of macA had no impact on response to oxidative stress. However, the capacity for Fe(III) reduction was greatly diminished, indicating that MacA, which is predicted to be localized in the periplasm, is a key intermediate in electron transfer to Fe(III).}, keywords = {Amino Acid Sequence, Bacterial Proteins, Cytochrome c Group, Deltaproteobacteria, Electron Transport, Ferric Compounds, Gene Deletion, Molecular Sequence Data, Oxidation-Reduction}, issn = {0021-9193}, doi = {10.1128/JB.186.12.4042-4045.2004}, author = {Butler, Jessica E and Kaufmann, Franz and Coppi, Maddalena V and N{\'u}{\~n}ez, Cinthia and Lovley, Derek R} } @article {544, title = {Microbial communities associated with electrodes harvesting electricity from a variety of aquatic sediments.}, journal = {Microb Ecol}, volume = {48}, year = {2004}, month = {2004 Aug}, pages = {178-90}, abstract = {The microbial communities associated with electrodes from underwater fuel cells harvesting electricity from five different aquatic sediments were investigated. Three fuel cells were constructed with marine, salt-marsh, or freshwater sediments incubated in the laboratory. Fuel cells were also deployed in the field in salt marsh sediments in New Jersey and estuarine sediments in Oregon, USA. All of the sediments produced comparable amounts of power. Analysis of 16S rRNA gene sequences after 3-7 months of incubation demonstrated that all of the energy-harvesting anodes were highly enriched in microorganisms in the delta-Proteobacteria when compared with control electrodes not connected to a cathode. Geobacteraceae accounted for the majority of delta-Proteobacterial sequences or all of the energy-harvesting anodes, except the one deployed at the Oregon estuarine site. Quantitative PCR analysis of 16S rRNA genes and culturing studies indicated that Geobacteraceae were 100-fold more abundant on the marine-deployed anodes versus controls. Sequences most similar to microorganisms in the family Desulfobulbaceae predominated on the anode deployed in the estuarine sediments, and a significant proportion of the sequences recovered from the freshwater anodes were closely related to the Fe(III)-reducing isolate, Geothrix fermentans. There was also a specific enrichment of microorganisms on energy harvesting cathodes, but the enriched populations varied with the sediment/water source. Thus, future studies designed to help optimize the harvesting of electricity from aquatic sediments or waste organic matter should focus on the electrode interactions of these microorganisms which are most competitive in colonizing anodes and cathodes.}, keywords = {Base Sequence, Biodiversity, Bioelectric Energy Sources, Cloning, Molecular, DNA Primers, Electrodes, Gammaproteobacteria, Geologic Sediments, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Restriction Mapping, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {0095-3628}, doi = {10.1007/s00248-003-0004-4}, author = {Holmes, D E and Bond, D R and O{\textquoteright}Neil, R A and Reimers, C E and Tender, L R and Lovley, D R} } @article {546, title = {Potential role of a novel psychrotolerant member of the family Geobacteraceae, Geopsychrobacter electrodiphilus gen. nov., sp. nov., in electricity production by a marine sediment fuel cell.}, journal = {Appl Environ Microbiol}, volume = {70}, year = {2004}, month = {2004 Oct}, pages = {6023-30}, abstract = {Previous studies have shown that members of the family Geobacteraceae that attach to the anodes of sediment fuel cells are directly involved in harvesting electricity by oxidizing organic compounds to carbon dioxide and transferring the electrons to the anode. In order to learn more about this process, microorganisms from the anode surface of a marine sediment fuel cell were enriched and isolated with Fe(III) oxide. Two unique marine isolates were recovered, strains A1(T) and A2. They are gram-negative, nonmotile rods, with abundant c-type cytochromes. Phylogenetic analysis of the 16S rRNA, recA, gyrB, fusA, rpoB, and nifD genes indicated that strains A1(T) and A2 represent a unique phylogenetic cluster within the Geobacteraceae. Both strains were able to grow with an electrode serving as the sole electron acceptor and transferred ca. 90\% of the electrons available in their organic electron donors to the electrodes. These organisms are the first psychrotolerant members of the Geobacteraceae reported thus far and can grow at temperatures between 4 and 30 degrees C, with an optimum temperature of 22 degrees C. Strains A1(T) and A2 can utilize a wide range of traditional electron acceptors, including all forms of soluble and insoluble Fe(III) tested, anthraquinone 2,6-disulfonate, and S(0). In addition to acetate, both strains can utilize a number of other organic acids, amino acids, long-chain fatty acids, and aromatic compounds to support growth with Fe(III) nitrilotriacetic acid as an electron acceptor. The metabolism of these organisms differs in that only strain A1(T) can use acetoin, ethanol, and hydrogen as electron donors, whereas only strain A2 can use lactate, propionate, and butyrate. The name Geopsychrobacter electrodiphilus gen. nov., sp. nov., is proposed for strains A1(T) and A2, with strain A1(T) (ATCC BAA-880(T); DSM 16401(T); JCM 12469) as the type strain. Strains A1(T) and A2 (ATCC BAA-770; JCM 12470) represent the first organisms recovered from anodes that can effectively couple the oxidation of organic compounds to an electrode. Thus, they may serve as important model organisms for further elucidation of the mechanisms of microbe-electrode electron transfer in sediment fuel cells.}, keywords = {Bioelectric Energy Sources, Cytochromes, Deltaproteobacteria, Electron Transport, Genes, Bacterial, Geologic Sediments, Microscopy, Electron, Molecular Sequence Data, Phylogeny, RNA, Bacterial, RNA, Ribosomal, 16S, Temperature}, issn = {0099-2240}, doi = {10.1128/AEM.70.10.6023-6030.2004}, author = {Holmes, Dawn E and Nicoll, Julie S and Bond, Daniel R and Lovley, Derek R} } @article {774, title = {Quorum sensing in Staphylococci is regulated via phosphorylation of three conserved histidine residues.}, journal = {J Biol Chem}, volume = {279}, year = {2004}, month = {2004 Apr 9}, pages = {14665-72}, abstract = {Staphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP(-) strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP(-) strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers.}, keywords = {Amino Acid Sequence, Animals, Blotting, Northern, Cell Communication, DNA, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Histidine, Mice, Molecular Sequence Data, Mutagenesis, Mutagenesis, Site-Directed, Mutation, Phenotype, Phosphorylation, Phylogeny, Plasmids, Protein Structure, Tertiary, RNA, Antisense, RNA, Bacterial, Sequence Homology, Amino Acid, Signal Transduction, Staphylococcus aureus, Time Factors}, issn = {0021-9258}, doi = {10.1074/jbc.M311106200}, author = {Gov, Yael and Borovok, Ilya and Korem, Moshe and Singh, Vineet K and Jayaswal, Radheshyam K and Wilkinson, Brian J and Rich, Stephen M and Balaban, Naomi} } @article {549, title = {The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {186}, year = {2004}, month = {2004 Aug}, pages = {5543-6}, abstract = {Geobacter sulfurreducens RpoS sigma factor was shown to contribute to survival in stationary phase and upon oxygen exposure. Furthermore, a mutation in rpoS decreased the rate of reduction of insoluble Fe(III) but not of soluble forms of iron. This study suggests that RpoS plays a role in regulating metabolism of Geobacter under suboptimal conditions in subsurface environments.}, keywords = {Adaptation, Physiological, Bacterial Proteins, Base Sequence, Ferric Compounds, Gene Expression Regulation, Bacterial, Genes, Bacterial, Geobacter, Molecular Sequence Data, Mutation, Oxidation-Reduction, Oxidative Stress, Sigma Factor, Transcription Initiation Site}, issn = {0021-9193}, doi = {10.1128/JB.186.16.5543-5546.2004}, author = {N{\'u}{\~n}ez, Cinthia and Adams, Lorrie and Childers, Susan and Lovley, Derek R} } @article {541, title = {The structure of the core region of the lipopolysaccharide from Geobacter sulfurreducens.}, journal = {Carbohydr Res}, volume = {339}, year = {2004}, month = {2004 Dec 27}, pages = {2901-4}, abstract = {The structure of the core part of the LPS from Geobacter sulfurreducens was analysed. The LPS contained no O-specific polysaccharide (O-side chain) and upon mild hydrolysis gave a core oligosaccharide, which was isolated by gel chromatography. It was studied by chemical methods, NMR and mass spectrometry, and the following structure was proposed. [carbohydrate structure: see text] where Q = 3-O-Me-alpha-L-QuiNAc-(1-->or H (approximately 3:2).}, keywords = {Carbohydrate Sequence, Geobacter, Lipopolysaccharides, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Oligosaccharides}, issn = {0008-6215}, doi = {10.1016/j.carres.2004.10.004}, author = {Vinogradov, Evgeny and Korenevsky, Anton and Lovley, Derek R and Beveridge, Terry J} } @article {576, title = {Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens.}, journal = {Biochem J}, volume = {369}, year = {2003}, month = {2003 Jan 1}, pages = {153-61}, abstract = {A 9.6 kDa periplasmic c -type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfurreducens and characterized. The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c (7) cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans. The gene encoding PpcA was identified from the G. sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58 kDa) with 49\% identity to the c (7) cytochrome of D. acetoxidans. In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method. Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant. When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored. The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered. The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor. When taken together with previous studies on other electron transport proteins in G. sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.}, keywords = {Amino Acid Sequence, Base Sequence, Cytochrome c Group, DNA, Bacterial, Molecular Sequence Data, Periplasm, Proteobacteria, Sequence Homology, Amino Acid}, issn = {0264-6021}, doi = {10.1042/BJ20020597}, author = {Lloyd, Jon R and Leang, Ching and Hodges Myerson, Allison L and Coppi, Maddalena V and Cuifo, Stacey and Methe, Barb and Sandler, Steven J and Lovley, Derek R} } @article {42, title = {Expression of the yeast PIS1 gene requires multiple regulatory elements including a Rox1p binding site.}, journal = {J Biol Chem}, volume = {278}, year = {2003}, month = {2003 Oct 3}, pages = {38646-52}, abstract = {The PIS1 gene is required for de novo synthesis of phosphatidylinositol (PI), an essential phospholipid in Saccharomyces cerevisiae. PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline. Relatively little is known about regulation of transcription of the PIS1 gene. We reported previously that PIS1 transcription is sensitive to carbon source. To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1-3). Deletion of either UAS1 or UAS2 resulted in an approximately 45\% reduction in expression, whereas removal of UAS3 yielded an 84\% decrease in expression. A comparison of promoters among several Saccharomyces species shows that these sequences are highly conserved. Curiously, the UAS3 element region (-149 to -138) includes a Rox1p binding site. Rox1p is a repressor of hypoxic genes under aerobic growth conditions. Consistent with this, we have found that expression of a PIS1-cat reporter was repressed under aerobic conditions, and this repression was dependent on both Rox1p and its binding site. Furthermore, PI levels were elevated under anaerobic conditions. This is the first evidence that PI levels are affected by regulation of PIS1 transcription.