@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 {3138, title = {Field evidence of selenium bioreduction in a uranium-contaminated aquifer.}, journal = {Environ Microbiol Rep}, volume = {5}, year = {2013}, month = {2013 Jun}, pages = {444-52}, abstract = {

Removal of selenium from groundwater was documented during injection of acetate into a uranium-contaminated aquifer near Rifle, Colorado (USA). Bioreduction of aqueous selenium to its elemental form (Se0) concentrated it within mineralized biofilms affixed to tubing used to circulate acetate-amended groundwater. Scanning and transmission electron microscopy revealed close association between Se0 precipitates and cell surfaces, with Se0 aggregates having a diameter of 50-60 nm. Accumulation of Se0 within biofilms occurred over a three-week interval at a rate of c. 9 mg Se0 m(-2) tubing day(-1). Removal was inferred to result from the activity of a mixed microbial community within the biofilms capable of coupling acetate oxidation to the reduction of oxygen, nitrate and selenate. Phylogenetic analysis of the biofilm revealed a community dominated by strains of Dechloromonas sp. and Thauera sp., with isolates exhibiting genetic similarity to the latter known to reduce selenate to Se0. Enrichment cultures of selenate-respiring microorganisms were readily established using Rifle site groundwater and acetate, with cultures dominated by strains closely related to D. aromatica (96-99\% similarity). Predominance of Dechloromonas sp. in recovered biofilms and enrichments suggests this microorganism may play a role in the removal of selenium oxyanions present in Se-impacted groundwaters and sediments.

}, keywords = {Acetates, Betaproteobacteria, Biodegradation, Environmental, Biofilms, Colorado, Groundwater, Humans, Microbial Consortia, Oxidation-Reduction, Oxygen, Phylogeny, RNA, Ribosomal, 16S, Selenic Acid, Selenium, Selenium Compounds, Thauera, Uranium, Water Pollutants, Chemical}, issn = {1758-2229}, doi = {10.1111/1758-2229.12032}, author = {Williams, Kenneth H and Wilkins, Michael J and N{\textquoteright}Guessan, A Lucie and Arey, Bruce and Dodova, Elena and Dohnalkova, Alice and Holmes, Dawn and Lovley, Derek R and Long, Philip E} } @article {1564, title = {A field trial of nutrient stimulation of methanotrophs to reduce greenhouse gas emissions from landfill cover soils.}, journal = {J Air Waste Manag Assoc}, volume = {63}, year = {2013}, month = {2013 Mar}, pages = {300-9}, abstract = {

UNLABELLED: Landfills are among the major sources of anthropogenic methane (CH4) estimated to reach 40 x 10(9) kg per year worldwide by 2015 (IPCC, 2007). A 2 1/2-year field experiment was conducted at a closed landfill in western Michigan where methanotrophs, methane-consuming bacteria, were stimulated by nutrient addition to the soil without significantly increasing biogenic nitrous oxide (N2O) production. The effects of the nitrogen amendments (KNO3 and NH4Cl), phenylacetylene (a selective inhibitor of nitrifying bacteria that contribute to N2O production), and a canopy (to reduce direct water infiltration) on the vertical soil gas profiles of CH4, CO2, and O2 were measured in the top meter of the soil. Methane and nitrous oxide fluxes were calculated from the corresponding soil gas concentration gradients with respect to depth and a Millington-Quirk diffusivity coefficient in soil derived empirically from soil porosity, water content, and diffusivity coefficients in air from the literature. Methane flux estimates were as high as 218.4 g m(-2) day(-1) in the fall and 12.8 g/m(-2) day(-1) in the summer. During the spring and summer CH4 fluxes were reduced by more than half by adding KNO3 and NH4Cl into the soil as compared to control plots, while N2O fluxes increased substantially. The concurrent addition of phenylacetylene to the amendment decreased peak N2O production by half and the rate of peak methane oxidation by about one-third. The seasonal average methane and N2O flux data were extrapolated to estimate the reduction of CH4 and N2O fluxes into the atmosphere by nitrogen and inhibitor addition to the cover soils. The results suggest that such additions coupled with soil moisture management may provide a potential strategy to significantly reduce greenhouse gas emissions from landfills.

IMPLICATIONS: The results of a 2 1/2-year study of effects of nutrient stimulation on methane oxidation in landfill cover soils demonstrates that nutrient addition does decrease methane emissions. The work further underscores the control which soil moisture exerts on methane oxidation. Water management is critical to the success of methane oxidation strategies.

