@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 {3120, title = {Evidence of Geobacter-associated phage in a uranium-contaminated aquifer.}, journal = {ISME J}, volume = {9}, year = {2015}, month = {2015 Feb}, pages = {333-46}, abstract = {

Geobacter species may be important agents in the bioremediation of organic and metal contaminants in the subsurface, but as yet unknown factors limit the in situ growth of subsurface Geobacter well below rates predicted by analysis of gene expression or in silico metabolic modeling. Analysis of the genomes of five different Geobacter species recovered from contaminated subsurface sites indicated that each of the isolates had been infected with phage. Geobacter-associated phage sequences were also detected by metagenomic and proteomic analysis of samples from a uranium-contaminated aquifer undergoing in situ bioremediation, and phage particles were detected by microscopic analysis in groundwater collected from sediment enrichment cultures. Transcript abundance for genes from the Geobacter-associated phage structural proteins, tail tube Gp19 and baseplate J, increased in the groundwater in response to the growth of Geobacter species when acetate was added, and then declined as the number of Geobacter decreased. Western blot analysis of a Geobacter-associated tail tube protein Gp19 in the groundwater demonstrated that its abundance tracked with the abundance of Geobacter species. These results suggest that the enhanced growth of Geobacter species in the subsurface associated with in situ uranium bioremediation increased the abundance and activity of Geobacter-associated phage and show that future studies should focus on how these phages might be influencing the ecology of this site.

}, keywords = {Bacteriophages, Biodegradation, Environmental, Genes, Viral, Geobacter, Groundwater, Metagenome, Proteomics, Transcriptome, Uranium, Viral Proteins, Water Pollutants, Radioactive}, issn = {1751-7370}, doi = {10.1038/ismej.2014.128}, author = {Holmes, Dawn E and Giloteaux, Ludovic and Chaurasia, Akhilesh K and Williams, Kenneth H and Luef, Birgit and Wilkins, Michael J and Wrighton, Kelly C and Thompson, Courtney A and Comolli, Luis R and Lovley, Derek R} } @article {3126, title = {Going wireless: Fe(III) oxide reduction without pili by Geobacter sulfurreducens strain JS-1.}, journal = {Appl Environ Microbiol}, volume = {80}, year = {2014}, month = {2014 Jul}, pages = {4331-40}, abstract = {

Previous studies have suggested that the conductive pili of Geobacter sulfurreducens are essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50\% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport through G. sulfurreducens biofilms is more effective via pili.

}, keywords = {Adaptation, Physiological, Biofilms, DNA, Bacterial, Electron Transport, Ferric Compounds, Fimbriae Proteins, Fimbriae, Bacterial, Gene Deletion, Geobacter, Oligonucleotide Array Sequence Analysis, Proteomics, Sequence Analysis, DNA}, issn = {1098-5336}, doi = {10.1128/AEM.01122-14}, author = {Smith, Jessica A and Tremblay, Pier-Luc and Shrestha, Pravin Malla and Snoeyenbos-West, Oona L and Franks, Ashley E and Nevin, Kelly P and Lovley, Derek R} } @article {3153, title = {Bioremediation of uranium-contaminated groundwater: a systems approach to subsurface biogeochemistry.}, journal = {Curr Opin Biotechnol}, volume = {24}, year = {2013}, month = {2013 Jun}, pages = {489-97}, abstract = {

Adding organic electron donors to stimulate microbial reduction of highly soluble U(VI) to less soluble U(IV) is a promising strategy for immobilizing uranium in contaminated subsurface environments. Studies suggest that diagnosing the in situ physiological status of the subsurface community during uranium bioremediation with environmental transcriptomic and proteomic techniques can identify factors potentially limiting U(VI) reduction activity. Models which couple genome-scale in silico representations of the metabolism of key microbial populations with geochemical and hydrological models may be able to predict the outcome of bioremediation strategies and aid in the development of new approaches. Concerns remain about the long-term stability of sequestered U(IV) minerals and the release of co-contaminants associated with Fe(III) oxides, which might be overcome through targeted delivery of electrons to select microorganisms using in situ electrodes.

}, keywords = {Biodegradation, Environmental, Electrodes, Ferric Compounds, Genomics, Groundwater, Proteomics, Uranium, Water Pollutants, Radioactive}, issn = {1879-0429}, doi = {10.1016/j.copbio.2012.10.008}, author = {Williams, Kenneth H and Bargar, John R and Lloyd, Jonathan R and Lovley, Derek R} } @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 {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 {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 {495, title = {Steady state protein levels in Geobacter metallireducens grown with iron (III) citrate or nitrate as terminal electron acceptor.}, journal = {Proteomics}, volume = {7}, year = {2007}, month = {2007 Nov}, pages = {4148-57}, abstract = {Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.}, keywords = {Bacterial Proteins, Cell Proliferation, Electrons, Electrophoresis, Gel, Two-Dimensional, Ferric Compounds, Geobacter, Hydrogen-Ion Concentration, Nitrates, Oxidation-Reduction, Proteomics, Tandem Mass Spectrometry}, issn = {1615-9853}, doi = {10.1002/pmic.200600955}, author = {Ahrendt, Angela J and Tollaksen, Sandra L and Lindberg, Carl and Zhu, Wenhong and Yates, John R and Nevin, Kelly P and Babnigg, Gy{\"o}rgy and Lovley, Derek R and Giometti, Carol S} } @article {515, title = {c-Type cytochromes in Pelobacter carbinolicus.}, journal = {Appl Environ Microbiol}, volume = {72}, year = {2006}, month = {2006 Nov}, pages = {6980-5}, abstract = {Previous studies failed to detect c-type cytochromes in Pelobacter species despite the fact that other close relatives in the Geobacteraceae, such as Geobacter and Desulfuromonas species, have abundant c-type cytochromes. Analysis of the recently completed genome sequence of Pelobacter carbinolicus revealed 14 open reading frames that could encode c-type cytochromes. Transcripts for all but one of these open reading frames were detected in acetoin-fermenting and/or Fe(III)-reducing cells. Three putative c-type cytochrome genes were expressed specifically during Fe(III) reduction, suggesting that the encoded proteins may participate in electron transfer to Fe(III). One of these proteins was a periplasmic triheme cytochrome with a high level of similarity to PpcA, which has a role in Fe(III) reduction in Geobacter sulfurreducens. Genes for heme biosynthesis and system II cytochrome c biogenesis were identified in the genome and shown to be expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of protein extracted from acetoin-fermenting P. carbinolicus cells contained three heme-staining bands which were confirmed by mass spectrometry to be among the 14 predicted c-type cytochromes. The number of cytochrome genes, the predicted amount of heme c per protein, and the ratio of heme-stained protein to total protein were much smaller in P. carbinolicus than in G. sulfurreducens. Furthermore, many of the c-type cytochromes that genetic studies have indicated are required for optimal Fe(III) reduction in G. sulfurreducens were not present in the P. carbinolicus genome. These results suggest that further evaluation of the functions of c-type cytochromes in the Geobacteraceae is warranted.}, keywords = {Bacterial Proteins, Cytochromes c, Deltaproteobacteria, Heme, Polymerase Chain Reaction, Proteomics, Reverse Transcriptase Polymerase Chain Reaction, RNA, Messenger}, issn = {0099-2240}, doi = {10.1128/AEM.01128-06}, author = {Haveman, Shelley A and Holmes, Dawn E and Ding, Yan-Huai R and Ward, Joy E and Didonato, Raymond J and Lovley, Derek R} }