}, keywords = {Anoxia, Base Sequence, Binding Sites, Carbon, Chloramphenicol O-Acetyltransferase, Choline, Chromatography, Thin Layer, Conserved Sequence, DNA, DNA, Complementary, DNA-Binding Proteins, Gene Deletion, Gene Expression Regulation, Fungal, Genes, Reporter, Inositol, Lipid Metabolism, Models, Biological, Models, Genetic, Molecular Sequence Data, Oxygen, Phospholipids, Plasmids, Promoter Regions, Genetic, Protein Binding, Repressor Proteins, RNA, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Transferases (Other Substituted Phosphate Groups)}, issn = {0021-9258}, doi = {10.1074/jbc.M305251200}, author = {Gardocki, Mary Elizabeth and Lopes, John M} } @article {570, title = {Geobacter sulfurreducens has two autoregulated lexA genes whose products do not bind the recA promoter: differing responses of lexA and recA to DNA damage.}, journal = {J Bacteriol}, volume = {185}, year = {2003}, month = {2003 Apr}, pages = {2493-502}, abstract = {The Escherichia coli LexA protein was used as a query sequence in TBLASTN searches to identify the lexA gene of the delta-proteobacterium Geobacter sulfurreducens from its genome sequence. The results of the search indicated that G. sulfurreducens has two independent lexA genes designated lexA1 and lexA2. A copy of a dinB gene homologue, which in E. coli encodes DNA polymerase IV, is present downstream of each lexA gene. Reverse transcription-PCR analyses demonstrated that, in both cases, lexA and dinB constitute a single transcriptional unit. Electrophoretic mobility shift assays with purified LexA1 and LexA2 proteins have shown that both proteins bind the imperfect palindrome GGTTN(2)CN(4)GN(3)ACC found in the promoter region of both lexA1 and lexA2. This sequence is also present upstream of the Geobacter metallireducens lexA gene, indicating that it is the LexA box of this bacterial genus. This palindrome is not found upstream of either the G. sulfurreducens or the G. metallireducens recA genes. Furthermore, DNA damage induces expression of the lexA-dinB transcriptional unit but not that of the recA gene. However, the basal level of recA gene expression is dramatically higher than that of the lexA gene. Likewise, the promoters of the G. sulfurreducens recN, ruvAB, ssb, umuDC, uvrA, and uvrB genes do not contain the LexA box and are not likely to bind to the LexA1 or LexA2 proteins. G. sulfurreducens is the first bacterial species harboring a lexA gene for which a constitutive expression of its recA gene has been described.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Consensus Sequence, DNA Damage, DNA Polymerase beta, DNA Repair, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Gene Expression, Genes, Bacterial, Molecular Sequence Data, Promoter Regions, Genetic, Proteobacteria, Rec A Recombinases, Sequence Alignment, Serine Endopeptidases, Transcription, Genetic}, issn = {0021-9193}, author = {Jara, M{\'o}nica and N{\'u}{\~n}ez, Cinthia and Campoy, Susana and Fern{\'a}ndez de Henestrosa, Antonio R and Lovley, Derek R and Barb{\'e}, Jordi} } @article {1215, title = {Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry.}, journal = {J Am Soc Mass Spectrom}, volume = {14}, year = {2003}, month = {2003 Sep}, pages = {957-70}, abstract = {

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100\% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97\% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50\% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50\% for approximately 70\% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

}, keywords = {Amino Acid Sequence, Chromatography, Liquid, Databases, Protein, Electrophoresis, Gel, Two-Dimensional, Methanococcus, Molecular Sequence Data, Molecular Weight, Peptide Mapping, Proteins, Pyrococcus furiosus, Sensitivity and Specificity, Software, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Trypsin}, issn = {1044-0305}, author = {Lim, Hanjo and Eng, Jimmy and Yates, John R and Tollaksen, Sandra L and Giometti, Carol S and Holden, James F and Adams, Michael W W and Reich, Claudia I and Olsen, Gary J and Hays, Lara G} } @article {571, title = {OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {185}, year = {2003}, month = {2003 Apr}, pages = {2096-103}, abstract = {Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79\% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Bacterial Proteins, Cytochrome c Group, Deltaproteobacteria, Gene Expression Regulation, Bacterial, Iron, Molecular Sequence Data, Multigene Family, Mutation, Oxidation-Reduction, Sequence Homology, Amino Acid}, issn = {0021-9193}, author = {Leang, Ching and Coppi, M V and Lovley, D R} } @article {567, title = {Rhodoferax ferrireducens sp. nov., a psychrotolerant, facultatively anaerobic bacterium that oxidizes acetate with the reduction of Fe(III).}, journal = {Int J Syst Evol Microbiol}, volume = {53}, year = {2003}, month = {2003 May}, pages = {669-73}, abstract = {To further investigate the diversity of micro-organisms capable of conserving energy to support growth from dissimilatory Fe(III) reduction, Fe(III)-reducing micro-organisms were enriched and isolated from subsurface sediments collected in Oyster Bay, VA, USA. A novel isolate, designated T118(T), was recovered in a medium with lactate as the sole electron donor and Fe(III) as the sole electron acceptor. Cells of T1 18(T) were Gram-negative, motile, short rods with a single polar flagellum. Strain T1 18(T) grew between pH 6.7 and 7.1, with a temperature range of 4-30 degrees C. The optimal growth temperature was 25 degrees C. Electron donors utilized by strain T1 18(T) with Fe(III) as the sole electron acceptor included acetate, lactate, malate, propionate, pyruvate, succinate and benzoate. None of the compounds tested was fermented. Electron acceptors utilized with either acetate or lactate as the electron donor included Fe(III)-NTA (nitrilotriacetic acid), Mn(IV) oxide, nitrate, fumarate and oxygen. Phylogenetic analysis demonstrated that strain T1 18(T) is most closely related to the genus Rhodoferax. Unlike other species in this genus, strain T1 18(T) is not a phototroph and does not ferment fructose. However, phototrophic genes may be present but not expressed under the experimental conditions tested. No Rhodoferax species have been reported to grow via dissimilatory Fe(III) reduction. Based on these physiological and phylogenetic differences, strain T1 18(T) (=ATCC BAA-621(T) = DSM 15236(T)) is proposed as a novel species, Rhodoferax ferrireducens sp. nov.}, keywords = {Acetates, Anaerobiosis, Bacterial Typing Techniques, Betaproteobacteria, Cold Temperature, Culture Media, DNA, Ribosomal, Electron Transport, Ferric Compounds, Fresh Water, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {1466-5026}, author = {Finneran, Kevin T and Johnsen, Claudia V and Lovley, Derek R} } @article {777, title = {Sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-1 (MSP-1) of Plasmodium falciparum.}, journal = {Gene}, volume = {304}, year = {2003}, month = {2003 Jan 30}, pages = {65-75}, abstract = {The merozoite surface protein-1 (MSP-1) of the malaria parasite Plasmodium falciparum is a major blood-stage antigen containing highly polymorphic tripeptide repeats in the domain known as block 2 and several non-repetitive domains that are essentially dimorphic. We have analyzed sequence variation in block 2 repeats and in non-repetitive block 17, as well as other polymorphisms within the MSP-1 gene, in clinical isolates of P. falciparum. Repeat haplotypes were defined as unique combinations of repeat motifs within block 2, whereas block 17 haplotypes were defined as unique combinations of single nucleotide replacements in this domain. A new block 17 haplotype, E-TNG-L, was found in one isolate from Vietnam. MSP-1 alleles, defined as unique combinations of haplotypes in blocks 2 and 17 and other polymorphisms within the molecule, were characterized in 60 isolates from hypoendemic Brazil and 37 isolates from mesoendemic Vietnam. Extensive diversity has been created in block 2 and elsewhere in the molecule, while maintaining significant linkage disequilibrium between polymorphisms across the non-telomeric MSP-1 locus separated by a map distance of more than 4 kb, suggesting that low meiotic recombination rates occur in both parasite populations. These results indicate a role for non-homologous recombination, such as strand-slippage mispairing during mitosis and gene conversion, in creating variation in a malarial antigen under strong diversifying selection.}, keywords = {Alleles, Animals, Brazil, DNA, Protozoan, Evolution, Molecular, Genetic Variation, Haplotypes, Linkage Disequilibrium, Malaria Vaccines, Merozoite Surface Protein 1, Molecular Sequence Data, Plasmodium falciparum, Polymorphism, Single Nucleotide, Recombination, Genetic, Sequence Analysis, DNA, Tanzania, Thailand, Vietnam}, issn = {0378-1119}, author = {Ferreira, Marcelo U and Ribeiro, Weber L and Tonon, Angela P and Kawamoto, Fumihiko and Rich, Stephen M} } @article {569, title = {Thermophily in the Geobacteraceae: Geothermobacter ehrlichii gen. nov., sp. nov., a novel thermophilic member of the Geobacteraceae from the "Bag City" hydrothermal vent.}, journal = {Appl Environ Microbiol}, volume = {69}, year = {2003}, month = {2003 May}, pages = {2985-93}, abstract = {Little is known about the microbiology of the "Bag City" hydrothermal vent, which is part of a new eruption site on the Juan de Fuca Ridge and which is notable for its accumulation of polysaccharide on the sediment surface. A pure culture, designated strain SS015, was recovered from a vent fluid sample from the Bag City site through serial dilution in liquid medium with malate as the electron donor and Fe(III) oxide as the electron acceptor and then isolation of single colonies on solid Fe(III) oxide medium. The cells were gram-negative rods, about 0.5 micro m by 1.2 to 1.5 micro m, and motile and contained c-type cytochromes. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain SS015 placed it in the family Geobacteraceae in the delta subclass of the Proteobacteria. Unlike previously described members of the Geobacteraceae, which are mesophiles, strain SS015 was a thermophile and grew at temperatures of between 35 and 65 degrees C, with an optimum temperature of 55 degrees C. Like many previously described members of the Geobacteraceae, strain SS015 grew with organic acids as the electron donors and Fe(III) or nitrate as the electron acceptor, with nitrate being reduced to ammonia. Strain SS015 was unique among the Geobacteraceae in its ability to use sugars, starch, or amino acids as electron donors for Fe(III) reduction. Under stress conditions, strain SS015 produced copious quantities of extracellular polysaccharide, providing a model for the microbial production of the polysaccharide accumulation at the Bag City site. The 16S rDNA sequence of strain SS015 was less than 94\% similar to the sequences of previously described members of the Geobacteraceae; this fact, coupled with its unique physiological properties, suggests that strain SS015 represents a new genus in the family Geobacteraceae. The name Geothermobacter ehrlichii gen. nov., sp. nov., is proposed (ATCC BAA-635 and DSM 15274). Although strains of Geobacteraceae are known to be the predominant Fe(III)-reducing microorganisms in a variety of Fe(III)-reducing environments at moderate temperatures, strain SS015 represents the first described thermophilic member of the Geobacteraceae and thus extends the known environmental range of this family to hydrothermal environments.}, keywords = {Base Composition, Cytochromes, Deltaproteobacteria, DNA, Bacterial, DNA, Ribosomal, Drug Resistance, Bacterial, Ecosystem, Electron Transport, Geologic Sediments, Hot Temperature, Hydrogen-Ion Concentration, Iron, Microscopy, Electron, Molecular Sequence Data, Oxidation-Reduction, Pacific Ocean, Phylogeny, Seawater, Sodium Chloride, Species Specificity}, issn = {0099-2240}, author = {Kashefi, Kazem and Holmes, Dawn E and Baross, John A and Lovley, Derek R} } @article {780, title = {Ancient lineages of non-LTR retrotransposons in the primitive eukaryote, Giardia lamblia.