}, keywords = {Biodegradation, Environmental, Gases, Greenhouse Effect, Methane, Oxidation-Reduction, Soil, Soil Microbiology, Waste Management}, issn = {1096-2247}, author = {Lizik, William and Im, Jeongdae and Semrau, Jeremy D and Barcelona, Michael J} } @article {3143, title = {Fluctuations in species-level protein expression occur during element and nutrient cycling in the subsurface.}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013}, pages = {e57819}, abstract = {

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

}, keywords = {Biomass, Carbon, Environment, Gene Expression Regulation, Bacterial, Geobacter, Groundwater, Humic Substances, Iron, Oxidation-Reduction, Phosphates, Plankton, Proteomics, RNA, Ribosomal, 16S, Tandem Mass Spectrometry, Uranium, Vanadium, Water Microbiology}, issn = {1932-6203}, doi = {10.1371/journal.pone.0057819}, author = {Wilkins, Michael J and Wrighton, Kelly C and Nicora, Carrie D and Williams, Kenneth H and McCue, Lee Ann and Handley, Kim M and Miller, Chris S and Giloteaux, Ludovic and Montgomery, Alison P and Lovley, Derek R and Banfield, Jillian F and Long, Philip E and Lipton, Mary S} } @article {802, title = {Factors Limiting SOS Expression in Log-Phase Cells of Escherichia coli.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Oct}, pages = {5325-33}, abstract = {In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25\%) log-phase cells have RecA filaments, few (about 1\%) are induced for SOS. It is hypothesized that RecA{\textquoteright}s ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24\% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91\%) nearly equals the percentage with at least one RecA-GFP focus (83\%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.}, issn = {1098-5530}, doi = {10.1128/JB.00674-12}, author = {Massoni, Shawn C and Leeson, Michael C and Long, Jarukit Edward and Gemme, Kristin and Mui, Alice and Sandler, Steven J} } @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 {737, title = {Fast disinfecting antimicrobial surfaces.}, journal = {Langmuir}, volume = {25}, year = {2009}, month = {2009 Jan 20}, pages = {1060-7}, abstract = {Silicon wafers and glass surfaces were functionalized with facially amphiphilic antimicrobial copolymers using the "grafting from" technique. Surface-initiated atom transfer radical polymerization (ATRP) was used to grow poly(butylmethacrylate)-co-poly(Boc-aminoethyl methacrylate) from the surfaces. Upon Boc-deprotection, these surfaces became highly antimicrobial and killed S. aureus and E. coli 100\% in less than 5 min. The molecular weight and grafting density of the polymer were controlled by varying the polymerization time and initiator surface density. Antimicrobial studies showed that the killing efficiency of these surfaces was independent of polymer layer thickness or grafting density within the range of surfaces studied.}, keywords = {Anti-Bacterial Agents, Escherichia coli, Glass, Microbial Sensitivity Tests, Molecular Structure, Particle Size, Polymethacrylic Acids, Silicon, Staphylococcus aureus, Surface Properties}, issn = {0743-7463}, doi = {10.1021/la802953v}, author = {Madkour, Ahmad E and Dabkowski, Jeffery M and Nusslein, Klaus and Tew, Gregory N} } @article {465, title = {Future shock from the microbe electric.}, journal = {Microb Biotechnol}, volume = {2}, year = {2009}, month = {2009 Mar}, pages = {139-41}, keywords = {Bacteria, Bioelectric Energy Sources, Biotechnology, Electricity, Energy-Generating Resources}, issn = {1751-7915}, doi = {10.1111/j.1751-7915.2009.00090_9.x}, author = {Lovley, Derek R} } @article {492, title = {Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens.}, journal = {Environ Microbiol}, volume = {10}, year = {2008}, month = {2008 Feb}, pages = {497-505}, abstract = {A novel fluorescence technique for monitoring the redox status of c-type cytochromes in Geobacter sulfurreducens was developed in order to evaluate the capacity of these extracytoplasmic cytochromes to store electrons during periods in which an external electron acceptor is not available. When intact cells in which the cytochromes were in a reduced state were excited at a wavelength of 350 nm, they fluoresced with maxima at 402 and 437 nm. Oxidation of the cytochromes resulted in a loss of fluorescence. This method was much more sensitive than the traditional approach of detecting c-type cytochromes via visible light absorbance. Furthermore, fluorescence of reduced cytochromes in individual cells could be detected via fluorescence microscopy, and the cytochromes in a G. sulfurreducens biofilm, remotely excited with an optical fibre, could be detected at distances as far as 5 cm. Fluorescence analysis of cytochrome oxidation and reduction of the external electron acceptor, anthraquinone-2,6-disulfonate, suggested that the extracytoplasmic cytochromes of G. sulfurreducens could store approximately 10(7) electrons per cell. Independent analysis of the haem content of the cells determined from analysis of incorporation of (55)Fe into cytochromes provided a similar estimate of cytochrome electron-storage capacity. This electron-storage capacity could, in the absence of an external electron acceptor, permit continued electron transfer across the inner membrane sufficient to supply the maintenance energy requirements for G. sulfurreducens for up to 8 min or enough proton motive force to power flagella motors for G. sulfurreducens motility. The fluorescence approach described here provides a sensitive method for evaluating the redox status of Geobacter species in culture and/or its environments. Furthermore, these results suggest that the periplasmic and outer-membrane cytochromes of Geobacter species act as capacitors, allowing continued electron transport, and thus viability and motility, for Geobacter species as they move between heterogeneously dispersed Fe(III) oxides during growth in the subsurface.