}, journal = {Mol Biol Evol}, volume = {19}, year = {2002}, month = {2002 May}, pages = {619-30}, abstract = {Mobile elements that use reverse transcriptase to make new copies of themselves are found in all major lineages of eukaryotes. The non-long terminal repeat (non-LTR) retrotransposons have been suggested to be the oldest of these eukaryotic elements. Phylogenetic analysis of non-LTR elements suggests that they have predominantly undergone vertical transmission, as opposed to the frequent horizontal transmissions found for other mobile elements. One prediction of this vertical model of inheritance is that the oldest lineages of eukaryotes should exclusively harbor the oldest lineages of non-LTR retrotransposons. Here we characterize the non-LTR retrotransposons present in one of the most primitive eukaryotes, the diplomonad Giardia lamblia. Two families of elements were detected in the WB isolate of G. lamblia currently being used for the genome sequencing project. These elements are clearly distinct from all other previously described non-LTR lineages. Phylogenetic analysis indicates that these Genie elements (for Giardia early non-LTR insertion element) are among the oldest known lineages of non-LTR elements consistent with strict vertical descent. Genie elements encode a single open reading frame with a carboxyl terminal endonuclease domain. Genie 1 is site specific, as seven to eight copies are present in a single tandem array of a 771-bp repeat near the telomere of one chromosome. The function of this repeat is not known. One additional, highly divergent, element within the Genie 1 lineage is not located in this tandem array but is near a second telomere. Four different telomere addition sites could be identified within or near the Genie elements on each of these chromosomes. The second lineage of non-LTR elements, Genie 2, is composed of about 10 degenerate copies. Genie 2 elements do not appear to be site specific in their insertion. An unusual aspect of Genie 2 is that all copies contain inverted repeats up to 172 bp in length.}, keywords = {Animals, Base Sequence, DNA, Protozoan, Evolution, Molecular, Giardia lamblia, Long Interspersed Nucleotide Elements, Molecular Sequence Data, Open Reading Frames, Phylogeny, Repetitive Sequences, Nucleic Acid, Sequence Homology, Nucleic Acid, Telomere}, issn = {0737-4038}, author = {Burke, William D and Malik, Harmit S and Rich, Stephen M and Eickbush, Thomas H} } @article {574, title = {Desulfitobacterium metallireducens sp. nov., an anaerobic bacterium that couples growth to the reduction of metals and humic acids as well as chlorinated compounds.}, journal = {Int J Syst Evol Microbiol}, volume = {52}, year = {2002}, month = {2002 Nov}, pages = {1929-35}, abstract = {Strain 853-15A(T) was enriched and isolated from uranium-contaminated aquifer sediment by its ability to grow under anaerobic conditions via the oxidation of lactate coupled to the reduction of anthraquinone-2,6-disulfonate (AQDS) to anthrahydroquinone-2,6-disulfonate (AHQDS). Lactate was oxidized incompletely to acetate and carbon dioxide according to the reaction CH3CHOHCOO(-)+ 2AQDS+H2O --> CH3COO(-)+ 2AHQDS+CO2. Additional electron donors utilized included formate, ethanol, butanol, butyrate, malate and pyruvate. Lactate also supported growth with Fe(III) citrate, Mn(IV) oxide, humic substances, elemental sulfur, 3-chloro-4-hydroxyphenylacetate, trichloroethylene or tetrachloroethylene serving as the electron acceptor. Growth was not observed with sulfate, sulfite, nitrate or fumarate as the terminal electron acceptor. The temperature optimum for growth was 30 degrees C, but growth was also observed at 20 and 37 degrees C. The pH optimum was approximately 7.0. The 16S rDNA sequence of strain 853-15A(T) suggested that it was most closely related to Desulfitobacterium dehalogenans and closely related to Desulfitobacterium chlororespirans and Desulfitobacterium frappieri. The phylogenetic and physiological properties exhibited by strain 853-15A(T) (= ATCC BAA-636(T)) place it within the genus Desulfitobacterium as the type strain of a novel species, Desulfitobacterium metallireducens sp. nov.}, keywords = {Anaerobiosis, DNA, Bacterial, DNA, Ribosomal, Ecosystem, Electron Transport, Geologic Sediments, Humic Substances, Metals, Microscopy, Electron, Molecular Sequence Data, Oxidation-Reduction, Peptococcaceae, Phenotype, Phylogeny, RNA, Bacterial, RNA, Ribosomal, 16S, Species Specificity}, issn = {1466-5026}, author = {Finneran, Kevin T and Forbush, Heather M and VanPraagh, Catherine V Gaw and Lovley, Derek R} } @article {581, title = {Geoglobus ahangari gen. nov., sp. nov., a novel hyperthermophilic archaeon capable of oxidizing organic acids and growing autotrophically on hydrogen with Fe(III) serving as the sole electron acceptor.}, journal = {Int J Syst Evol Microbiol}, volume = {52}, year = {2002}, month = {2002 May}, pages = {719-28}, abstract = {A novel, regular to irregular, coccoid-shaped, anaerobic, Fe(III)-reducing microorganism was isolated from the Guaymas Basin hydrothermal system at a depth of 2000 m. Isolation was carried out with a new technique using Fe(III) oxide as the electron acceptor for the recovery of colonies on solid medium. The isolate, designated strain 234T, was strictly anaerobic and exhibited a tumbling motility. The cells had a single flagellum. Strain 234T grew at temperatures between 65 and 90 degrees C, with an optimum at about 88 degrees C. The optimal salt concentration for growth was around 19 g l(-1). The isolate was capable of growth with H2 as the sole electron donor coupled to the reduction of Fe(III) without the need for an organic carbon source. This is the first example of a dissimilatory Fe(III)-reducing micro-organism capable of growing autotrophically on hydrogen. In addition to molecular hydrogen, strain 234T oxidizes pyruvate, acetate, malate, succinate, peptone, formate, fumarate, yeast extract, glycerol, isoleucine, arginine, serine, glutamine, asparagine, stearate, palmitate, valerate, butyrate and propionate with the reduction of Fe(III). This isolate is the first example of a hyperthermophile capable of oxidizing long-chain fatty acids anaerobically. Isolate 234T grew exclusively with Fe(III) as the sole electron acceptor. The G+C content was 58.7 mol\%. Based on detailed analysis of its 16S rDNA sequence, G+C content, distinguishing physiological features and metabolism, strain 234T is proposed to represent a novel genus within the Archaeoglobales. The name proposed for strain 234T is Geoglobus ahangari gen. nov., sp. nov..}, keywords = {Anaerobiosis, Archaea, Bacterial Typing Techniques, DNA, Ribosomal, Electron Transport, Fatty Acids, Ferric Compounds, Hot Temperature, Hydrogen, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Water Microbiology}, issn = {1466-5026}, author = {Kashefi, Kazem and Tor, Jason M and Holmes, Dawn E and Gaw Van Praagh, Catherine V and Reysenbach, Anna-Louise and Lovley, Derek R} } @article {580, title = {Harnessing microbially generated power on the seafloor.}, journal = {Nat Biotechnol}, volume = {20}, year = {2002}, month = {2002 Aug}, pages = {821-5}, abstract = {In many marine environments, a voltage gradient exists across the water sediment interface resulting from sedimentary microbial activity. Here we show that a fuel cell consisting of an anode embedded in marine sediment and a cathode in overlying seawater can use this voltage gradient to generate electrical power in situ. Fuel cells of this design generated sustained power in a boat basin carved into a salt marsh near Tuckerton, New Jersey, and in the Yaquina Bay Estuary near Newport, Oregon. Retrieval and analysis of the Tuckerton fuel cell indicates that power generation results from at least two anode reactions: oxidation of sediment sulfide (a by-product of microbial oxidation of sedimentary organic carbon) and oxidation of sedimentary organic carbon catalyzed by microorganisms colonizing the anode. These results demonstrate in real marine environments a new form of power generation that uses an immense, renewable energy reservoir (sedimentary organic carbon) and has near-immediate application.}, keywords = {Bacteria, Bioelectric Energy Sources, Biotechnology, Carbon, Conservation of Energy Resources, DNA, Ribosomal, Electricity, Electrodes, Environmental Microbiology, Geologic Sediments, Molecular Sequence Data, New Jersey, Oceans and Seas, Oregon, Oxidation-Reduction, RNA, Bacterial, RNA, Ribosomal, 16S, Sulfides}, issn = {1087-0156}, doi = {10.1038/nbt716}, author = {Tender, Leonard M and Reimers, Clare E and Stecher, Hilmar A and Holmes, Dawn E and Bond, Daniel R and Lowy, Daniel A and Pilobello, Kanoelani and Fertig, Stephanie J and Lovley, Derek R} } @article {589, title = {A hydrogen-based subsurface microbial community dominated by methanogens.}, journal = {Nature}, volume = {415}, year = {2002}, month = {2002 Jan 17}, pages = {312-5}, abstract = {The search for extraterrestrial life may be facilitated if ecosystems can be found on Earth that exist under conditions analogous to those present on other planets or moons. It has been proposed, on the basis of geochemical and thermodynamic considerations, that geologically derived hydrogen might support subsurface microbial communities on Mars and Europa in which methanogens form the base of the ecosystem. Here we describe a unique subsurface microbial community in which hydrogen-consuming, methane-producing Archaea far outnumber the Bacteria. More than 90\% of the 16S ribosomal DNA sequences recovered from hydrothermal waters circulating through deeply buried igneous rocks in Idaho are related to hydrogen-using methanogenic microorganisms. Geochemical characterization indicates that geothermal hydrogen, not organic carbon, is the primary energy source for this methanogen-dominated microbial community. These results demonstrate that hydrogen-based methanogenic communities do occur in Earth{\textquoteright}s subsurface, providing an analogue for possible subsurface microbial ecosystems on other planets.}, keywords = {Bacteria, DNA, Archaeal, DNA, Bacterial, Ecosystem, Euryarchaeota, Exobiology, Hydrogen, Molecular Sequence Data, Phylogeny, RNA, Ribosomal, 16S, Water Microbiology}, issn = {0028-0836}, doi = {10.1038/415312a}, author = {Chapelle, Francis H and O{\textquoteright}Neill, Kathleen and Bradley, Paul M and Meth{\'e}, Barbara A and Ciufo, Stacy A and Knobel, LeRoy L and Lovley, Derek R} } @article {388, title = {Multiple mitochondrial DNA polymerases in Trypanosoma brucei.}, journal = {Mol Cell}, volume = {10}, year = {2002}, month = {2002 Jul}, pages = {175-86}, abstract = {Kinetoplast DNA (kDNA), the unusual mitochondrial DNA of Trypanosoma brucei, is a network containing thousands of catenated circles. Database searching for a kDNA replicative polymerase (pol) revealed no mitochondrial pol gamma homolog. Instead, we identified four proteins (TbPOLIA, IB, IC, and ID) related to bacterial pol I. Remarkably, all four localized to the mitochondrion. TbPOLIB and TbPOLIC localized beside the kDNA where replication occurs, and their knockdown by RNA interference caused kDNA network shrinkage. Furthermore, silencing of TbPOLIC caused loss of both minicircles and maxicircles and accumulation of minicircle replication intermediates, consistent with a role in replication. While typical mitochondria contain one DNA polymerase, pol gamma, trypanosome mitochondria contain five such enzymes, including the previously characterized pol beta.}, keywords = {Amino Acid Sequence, Animals, DNA, Kinetoplast, DNA-Directed DNA Polymerase, Mitochondria, Molecular Sequence Data, Phylogeny, Protein Structure, Tertiary, Protein Transport, RNA, Double-Stranded, RNA, Messenger, Sequence Homology, Amino Acid, Trypanosoma brucei brucei}, issn = {1097-2765}, author = {Klingbeil, Michele M and Motyka, Shawn A and Englund, Paul T} } @article {587, title = {Use of Fe(III) as an electron acceptor to recover previously uncultured hyperthermophiles: isolation and characterization of Geothermobacterium ferrireducens gen. nov., sp. nov.}, journal = {Appl Environ Microbiol}, volume = {68}, year = {2002}, month = {2002 Apr}, pages = {1735-42}, abstract = {It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor. As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. A pure culture was recovered on solidified, Fe(III) oxide medium. The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide. Organic carbon is not required for growth. Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated. The cells are rod shaped, about 0.5 microm by 1.0 to 1.2 microm, and motile and have a single flagellum. Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100 degrees C with an optimum of 85 to 90 degrees C. To our knowledge this is the highest temperature optimum of any organism in the Bacteria. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the Bacteria, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland. While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers. These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences. Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the Bacteria. The name Geothermobacterium ferrireducens gen. nov., sp. nov., is proposed (ATCC BAA-426).}, keywords = {Bacterial Typing Techniques, Culture Media, DNA, Ribosomal, Electron Transport, Ferric Compounds, Fresh Water, Geologic Sediments, Gram-Negative Anaerobic Bacteria, Hot Temperature, Molecular Sequence Data, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {0099-2240}, author = {Kashefi, Kazem and Holmes, Dawn E and Reysenbach, Anna-Louise and Lovley, Derek R} } @article {601, title = {Geobacter hydrogenophilus, Geobacter chapellei and Geobacter grbiciae, three new, strictly anaerobic, dissimilatory Fe(III)-reducers.}, journal = {Int J Syst Evol Microbiol}, volume = {51}, year = {2001}, month = {2001 Mar}, pages = {581-8}, abstract = {Recent studies on the diversity and ubiquity of Fe(III)-reducing organisms in different environments led to the isolation and identification of four new dissimilatory Fe(III)-reducers (strains H-2T, 172T, TACP-2T and TACP-5). All four isolates are non-motile, Gram-negative, freshwater, mesophilic, strict anaerobes with morphology identical to that of Geobacter metallireducens strain GS-15T. Analysis of the 16S rRNA sequences indicated that the new isolates belong to the genus Geobacter, in the delta-Proteobacteria. Significant differences in phenotypic characteristics, DNA-DNA homology and G+C content indicated that the four isolates represent three new species of the genus. The names Geobacter hydrogenophilus sp. nov. (strain H-2T), Geobacter chapellei sp. nov. (strain 172T) and Geobacter grbiciae sp. nov. (strains TACP-2T and TACP-5) are proposed. Geobacter hydrogenophilus and Geobacter chapellei were isolated from a petroleum-contaminated aquifer and a pristine, deep, subsurface aquifer, respectively. Geobacter grbiciae was isolated from aquatic sediments. All of the isolates can obtain energy for growth by coupling the oxidation of acetate to the reduction of Fe(III). The four isolates also coupled Fe(III) reduction to the oxidation of other simple, volatile fatty acids. In addition, Geobacter hydrogenophilus and Geobacter grbiciae were able to oxidize aromatic compounds such as benzoate, whilst Geobacter grbiciae was also able to use the monoaromatic hydrocarbon toluene.}, keywords = {Base Composition, Deltaproteobacteria, Ferric Compounds, Gram-Negative Anaerobic Straight, Curved, and Helical Rods, Hydrocarbons, Iron, Molecular Sequence Data, Nucleic Acid Hybridization, Oxidation-Reduction, RNA, Ribosomal, 16S}, issn = {1466-5026}, author = {Coates, J D and Bhupathiraju, V K and Achenbach, L A and Mclnerney, M J and Lovley, D R} } @article {592, title = {Isolation and characterization of a soluble NADPH-dependent Fe(III) reductase from Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {183}, year = {2001}, month = {2001 Aug}, pages = {4468-76}, abstract = {NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30\% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Deltaproteobacteria, Ferric Compounds, FMN Reductase, Molecular Sequence Data, NADH, NADPH Oxidoreductases, NADP, Nitrilotriacetic Acid, Sequence Analysis, Protein, Sequence Homology, Amino Acid, Solubility}, issn = {0021-9193}, doi = {10.1128/JB.183.15.4468-4476.2001}, author = {Kaufmann, F and Lovley, D R} } @article {590, title = {Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens.}, journal = {Biochem J}, volume = {359}, year = {2001}, month = {2001 Oct 1}, pages = {147-52}, abstract = {Geobacter sulfurreducens is capable of anaerobic respiration with Fe(III) as a terminal electron acceptor via a membrane-bound Fe(III) reductase activity associated with a large molecular mass cytochrome c. This cytochrome was purified by detergent extraction of the membrane fraction, Q-Sepharose ion-exchange chromatography, preparative electrophoresis, and MonoQ ion-exchange chromatography. Spectrophotometric analysis of the purified cytochrome reveals a c-type haem, with no evidence of haem a, haem b or sirohaem. The cytochrome has an M(r) of 89000 as determined by denaturing PAGE, and has an isoelectric point of 5.2 as determined by analytical isoelectric focusing. Dithionite-reduced cytochrome can donate electrons to Fe(III)-nitrilotriacetic acid and synthetic ferrihydrite, thus demonstrating that the cytochrome has redox and thermodynamic properties required for reduction of Fe(III). Analysis using cyclic voltammetry confirmed that the reduced cytochrome can catalytically transfer electrons to ferrihydrite, further demonstrating its ability to be an electron transport mediator in anaerobic Fe(III) respiration. Sequence analysis of a cloned chromosomal DNA fragment revealed a 2307 bp open reading frame (ferA) encoding a 768 amino acid protein corresponding to the 89 kDa cytochrome. The deduced amino acid sequence (FerA) translated from the open reading frame contained 12 putative haem-binding motifs, as well as a hydrophobic N-terminal membrane anchor sequence, a lipid-attachment site and an ATP/GTP-binding site. FerA displayed 20\% or less identity with amino acid sequences of other known cytochromes, although it does share some features with characterized polyhaem cytochromes c.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Chromatography, Ion Exchange, Cloning, Molecular, Cytochrome c Group, Deltaproteobacteria, DNA Primers, Electron Transport, Electrophoresis, Polyacrylamide Gel, Ferric Compounds, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Molecular Weight, Nitrilotriacetic Acid, Oxidation-Reduction, Polymerase Chain Reaction, Sequence Homology, Amino Acid}, issn = {0264-6021}, author = {Magnuson, T S and Isoyama, N and Hodges-Myerson, A L and Davidson, G and Maroney, M J and Geesey, G G and Lovley, D R} } @article {402, title = {Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation.}, journal = {Mol Microbiol}, volume = {40}, year = {2001}, month = {2001 Jun}, pages = {1141-54}, abstract = {SoxS is the direct transcriptional activator of at least 15 genes of the Escherichia coli superoxide regulon. SoxS is small (107 amino acids), binds DNA as a monomer and recognizes a highly degenerate DNA binding site, termed {\textquoteright}soxbox{\textquoteright}. Like other members of the AraC/XylS family, SoxS has two putative helix-turn-helix (HTH) DNA-binding motifs, and it has been proposed that each HTH motif recognizes a highly conserved recognition element of the soxbox. To determine which nucleotides are important for SoxS binding, we conducted a systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters and determined the effect of the soxbox mutations on SoxS DNA binding and transcription activation in vivo by measuring beta-galactosidase activity in strains with fusions to lacZ. We found that the sequences GCAC and CAAA, termed recognition elements 1 and 2 (RE 1 and RE 2), respectively, are critical for SoxS binding, as mutations within these elements severely hinder or eliminate SoxS-dependent transcription activation; substitutions within RE 2 (CAAA), however, are tolerated better than changes within RE 1 (GCAC). Although substitutions at the seven positions separating the two REs had only a modest effect on SoxS binding, AT basepairs were favoured within this {\textquoteright}spacer{\textquoteright} region, presumably because, by facilitating DNA bending, they help bring the two recognition elements into proper juxtaposition. We also found that the {\textquoteright}invariant A{\textquoteright} present at position 1 of 14/15 functional soxboxes identified thus far is important for SoxS binding, as a change to any other nucleotide at this position reduced SoxS-dependent transcription by approximately 50\%. In addition, positions surrounding the REs seem to show a context effect, in that certain substitutions there have little or no effect when the RE has the optimal binding sequence, but produce a pronounced effect when the RE has a suboptimal sequence. We propose that these nucleotides play an important role in effecting differential expression from the various promoters. Lastly, we used gel retardation assays to show that alterations in transcription activation in vivo are caused by effects on DNA binding. Based on this exhaustive mutagenesis, we propose the following optimal sequence for SoxS binding: AnVGCACWWWnKRHCAAAHn (n = A, C, G, T; V = A, C, G; W = A, T; K = G, T; R = A, G; H = A, C, T).}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, Binding Sites, Carrier Proteins, DNA, Bacterial, Electrophoresis, Escherichia coli, Escherichia coli Proteins, Molecular Sequence Data, Mutagenesis, Promoter Regions, Genetic, Trans-Activators, Transcription Factors, Transcriptional Activation}, issn = {0950-382X}, author = {Griffith, K L and Wolf, R E} } @article {789, title = {Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.}, journal = {Mol Biochem Parasitol}, volume = {108}, year = {2000}, month = {2000 May}, pages = {187-97}, abstract = {Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2\% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95\% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.}, keywords = {Animals, Cryptosporidiosis, Cryptosporidium parvum, Genome, Protozoan, Genotype, Germ-Free Life, Humans, Mice, Mice, Knockout, Molecular Sequence Data, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Sequence Analysis, DNA, Swine}, issn = {0166-6851}, author = {Widmer, G and Akiyoshi, D and Buckholt, M A and Feng, X and Rich, S M and Deary, K M and Bowman, C A and Xu, P and Wang, Y and Wang, X and Buck, G A and Tzipori, S} } @article {787, title = {Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.}, journal = {Appl Environ Microbiol}, volume = {66}, year = {2000}, month = {2000 Aug}, pages = {3344-9}, abstract = {Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.}, keywords = {Animals, Base Sequence, Cattle, Cryptosporidiosis, Cryptosporidium parvum, DNA, Protozoan, Humans, Karyotyping, Mice, Mice, Knockout, Microsatellite Repeats, Molecular Sequence Data, Polymerase Chain Reaction, Polymorphism, Genetic, Sequence Analysis, DNA}, issn = {0099-2240}, author = {Feng, X and Rich, S M and Akiyoshi, D and Tumwine, J K and Kekitiinwa, A and Nabukeera, N and Tzipori, S and Widmer, G} } @article {784, title = {Genetic variation and the recent worldwide expansion of Plasmodium falciparum.}, journal = {Gene}, volume = {261}, year = {2000}, month = {2000 Dec 30}, pages = {161-70}, abstract = {Plasmodium falciparum, the agent of human malignant malaria, diverged from Plasmodium reichenowi, the chimpanzee parasite, about the time the human and chimpanzee lineages diverged from each other. The absence of synonymous nucleotide variation at ten loci indicates that the world populations of P. falciparum derive most recently from one single strain, or {\textquoteright}cenancestor,{\textquoteright} which lived a few thousand years ago. Antigenic genes of P. falciparum (such as Csp, Msp-1, and Msp-2) exhibit numerous polymorphisms that have been estimated to be millions of years old. We have discovered in these antigenic genes short repetitive sequences that distort the alignment of alleles and account for the apparent old age of the polymorphisms. The processes of intragenic recombination that generate the repeats occur at rates about 10(-3) to 10(-2), several orders of magnitude greater than the typical mutational process of nucleotide substitutions. We conclude that the antigenic polymorphisms of P. falciparum are consistent with a recent expansion of the world populations of the parasite from a cenancestor that lived in tropical Africa a few thousand years ago.