}, keywords = {Anthraquinones, Cytochromes c, Electron Transport, Ferric Compounds, Flagella, Fluorescence, Geobacter, Microscopy, Fluorescence, Oxidation-Reduction}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2007.01470.x}, author = {Esteve-N{\'u}{\~n}ez, Abraham and Sosnik, Julian and Visconti, Pablo and Lovley, Derek R} } @article {697, title = {Function of phosphatidylinositol in mycobacteria.}, journal = {J Biol Chem}, volume = {280}, year = {2005}, month = {2005 Mar 25}, pages = {10981-7}, abstract = {Phosphatidylinositol (PI) is an abundant phospholipid in the cytoplasmic membrane of mycobacteria and the precursor for more complex glycolipids, such as the PI mannosides (PIMs) and lipoarabinomannan (LAM). To investigate whether the large steady-state pools of PI and apolar PIMs are required for mycobacterial growth, we have generated a Mycobacterium smegmatis inositol auxotroph by disruption of the ino1 gene. The ino1 mutant displayed wild-type growth rates and steady-state levels of PI, PIM, and LAM when grown in the presence of 1 mM inositol. The non-dividing ino1 mutant was highly resistant to inositol starvation, reflecting the slow turnover of inositol lipids in this stage. In contrast, dilution of growing or stationary-phase ino1 mutant in inositol-free medium resulted in the rapid depletion of PI and apolar PIMs. Whereas depletion of these lipids was not associated with loss of viability, subsequent depletion of polar PIMs coincided with loss of major cell wall components and cell viability. Metabolic labeling experiments confirmed that the large pools of PI and apolar PIMs were used to sustain polar PIM and LAM biosynthesis during inositol limitation. They also showed that under non-limiting conditions, PI is catabolized via lyso-PI. These data suggest that large pools of PI and apolar PIMs are not essential for membrane integrity but are required to sustain polar PIM biosynthesis, which is essential for mycobacterial growth.}, keywords = {Inositol, Lipopolysaccharides, Mannose, Mycobacterium smegmatis, Phosphatidylinositols}, issn = {0021-9258}, doi = {10.1074/jbc.M413443200}, author = {Haites, Ruth E and Morita, Yasu S and McConville, Malcolm J and Billman-Jacobe, Helen} } @article {578, title = {Fulvic acid oxidation state detection using fluorescence spectroscopy.}, journal = {Environ Sci Technol}, volume = {36}, year = {2002}, month = {2002 Jul 15}, pages = {3170-5}, abstract = {Humic substances are a heterogeneous class of moderate molecular weight, yellow-colored biomolecules present in all soils, sediments, and natural waters. Although humic substances are generally resistant to microbial degradation under anaerobic conditions, some microorganisms in soils and sediments can use quinone moieties in humic substances as electron acceptors. Laboratory experiments have shown that humic substances can act as electron shuttles in the microbial reduction of ferric iron. Field studies of electron shuttling processes have been constrained by the lack of methods to characterize the oxidation state of quinone moieties in humic substances at natural concentrations. All humic substances have fluorescent properties, and fluorescence spectroscopy can indicate differences in precursor organic source of humic substances. Here we show that the quinone moieties responsible for electron transfer reactions contribute significantly to the fluorescence of humic substances. Further we use fluorescence spectroscopy to elucidate the oxidation state of quinone moieties in humic substances at natural concentrations found in sediment interstitial waters.}, keywords = {Benzopyrans, Environmental Monitoring, Iron, Oxidation-Reduction, Spectrometry, Fluorescence}, issn = {0013-936X}, author = {Klapper, Lisa and McKnight, Diane M and Fulton, J Robin and Blunt-Harris, Elizabeth L and Nevin, Kelly P and Lovley, Derek R and Hatcher, Patrick G} } @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 {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} } @article {1701, title = {Formate assay in body fluids: application in methanol poisoning.}, journal = {Biochem Med}, volume = {13}, year = {1975}, month = {1975 Jun}, pages = {117-26}, keywords = {Aldehyde Oxidoreductases, Animals, Body Fluids, Carbon Dioxide, Formates, Haplorhini, Humans, Hydrogen-Ion Concentration, Kinetics, Methanol, Methods, Pseudomonas}, issn = {0006-2944}, author = {Makar, A B and McMartin, K E and Palese, M and Tephly, T R} }