}, keywords = {Animals, Antigens, Protozoan, Base Sequence, Genetic Variation, Merozoite Surface Protein 1, Molecular Sequence Data, Phylogeny, Plasmodium, Plasmodium falciparum, Polymorphism, Genetic, Protozoan Proteins, Repetitive Sequences, Nucleic Acid, RNA, Ribosomal, Sequence Homology, Nucleic Acid, Species Specificity}, issn = {0378-1119}, author = {Ayala, F J and Rich, S M} } @article {786, title = {The origin of antigenic diversity in Plasmodium falciparum.}, journal = {Parasitol Today}, volume = {16}, year = {2000}, month = {2000 Sep}, pages = {390-6}, abstract = {Most studies of genetic variability of Plasmodium falciparum have focused on protein antigens and the genes that encode them. The consensus is that populations exhibit high levels of genetic polymorphism, most notably the genes encoding surface proteins of the merozoite (Msp1, Msp2) and the sporozoite (Csp). The age and derivation of this variation is a subject that warrants further careful consideration, as discussed here by Stephen Rich, Marcelo Ferreira and Francisco Ayala.}, keywords = {Amino Acid Sequence, Animals, Antigens, Protozoan, Base Sequence, Genes, Protozoan, Genetic Variation, Merozoite Surface Protein 1, Molecular Sequence Data, Plasmodium falciparum, Protozoan Proteins, Sequence Homology}, issn = {0169-4758}, author = {Rich, S M and Ferreira, M U and Ayala, F J} } @article {788, title = {Population structure and recent evolution of Plasmodium falciparum.}, journal = {Proc Natl Acad Sci U S A}, volume = {97}, year = {2000}, month = {2000 Jun 20}, pages = {6994-7001}, abstract = {Plasmodium falciparum is the agent of malignant malaria, one of mankind{\textquoteright}s most severe maladies. The parasite exhibits antigenic polymorphisms that have been postulated to be ancient. We have proposed that the extant world populations of P. falciparum have derived from one single parasite, a cenancestor, within the last 5, 000-50,000 years. This inference derives from the virtual or complete absence of synonymous nucleotide polymorphisms at genes not involved in immune or drug responses. Seeking to conciliate this claim with extensive antigenic polymorphism, we first note that allele substitutions or polymorphisms can arise very rapidly, even in a single generation, in large populations subject to strong natural selection. Second, new alleles can arise not only by single-nucleotide mutations, but also by duplication/deletion of short simple-repeat DNA sequences, a process several orders of magnitude faster than single-nucleotide mutation. We analyze three antigenic genes known to be extremely polymorphic: Csp, Msp-1, and Msp-2. We identify regions consisting of tandem or proximally repetitive short DNA sequences, including some previously unnoticed. We conclude that the antigenic polymorphisms are consistent with the recent origin of the world populations of P. falciparum inferred from the analysis of nonantigenic genes.}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Biological Evolution, Genes, Protozoan, Genetics, Population, Molecular Sequence Data, Plasmodium falciparum}, issn = {0027-8424}, author = {Rich, S M and Ayala, F J} } @article {832, title = {Diversity of radA genes from cultured and uncultured archaea: comparative analysis of putative RadA proteins and their use as a phylogenetic marker.}, journal = {J Bacteriol}, volume = {181}, year = {1999}, month = {1999 Feb}, pages = {907-15}, abstract = {Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of Crenarcheota. The two most deeply branching archaeal radA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.}, keywords = {Amino Acid Sequence, Archaea, Archaeal Proteins, Bacteria, Bacterial Proteins, Cloning, Molecular, DNA Primers, DNA Repair, DNA-Binding Proteins, Evolution, Molecular, Humans, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Recombinant Proteins, RNA, Ribosomal, 16S, Saccharomyces cerevisiae, Sequence Alignment}, issn = {0021-9193}, author = {Sandler, S J and Hugenholtz, P and Schleper, C and DeLong, E F and Pace, N R and Clark, A J} } @article {790, title = {Evolution of Plasmodium and the recent origin of the world populations of Plasmodium falciparum.}, journal = {Parassitologia}, volume = {41}, year = {1999}, month = {1999 Sep}, pages = {55-68}, abstract = {We have investigated the evolution of Plasmodium parasites by analyzing DNA sequences of several genes. We reach the following conclusions: (1) The four human parasites, P. falciparum, P. malariae, P. ovale, and P. vivax are very remotely related to each other, so that their evolutionary divergence predates the origin of the hominids; several of these parasites became associated with the human lineage by lateral transfer from other hosts. (2) P. falciparum diverged from P. reichenowi about 8 million years ago, consistently with the time of divergence of the human lineage from the apes; a parsimonious inference is that falciparum has been associated with humans since the origin of the hominids. (3) P. malariae is genetically indistinguishable from P. brasilianum, a parasite of New World monkeys; and, similarly. (4) P. vivax is genetically indistinguishable from the New World monkey parasite P. simium. We infer in each of these two cases a very recent lateral transfer between the human and monkey hosts, and explore alternative hypotheses about the direction of the transfer. We have also investigated the population structure of P. falciparum by analyzing 10 genes and conclude that the extant world populations of this parasite have evolved from a single strain within the last several thousand years. The extensive polymorphisms observed in the highly repetitive central region of the Csp gene, as well as the apparently very divergent two classes of alleles at the Msa-1 gene, are consistent with this conclusion.}, keywords = {Amino Acid Sequence, Animals, Base Sequence, DNA, Protozoan, Evolution, Molecular, Hominidae, Humans, Merozoite Surface Protein 1, Molecular Sequence Data, Plasmodium, Plasmodium falciparum, Polymorphism, Genetic, Sequence Alignment}, issn = {0048-2951}, author = {Ayala, F J and Escalante, A A and Rich, S M} } @article {616, title = {Geothrix fermentans gen. nov., sp. nov., a novel Fe(III)-reducing bacterium from a hydrocarbon-contaminated aquifer.}, journal = {Int J Syst Bacteriol}, volume = {49 Pt 4}, year = {1999}, month = {1999 Oct}, pages = {1615-22}, abstract = {In an attempt to understand better the micro-organisms involved in anaerobic degradation of aromatic hydrocarbons in the Fe(III)-reducing zone of petroleum-contaminated aquifers, Fe(III)-reducing micro-organisms were isolated from contaminated aquifer material that had been adapted for rapid oxidation of toluene coupled to Fe(III) reduction. One of these organisms, strain H-5T, was enriched and isolated on acetate/Fe(III) medium. Strain H-5T is a Gram-negative strict anaerobe that grows with various simple organic acids such as acetate, propionate, lactate and fumarate as alternative electron donors with Fe(III) as the electron acceptor. In addition, strain H-5T also oxidizes long-chain fatty acids such as palmitate with Fe(III) as the sole electron acceptor. Strain H-5T can also grow by fermentation of citrate or fumarate in the absence of an alternative electron acceptor. The primary end-products of citrate fermentation are acetate and succinate. In addition to various forms of soluble and insoluble Fe(III), strain H-5T grows with nitrate, Mn(IV), fumarate and the humic acid analogue 2,6-anthraquinone disulfonate as alternative electron acceptors. As with other organisms that can oxidize organic compounds completely with the reduction of Fe(III), cell suspensions of strain H-5T have absorbance maxima indicative of a c-type cytochrome(s). It is proposed that strain H-5T represents a novel genus in the Holophaga-Acidobacterium phylum and that it should be named Geothrix fermentans sp. nov., gen. nov.}, keywords = {Biodegradation, Environmental, DNA, Bacterial, DNA, Ribosomal, Ferric Compounds, Geologic Sediments, Gram-Negative Anaerobic Bacteria, Molecular Sequence Data, Oxidation-Reduction, Petroleum, Phylogeny, RNA, Ribosomal, 16S, Water Microbiology, Water Pollutants, Chemical, Water Supply}, issn = {0020-7713}, author = {Coates, J D and Ellis, D J and Gaw, C V and Lovley, D R} } @article {403, title = {Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters.}, journal = {Mol Microbiol}, volume = {34}, year = {1999}, month = {1999 Nov}, pages = {414-30}, abstract = {SoxS is the direct transcriptional activator of the member genes of the Escherichia coli superoxide regulon. At class I SoxS-dependent promoters, e.g. zwf and fpr, whose SoxS binding sites ({\textquoteright}soxbox{\textquoteright}) lie upstream of the -35 region of the promoter, activation requires the C-terminal domain of the RNA polymerase alpha-subunit, while at class II SoxS-dependent promoters, e.g. fumC and micF, whose binding sites overlap the -35 region, activation is independent of the alpha-CTD. To determine whether SoxS activation of its class I promoters shows the same helical phase-dependent spacing requirement as class I promoters activated by catabolite gene activator protein, we increased the 7 bp distance between the 20 bp zwf soxbox and the zwf -35 promoter hexamer by 5 bp and 11 bp, and we decreased the 15 bp distance between the 20 bp fpr soxbox and the fpr -35 promoter hexamer by the same amounts. In both cases, displacement of the binding site by a half or full turn of the DNA helix prevented transcriptional activation. With constructs containing the binding site of one gene fused to the promoter of the other, we demonstrated that the positional requirements are a function of the specific binding site, not the promoter. Supposing that opposite orientation of the SoxS binding site at the two promoters might account for the positional requirements, we placed the zwf and fpr soxboxes in the reverse orientation at the various positions upstream of the promoters and determined the effect of orientation on transcription activation. We found that reversing the orientation of the zwf binding site converts its positional requirement to that of the fpr binding site in its normal orientation, and vice versa. Analysis by molecular information theory of DNA sequences known to bind SoxS in vitro is consistent with the opposite orientation of the zwf and fpr soxboxes.}, keywords = {Bacterial Proteins, Base Sequence, Binding Sites, Escherichia coli, Escherichia coli Proteins, Molecular Sequence Data, Plasmids, Promoter Regions, Genetic, Sequence Analysis, DNA, Superoxides, Trans-Activators, Transcription Factors, Transcription, Genetic, Transcriptional Activation}, issn = {0950-382X}, author = {Wood, T I and Griffith, K L and Fawcett, W P and Jair, K W and Schneider, T D and Wolf, R E} } @article {619, title = {Microbial communities associated with anaerobic benzene degradation in a petroleum-contaminated aquifer.}, journal = {Appl Environ Microbiol}, volume = {65}, year = {1999}, month = {1999 Jul}, pages = {3056-63}, abstract = {Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number-PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site{\textquoteright}s capacity for anaerobic benzene degradation.}, keywords = {Anaerobiosis, Benzene, Biodegradation, Environmental, Culture Media, DNA, Bacterial, DNA, Ribosomal, Fresh Water, Geologic Sediments, Gram-Negative Anaerobic Bacteria, Molecular Sequence Data, Oxidation-Reduction, Petroleum, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Water Pollutants}, issn = {0099-2240}, author = {Rooney-Varga, J N and Anderson, R T and Fraga, J L and Ringelberg, D and Lovley, D R} } @article {831, title = {Replication fork assembly at recombination intermediates is required for bacterial growth.}, journal = {Proc Natl Acad Sci U S A}, volume = {96}, year = {1999}, month = {1999 Mar 30}, pages = {3552-5}, abstract = {PriA, a 3{\textquoteright} --> 5{\textquoteright} DNA helicase, directs assembly of a primosome on some bacteriophage and plasmid DNAs. Primosomes are multienzyme replication machines that contribute both the DNA-unwinding and Okazaki fragment-priming functions at the replication fork. The role of PriA in chromosomal replication is unclear. The phenotypes of priA null mutations suggest that the protein participates in replication restart at recombination intermediates. We show here that PriA promotes replication fork assembly at a D loop, an intermediate formed during initiation of homologous recombination. We also show that DnaC810, encoded by a naturally arising intergenic suppressor allele of the priA2::kan mutation, bypasses the need for PriA during replication fork assembly at D loops in vitro. These findings underscore the essentiality of replication fork restart at recombination intermediates under normal growth conditions in bacteria.}, keywords = {Bacteria, Bacteriophage phi X 174, Base Sequence, DNA Polymerase III, DNA Replication, DNA-Binding Proteins, Escherichia coli, Molecular Sequence Data, Oligodeoxyribonucleotides, Open Reading Frames, Recombination, Genetic, Replication Protein A, Templates, Genetic}, issn = {0027-8424}, author = {Liu, J and Xu, L and Sandler, S J and Marians, K J} } @article {753, title = {Soil bacterial community shift correlated with change from forest to pasture vegetation in a tropical soil.}, journal = {Appl Environ Microbiol}, volume = {65}, year = {1999}, month = {1999 Aug}, pages = {3622-6}, abstract = {The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35\% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63\% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61\%, while the DNA of the pasture soil had a peak in G+C content of 67\%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63\% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium-Agrobacterium assemblages in the pasture soil.}, keywords = {Agriculture, Bacteria, Base Composition, DNA, Bacterial, Ecosystem, Molecular Sequence Data, Soil Microbiology, Trees, Tropical Climate}, issn = {0099-2240}, author = {N{\"u}sslein, K and Tiedje, J M} } @article {618, title = {Sulfurospirillum barnesii sp. nov. and Sulfurospirillum arsenophilum sp. nov., new members of the Sulfurospirillum clade of the epsilon Proteobacteria.}, journal = {Int J Syst Bacteriol}, volume = {49 Pt 3}, year = {1999}, month = {1999 Jul}, pages = {1177-80}, abstract = {Two strains of dissimilatory arsenate-reducing vibrio-shaped bacteria are assigned to the genus Sulfurospirillum. These two new species, Sulfurospirillum barnesii strain SES-3T and Sulfurospirillum arsenophilum strain MIT-13T, in addition to Sulfurospirillum sp. SM-5, two strains of Sulfurospirillum deleyianum, and Sulfurospirillum arcachonense, form a distinct clade within the epsilon subclass of the Proteobacteria based on 16S rRNA analysis.}, keywords = {Arsenates, Bacterial Typing Techniques, DNA, Ribosomal, Genes, rRNA, Gram-Negative Bacteria, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Selenium Compounds, Sequence Analysis, DNA}, issn = {0020-7713}, author = {Stolz, J F and Ellis, D J and Blum, J S and Ahmann, D and Lovley, D R and Oremland, R S} } @article {625, title = {Recovery of humic-reducing bacteria from a diversity of environments.}, journal = {Appl Environ Microbiol}, volume = {64}, year = {1998}, month = {1998 Apr}, pages = {1504-9}, abstract = {To evaluate which microorganisms might be responsible for microbial reduction of humic substances in sedimentary environments, humic-reducing bacteria were isolated from a variety of sediment types. These included lake sediments, pristine and contaminated wetland sediments, and marine sediments. In each of the sediment types, all of the humic reducers recovered with acetate as the electron donor and the humic substance analog, 2,6-anthraquinone disulfonate (AQDS), as the electron acceptor were members of the family Geobacteraceae. This was true whether the AQDS-reducing bacteria were enriched prior to isolation on solid media or were recovered from the highest positive dilutions of sediments in liquid media. All of the isolates tested not only conserved energy to support growth from acetate oxidation coupled to AQDS reduction but also could oxidize acetate with highly purified soil humic acids as the sole electron acceptor. All of the isolates tested were also able to grow with Fe(III) serving as the sole electron acceptor. This is consistent with previous studies that have suggested that the capacity for Fe(III) reduction is a common feature of all members of the Geobacteraceae. These studies demonstrate that the potential for microbial humic substance reduction can be found in a wide variety of sediment types and suggest that Geobacteraceae species might be important humic-reducing organisms in sediments.}, keywords = {Acetic Acid, Anthraquinones, Base Sequence, DNA Primers, Electron Transport, Fresh Water, Gram-Negative Anaerobic Bacteria, Humic Substances, Iron, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, RNA, Bacterial, RNA, Ribosomal, 16S, Seawater, Sulfur-Reducing Bacteria, Water Microbiology}, issn = {0099-2240}, author = {Coates, J D and Ellis, D J and Blunt-Harris, E L and Gaw, C V and Roden, E E and Lovley, D R} } @article {627, title = {Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.}, journal = {Arch Microbiol}, volume = {168}, year = {1997}, month = {1997 Nov}, pages = {380-8}, abstract = {A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO42. H2O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the epsilon-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed.}, keywords = {Arsenates, Bacteria, Anaerobic, Biotransformation, Geologic Sediments, Gram-Positive Endospore-Forming Rods, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Substrate Specificity, Sulfates, Sulfides, Sulfur-Reducing Bacteria, Water Microbiology}, issn = {0302-8933}, author = {Newman, D K and Kennedy, E K and Coates, J D and Ahmann, D and Ellis, D J and Lovley, D R and Morel, F M} } @article {834, title = {Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms.}, journal = {J Mol Evol}, volume = {44}, year = {1997}, month = {1997 May}, pages = {528-41}, abstract = {Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes. The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups. A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences. The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids. Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions. Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes. We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively.}, keywords = {Amino Acid Sequence, Animals, Archaeal Proteins, Bacteria, Bacterial Proteins, Cell Cycle Proteins, Consensus Sequence, Conserved Sequence, DNA-Binding Proteins, Escherichia coli Proteins, Evolution, Molecular, Humans, Molecular Sequence Data, Phylogeny, Rad51 Recombinase, Rec A Recombinases, Sequence Alignment}, issn = {0022-2844}, author = {Brendel, V and Brocchieri, L and Sandler, S J and Clark, A J and Karlin, S} } @article {795, title = {Heterogeneity of the internal transcribed spacer (ITS-2) region within individual deer ticks.}, journal = {Insect Mol Biol}, volume = {6}, year = {1997}, month = {1997 May}, pages = {123-9}, abstract = {To determine whether nuclear rDNA sequences provide a useful means for assessing the structure of populations of Ixodes ticks, we compared variability among copies of an internal transcribed spacer (ITS-2) sequence within individual ticks to the variability between ticks. At least 4\% of the nucleotides comprising this sequence vary among the copies present within individual ticks. ITS-2 diversity in each of two ticks is nearly half as great as that reported between ticks from geographically disparate populations. Because individual ticks retain ancestral polymorphism, ITS-2 variation does not accurately reflect descent relationships among these ticks. Sequencing single copies of PCR-amplified ITS-2 therefore does not permit assessment of the phylogenetic relationships among the I. ricinus-like ticks in eastern North America. We recommend caution in future analyses, and emphasize the importance of procedures designed to ensure that the many paralogous copies of the rDNA cistron have been sufficiently homogenized by concerted evolutionary processes. Such precautionary measures will make certain that phylogenetic trees based on these gene sequences reflect the phyletic relatedness of the biological species.}, keywords = {Animals, Base Sequence, Deer, DNA, DNA, Ribosomal, Genetic Heterogeneity, Ixodes, Molecular Sequence Data}, issn = {0962-1075}, author = {Rich, S M and Rosenthal, B M and Telford, S R and Spielman, A and Hartl, D L and Ayala, F J} } @article {794, title = {Plasmodium falciparum antigenic diversity: evidence of clonal population structure.}, journal = {Proc Natl Acad Sci U S A}, volume = {94}, year = {1997}, month = {1997 Nov 25}, pages = {13040-5}, abstract = {Plasmodium falciparum, the agent of malignant malaria, is one of mankind{\textquoteright}s most severe scourges. Efforts to develop preventive vaccines or remedial drugs are handicapped by the parasite{\textquoteright}s rapid evolution of drug resistance and protective antigens. We examine 25 DNA sequences of the gene coding for the highly polymorphic antigenic circumsporozoite protein. We observe total absence of silent nucleotide variation in the two nonrepeated regions of the gene. We propose that this absence reflects a recent origin (within several thousand years) of the world populations of P. falciparum from a single individual; the amino acid polymorphisms observed in these nonrepeat regions would result from strong natural selection. Analysis of these polymorphisms indicates that: (i) the incidence of recombination events does not increase with nucleotide distance; (ii) the strength of linkage disequilibrium between nucleotides is also independent of distance; and (iii) haplotypes in the two nonrepeat regions are correlated with one another, but not with the central repeat region they span. We propose two hypotheses: (i) variation in the highly polymorphic central repeat region arises by mitotic intragenic recombination, and (ii) the population structure of P. falciparum is clonal--a state of affairs that persists in spite of the necessary stage of physiological sexuality that the parasite must sustain in the mosquito vector to complete its life cycle.}, keywords = {Amino Acid Sequence, Animals, Antigens, Protozoan, Molecular Sequence Data, Phylogeny, Plasmodium falciparum, Polymorphism, Genetic, Protozoan Proteins, Recombination, Genetic, Sequence Homology, Amino Acid}, issn = {0027-8424}, author = {Rich, S M and Hudson, R R and Ayala, F J} } @article {391, title = {Identification of a novel dihydrolipoyl dehydrogenase-binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode, Ascaris suum.}, journal = {J Biol Chem}, volume = {271}, year = {1996}, month = {1996 Mar 8}, pages = {5451-7}, abstract = {A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which lacks an amino-terminal lipoyl domain, p45, has been identified in the pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited significant similarity with internal E3-binding domains of dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution of a pyruvate dehydrogenase-depleted adult A. suum PDC in guanidine hydrochloride resulted in two E3-depleted E2 core preparations which were either enriched or substantially depleted of p45. Following reconstitution, the p45-enriched E2 core exhibited enhanced E3 binding, whereas, the p45-depleted E2 core exhibited dramatically reduced E3 binding. Reconstitution of either the bovine kidney or A. suum PDCs with the A. suum E3 suggested that the ascarid E3 was more sensitive to NADH inhibition when bound to the bovine kidney core. The expression of p45 was developmentally regulated and p45 was most abundant in anaerobic muscle. In contrast, E3s isolated from anaerobic muscle or aerobic second-stage larvae were identical. These results suggest that during the transition to anaerobic metabolism, E3 remains unchanged, but it appears that a novel E3BP, p45, is expressed which may help to maintain the activity of the PDC in the face of the elevated intramitochondrial NADH/NAD+ ratios associated with anaerobiosis.}, keywords = {Amino Acid Sequence, Anaerobiosis, Animals, Ascaris suum, Binding Sites, Carrier Proteins, Dihydrolipoamide Dehydrogenase, Electrophoresis, Polyacrylamide Gel, Flavin-Adenine Dinucleotide, Helminth Proteins, Kinetics, Larva, Molecular Sequence Data, NAD, Oxidation-Reduction, Pyruvate Dehydrogenase Complex, Sequence Homology, Amino Acid}, issn = {0021-9258}, author = {Klingbeil, M M and Walker, D J and Arnette, R and Sidawy, E and Hayton, K and Komuniecki, P R and Komuniecki, R} } @article {633, title = {Isolation of Geobacter species from diverse sedimentary environments.}, journal = {Appl Environ Microbiol}, volume = {62}, year = {1996}, month = {1996 May}, pages = {1531-6}, abstract = {In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram-negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria. Unlike the type strain, Geobacter metallireducens, three of the five isolates could use H2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process.}, keywords = {Base Sequence, Ferric Compounds, Gram-Negative Anaerobic Bacteria, Molecular Sequence Data, Phylogeny, RNA, Bacterial, RNA, Ribosomal, 16S, Water Microbiology}, issn = {0099-2240}, author = {Coates, J D and Phillips, E J and Lonergan, D J and Jenter, H and Lovley, D R} } @article {796, title = {Mosaic structure of plasmids from natural populations of Escherichia coli.}, journal = {Genetics}, volume = {143}, year = {1996}, month = {1996 Jul}, pages = {1091-100}, abstract = {The distribution of plasmids related to the fertility factor F was examined in the ECOR reference collection of Escherichia coli. Probes specific for four F-related genes were isolated and used to survey the collection by DNA hybridization. To estimate the genetic diversity of genes in F-like plasmids, DNA sequences were obtained for four plasmid genes. The phylogenetic relationships among the plasmids in the ECOR strains is very different from that of the strains themselves. This finding supports the view that plasmid transfer has been frequent within and between the major groups of ECOR. Furthermore, the sequences indicate that recombination between genes in plasmids takes place at a considerably higher frequency than that observed for chromosomal genes. The plasmid genes, and by inference the plasmids themselves, are mosaic in structure with different regions acquired from different sources. Comparison of gene sequences from a variety of naturally occurring plasmids suggested a plausible donor of some of the recombinant regions as well as implicating a chi site in the mechanism of genetic exchange. The relatively high rate of recombination in F-plasmid genes suggests that conjugational gene transfer may play a greater role in bacterial population structure than previously appreciated.}, keywords = {Bacterial Proteins, Base Sequence, DNA Helicases, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Evolution, Molecular, Membrane Proteins, Molecular Sequence Data, Mosaicism, Nucleic Acid Hybridization, Phylogeny, Plasmids, Polymorphism, Genetic, Proteins, Recombination, Genetic, Repressor Proteins, RNA-Binding Proteins, Trans-Activators}, issn = {0016-6731}, author = {Boyd, E F and Hill, C W and Rich, S M and Hartl, D L} } @article {797, title = {A new Borrelia infecting Lone Star ticks.}, journal = {Lancet}, volume = {347}, year = {1996}, month = {1996 Jan 6}, pages = {67-8}, keywords = {Animals, Base Sequence, Borrelia, Borrelia Infections, Deer, Disease Vectors, DNA Primers, Maryland, Molecular Sequence Data, Ticks}, issn = {0140-6736}, author = {Armstrong, P M and Rich, S M and Smith, R D and Hartl, D L and Spielman, A and Telford, S R} } @article {634, title = {Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria.}, journal = {J Bacteriol}, volume = {178}, year = {1996}, month = {1996 Apr}, pages = {2402-8}, abstract = {Evolutionary relationships among strictly anaerobic dissimilatory Fe(III)-reducing bacteria obtained from a diversity of sedimentary environments were examined by phylogenetic analysis of 16S rRNA gene sequences. Members of the genera Geobacter, Desulfuromonas, Pelobacter, and Desulfuromusa formed a monophyletic group within the delta subdivision of the class Proteobacteria. On the basis of their common ancestry and the shared ability to reduce Fe(III) and/or S0, we propose that this group be considered a single family, Geobacteraceae. Bootstrap analysis, characteristic nucleotides, and higher-order secondary structures support the division of Geobacteraceae into two subgroups, designated the Geobacter and Desulfuromonas clusters. The genus Desulfuromusa and Pelobacter acidigallici make up a distinct branch within the Desulfuromonas cluster. Several members of the family Geobacteraceae, none of which reduce sulfate, were found to contain the target sequences of probes that have been previously used to define the distribution of sulfate-reducing bacteria and sulfate-reducing bacterium-like microorganisms. The recent isolations of Fe(III)-reducing microorganisms distributed throughout the domain Bacteria suggest that development of 16S rRNA probes that would specifically target all Fe(III) reducers may not be feasible. However, all of the evidence suggests that if a 16S rRNA sequence falls within the family Geobacteraceae, then the organism has the capacity for Fe(III) reduction. The suggestion, based on geological evidence, that Fe(III) reduction was the first globally significant process for oxidizing organic matter back to carbon dioxide is consistent with the finding that acetate-oxidizing Fe(III) reducers are phylogenetically diverse.}, keywords = {Bacteria, Anaerobic, Base Sequence, DNA, Ribosomal, Ferric Compounds, Iron, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Sequence Homology, Nucleic Acid}, issn = {0021-9193}, author = {Lonergan, D J and Jenter, H L and Coates, J D and Phillips, E J and Schmidt, T M and Lovley, D R} } @article {835, title = {recA-like genes from three archaean species with putative protein products similar to Rad51 and Dmc1 proteins of the yeast Saccharomyces cerevisiae.}, journal = {Nucleic Acids Res}, volume = {24}, year = {1996}, month = {1996 Jun 1}, pages = {2125-32}, abstract = {The process of homologous recombination has been documented in bacterial and eucaryotic organisms. The Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins are the archetypal members of two related families of proteins that play a central role in this process. Using the PCR process primed by degenerate oligonucleotides designed to encode regions of the proteins showing the greatest degree of identity, we examined DNA from three organisms of a third phylogenetically divergent group, Archaea, for sequences encoding proteins similar to RecA and Rad51. The archaeans examined were a hyperthermophilic acidophile, Sulfolobus sofataricus (Sso); a halophile, Haloferax volcanii (Hvo); and a hyperthermophilic piezophilic methanogen, Methanococcus jannaschii (Mja). The PCR generated DNA was used to clone a larger genomic DNA fragment containing an open reading frame (orf), that we refer to as the radA gene, for each of the three archaeans. As shown by amino acid sequence alignments, percent amino acid identities and phylogenetic analysis, the putative proteins encoded by all three are related to each other and to both the RecA and Rad51 families of proteins. The putative RadA proteins are more similar to the Rad51 family (approximately 40\% identity at the amino acid level) than to the RecA family (approximately 20\%). Conserved sequence motifs, putative tertiary structures and phylogenetic analysis implied by the alignment are discussed. The 5{\textquoteright} ends of mRNA transcripts to the Sso radA were mapped. The levels of radA mRNA do not increase after treatment with UV irradiation as do recA and RAD51 transcripts in E.coli and S.cerevisiae. Hence it is likely that radA in this organism is a constitutively expressed gene and we discuss possible implications of the lack of UV-inducibility.}, keywords = {Amino Acid Sequence, Archaea, Base Sequence, Cell Cycle Proteins, DNA-Binding Proteins, Escherichia coli, Halobacteriaceae, Methanococcus, Models, Molecular, Molecular Sequence Data, Phylogeny, Rad51 Recombinase, Rec A Recombinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Alignment, Sulfolobus}, issn = {0305-1048}, author = {Sandler, S J and Satin, L H and Samra, H S and Clark, A J} } @article {798, title = {Discriminating between Ixodes ticks by means of mitochondrial DNA sequences.}, journal = {Mol Phylogenet Evol}, volume = {4}, year = {1995}, month = {1995 Dec}, pages = {361-5}, abstract = {Ticks of the genus Ixodes have recently assumed prominence because they frequently serve as vectors of important zoonoses, including Lyme disease and babesiosis. The morphological characteristics that have been used in their identification often are ambiguous and are useful solely at a particular stage of development. Here we report the DNA sequence of the mitochondrially encoded 16S rRNA gene of nine different Ixodes ticks and an outgroup from another genus, Dermacentor. The sequences readily discriminate between these ticks. Samples of I. dammini from the northeastern and upper midwestern United States differ from southeastern I. scapularis at about 2\% of the nucleotides. This difference is about half that separating other members of the I. ricinus group of species, but exceeds typical levels of intraspecific variation. Two major clades exist within the I. ricinus complex. One includes I. cookei, I. hexagonus, and I. angustus. The other includes I. persulcatus, I. pacificus, I. muris, I. ricinus, I. scapularis, and I. dammini. We conclude that mtDNA sequences are useful for unravelling the systematics of these important vectors of human disease.}, keywords = {Animals, Arachnid Vectors, Base Sequence, Dermacentor, DNA Primers, DNA, Mitochondrial, Genes, Humans, Ixodes, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Species Specificity}, issn = {1055-7903}, doi = {10.1006/mpev.1995.1033}, author = {Caporale, D A and Rich, S M and Spielman, A and Telford, S R and Kocher, T D} } @article {799, title = {Distribution of the Ixodes ricinus-like ticks of eastern North America.}, journal = {Proc Natl Acad Sci U S A}, volume = {92}, year = {1995}, month = {1995 Jul 3}, pages = {6284-8}, abstract = {We analyzed the geographic distribution of the Ixodes ricinus-like ticks in eastern North America by comparing the mitochondrial 16S rDNA sequences of specimens sampled directly from the field during the 1990s. Two distinct lineages are evident. The southern clade includes ticks from the southeastern and middle-eastern regions of the United States. The range of the northern clade, which appears to have been restricted to the northeastern region until the mid-1900s, now extends throughout the northeastern and middle-eastern regions. These phyletic units correspond to northern and southern taxa that have previously been assigned specific status as Ixodes dammini and Ixodes scapularis, respectively. The expanding range of I. dammini appears to drive the present outbreaks of zoonotic disease in eastern North America that include Lyme disease and human babesiosis.}, keywords = {Animals, Base Sequence, Demography, DNA Primers, DNA, Mitochondrial, DNA, Ribosomal, Geography, Mitochondria, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Population, RNA, Ribosomal, 16S, Ticks, United States}, issn = {0027-8424}, author = {Rich, S M and Caporale, D A and Telford, S R and Kocher, T D and Hartl, D L and Spielman, A} } @article {639, title = {Fe(III) and S0 reduction by Pelobacter carbinolicus.}, journal = {Appl Environ Microbiol}, volume = {61}, year = {1995}, month = {1995 Jun}, pages = {2132-8}, abstract = {There is a close phylogenetic relationship between Pelobacter species and members of the genera Desulfuromonas and Geobacter, and yet there has been a perplexing lack of physiological similarities. Pelobacter species have been considered to have a fermentative metabolism. In contrast, Desulfuromonas and Geobacter species have a respiratory metabolism with Fe(III) serving as the common terminal electron acceptor in all species. However, the ability of Pelobacter species to reduce Fe(III) had not been previously evaluated. When a culture of Pelobacter carbinolicus that had grown by fermentation of 2,3-butanediol was inoculated into the same medium supplemented with Fe(III), the Fe(III) was reduced. There was less accumulation of ethanol and more production of acetate in the presence of Fe(III). P. carbinolicus grew with ethanol as the sole electron donor and Fe(III) as the sole electron acceptor. Ethanol was metabolized to acetate. Growth was also possible on Fe(III) with the oxidation of propanol to propionate or butanol to butyrate if acetate was provided as a carbon source. P. carbinolicus appears capable of conserving energy to support growth from Fe(III) respiration as it also grew with H2 or formate as the electron donor and Fe(III) as the electron acceptor. Once adapted to Fe(III) reduction, P. carbinolicus could also grow on ethanol or H2 with S0 as the electron acceptor. P. carbinolicus did not contain detectable concentrations of the c-type cytochromes that previous studies have suggested are involved in electron transport to Fe(III) in other organisms that conserve energy to support growth from Fe(III) reduction.(ABSTRACT TRUNCATED AT 250 WORDS)}, keywords = {Bacteria, Anaerobic, Base Sequence, Butylene Glycols, Culture Media, Ethanol, Ferrous Compounds, Hydrogen, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sulfur}, issn = {0099-2240}, author = {Lovley, D R and Phillips, E J and Lonergan, D J and Widman, P K} } @article {642, title = {Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism.}, journal = {Appl Environ Microbiol}, volume = {60}, year = {1994}, month = {1994 Oct}, pages = {3752-9}, abstract = {A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PPi, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutyrate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metallireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter sulfurreducens, is proposed.}, keywords = {Acetic Acid, Acetic Acids, Base Sequence, DNA Primers, DNA, Bacterial, DNA, Ribosomal, Electron Transport, Gram-Negative Anaerobic Bacteria, Hydrogen, Metals, Microscopy, Electron, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Bacterial, RNA, Ribosomal, 16S, Soil Microbiology}, issn = {0099-2240}, author = {Caccavo, F and Lonergan, D J and Lovley, D R and Davis, M and Stolz, J F and McInerney, M J} } @article {840, title = {Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression.}, journal = {J Bacteriol}, volume = {176}, year = {1994}, month = {1994 Jul}, pages = {4011-6}, abstract = {The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF. In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence. Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation. When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally. We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible. In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.}, keywords = {Bacterial Proteins, Base Sequence, DNA Mutational Analysis, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Half-Life, Lac Operon, Molecular Sequence Data, Protein Biosynthesis, Recombinant Fusion Proteins, Regulatory Sequences, Nucleic Acid, Ribosomes}, issn = {0021-9193}, author = {Sandler, S J and Clark, A J} } @article {841, title = {RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.}, journal = {J Bacteriol}, volume = {176}, year = {1994}, month = {1994 Jun}, pages = {3661-72}, abstract = {The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.}, keywords = {Bacterial Proteins, Base Sequence, DNA Damage, DNA Repair, DNA-Binding Proteins, Epistasis, Genetic, Escherichia coli, Escherichia coli Proteins, Models, Genetic, Molecular Sequence Data, Phenotype, Plasmids, SOS Response (Genetics), Suppression, Genetic, Ultraviolet Rays}, issn = {0021-9193}, author = {Sandler, S J and Clark, A J} } @article {839, title = {Studies on the mechanism of reduction of UV-inducible sulAp expression by recF overexpression in Escherichia coli K-12.}, journal = {Mol Gen Genet}, volume = {245}, year = {1994}, month = {1994 Dec 15}, pages = {741-9}, abstract = {UV-inducible sulAp expression, an indicator of the SOS response, is reduced by recF+ overexpression in vivo. Different DNA-damaging agents and amounts of RecO and RecR were tested for their effects on this phenotype. It was found that recF+ overexpression reduced sulAp expression after DNA damage by mitomycin C or nalidixic acid, recO+ and recR+ overexpression partially suppressed the reduction of UV-induced sulAp expression caused by recF+ overexpression. The requirement for ATP binding to RecF to produce the phenotype was tested by genetically altering the putative phosphate binding cleft of recF in a way that should prevent the mutant recF protein from binding ATP. It was found that a change of lysine to glutamine at codon 36 results in a mutant recF protein (RecF4115) that is unable to reduce UV-inducible sulAp expression when overproduced. It is inferred from these results that recF overexpression may reduce UV-inducible sulAp expression by a mechanism that is sensitive to the ability of RecF to bind ATP and to the levels of RecO and RecR (RecOR) in the cell, but not to the type of DNA damage per se. Models are explored that can explain how recF+ overexpression reduces UV induction of sulAp and how RecOR overproduction might suppress this phenotype.}, keywords = {Bacterial Proteins, Base Sequence, DNA Primers, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Mutagenesis, Site-Directed, Restriction Mapping, RNA, Messenger, SOS Response (Genetics), Structure-Activity Relationship, Ultraviolet Rays}, issn = {0026-8925}, author = {Sandler, S J} } @article {392, title = {Characterization of cDNA clones for the 2-methyl branched-chain enoyl-CoA reductase. An enzyme involved in branched-chain fatty acid synthesis in anaerobic mitochondria of the parasitic nematode Ascaris suum.}, journal = {J Biol Chem}, volume = {268}, year = {1993}, month = {1993 Oct 25}, pages = {22391-6}, abstract = {The 2-methyl branched-chain enoyl-CoA reductase plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode Ascaris suum. An affinity-purified polyclonal anti-serum against the reductase was used to screen a cDNA library constructed in lambda gt11 with poly(A)+ RNA from adult A. suum muscle. A 1.2-kilobase partial cDNA clone was isolated, subcloned, and sequenced in both directions. Additional sequence at the 5{\textquoteright} end of the mRNA was determined by the RACE (rapid amplification of cDNA ends) procedure. Nucleotide sequence analysis of the cDNAs revealed the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1236 nucleotides and a 3{\textquoteright}-untranslated sequence of 109 nucleotides including a short poly(A) tail 14 nucleotides from a polyadenylation signal (AATAAA). The open reading frame encoded a 396-amino acid sequence (M(r) 43,046) including a 16-amino acid leader peptide. Two-dimensional gel electrophoresis of the purified reductase yielded multiple spots with two distinct but overlapping amino-terminal amino acid sequences. Both sequences overlapped with the sequence predicted from the mRNA, and one of the sequences was identical to the predicted sequence. Comparison of the ascarid sequence with that of mammalian acyl-CoA dehydrogenases revealed a high degree of sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene even though the ascarid enzyme functions as a reductase, not as a dehydrogenase. Immunoblotting of A. suum larval stages and adult tissues with antisera that cross-reacted with each of the spots separated on two-dimensional gels suggested that the reductase was only found in adult muscle. Northern blotting using the partial cDNA revealed a hybridization band of about 1.5 kilobases and also suggested that the enzyme was tissue-specific and developmentally regulated in agreement with the results of the immunoblotting.}, keywords = {Amino Acid Sequence, Anaerobiosis, Animals, Ascaris suum, Base Sequence, Cloning, Molecular, Consensus Sequence, DNA, DNA Primers, DNA, Complementary, Fatty Acid Desaturases, Gene Library, Humans, Mitochondria, Molecular Sequence Data, Oligonucleotides, Antisense, Oxidoreductases, Oxidoreductases Acting on CH-CH Group Donors, Poly A, RNA, RNA, Messenger, Sequence Homology, Amino Acid}, issn = {0021-9258}, author = {Duran, E and Komuniecki, R W and Komuniecki, P R and Wheelock, M J and Klingbeil, M M and Ma, Y C and Johnson, K R} } @article {843, title = {Use of high and low level overexpression plasmids to test mutant alleles of the recF gene of Escherichia coli K-12 for partial activity.}, journal = {Genetics}, volume = {135}, year = {1993}, month = {1993 Nov}, pages = {643-54}, abstract = {We showed that sufficient overexpression of the wild-type recF gene interfered with three normal cell functions: (1) UV induction of transcription from the LexA-protein-repressed sulA promoter, (2) UV resistance and (3) cell viability at 42 degrees. To show this, we altered a low-level overexpressing recF+ plasmid with a set of structurally neutral mutations that increased the rate of expression of recF. The resulting high-level overexpressing plasmid interfered with UV induction of the sulA promoter, as did the low-level overexpressing plasmid. It also reduced UV resistance more than its low level progenitor and decreased viability at 42 degrees, an effect not seen with the low-level plasmid. We used the high-level plasmid to test four recF structural mutations for residual activity. The structural alleles consisted of an insertion mutation, two single amino acid substitution mutations and a double amino acid substitution mutation. On the Escherichia coli chromosome the three substitution mutations acted similarly to a recF deletion in reducing UV resistance in a recB21 recC22 sbcB15 sbcC201 genetic background. By this test, therefore, all three appeared to be null alleles. Measurements of conjugational recombination revealed, however, that the three substitution mutations may have residual activity. On the high-level overexpressing plasmid all three substitution mutations definitely showed partial activity. By contrast, the insertion mutation on the high-level overexpressing plasmid showed no partial activity and can be considered a true null mutation. One of the substitutions, recF143, showed a property attributable to a leaky mutation. Another substitution, recF4101, may block selectively two of the three interference phenotypes, thus allowing us to infer a mechanism for them.}, keywords = {Alleles, Bacterial Proteins, Base Sequence, Chromosomes, Bacterial, DNA Repair, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression, Genes, Bacterial, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Phenotype, Plasmids, Radiation Tolerance, Recombination, Genetic, Ultraviolet Rays}, issn = {0016-6731}, author = {Sandler, S J and Clark, A J} } @article {844, title = {Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.}, journal = {Nucleic Acids Res}, volume = {20}, year = {1992}, month = {1992 Feb 25}, pages = {839-45}, abstract = {We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92\% and 38\% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20\% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations.}, keywords = {Amino Acid Sequence, Bacillus subtilis, Bacterial Proteins, Base Sequence, Binding Sites, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Genetic Complementation Test, Molecular Sequence Data, Nucleic Acid Conformation, Phosphates, Pseudomonas putida, Salmonella typhimurium, Sequence Alignment}, issn = {0305-1048}, author = {Sandler, S J and Chackerian, B and Li, J T and Clark, A J} } @article {845, title = {Factors affecting expression of the recF gene of Escherichia coli K-12.}, journal = {Gene}, volume = {86}, year = {1990}, month = {1990 Jan 31}, pages = {35-43}, abstract = {This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control. The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95\% of the dnaA gene and lambda bet, N (double amber) and 40\% of the exo gene. When most of this DNA was deleted, RecF became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.}, keywords = {Bacterial Proteins, Base Sequence, Cloning, Molecular, Escherichia coli, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Peptide Chain Initiation, Translational, Recombinant Fusion Proteins, Recombination, Genetic, Transcription, Genetic}, issn = {0378-1119}, author = {Sandler, S J and Clark, A J} }