@article {3085, title = {Potential for Methanosarcina to Contribute to Uranium Reduction during Acetate-Promoted Groundwater Bioremediation.}, journal = {Microb Ecol}, volume = {76}, year = {2018}, month = {2018 Oct}, pages = {660-667}, abstract = {

Previous studies of acetate-promoted bioremediation of uranium-contaminated aquifers focused on Geobacter because no other microorganisms that can couple the oxidation of acetate with U(VI) reduction had been detected in situ. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40~days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of Methanosarcina barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells; however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.

}, keywords = {Acetates, Biodegradation, Environmental, Geobacter, Groundwater, Methane, Methanosarcina, Oxidation-Reduction, Uranium, Water Pollutants, Chemical}, issn = {1432-184X}, doi = {10.1007/s00248-018-1165-5}, author = {Holmes, Dawn E and Orelana, Roberto and Giloteaux, Ludovic and Wang, Li-Ying and Shrestha, Pravin and Williams, Kenneth and Lovley, Derek R and Rotaru, Amelia-Elena} } @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 {3154, title = {Characterization and transcription of arsenic respiration and resistance genes during in situ uranium bioremediation.}, journal = {ISME J}, volume = {7}, year = {2013}, month = {2013 Feb}, pages = {370-83}, abstract = {

The possibility of arsenic release and the potential role of Geobacter in arsenic biogeochemistry during in situ uranium bioremediation was investigated because increased availability of organic matter has been associated with substantial releases of arsenic in other subsurface environments. In a field experiment conducted at the Rifle, CO study site, groundwater arsenic concentrations increased when acetate was added. The number of transcripts from arrA, which codes for the α-subunit of dissimilatory As(V) reductase, and acr3, which codes for the arsenic pump protein Acr3, were determined with quantitative reverse transcription-PCR. Most of the arrA (>60\%) and acr3-1 (>90\%) sequences that were recovered were most similar to Geobacter species, while the majority of acr3-2 (>50\%) sequences were most closely related to Rhodoferax ferrireducens. Analysis of transcript abundance demonstrated that transcription of acr3-1 by the subsurface Geobacter community was correlated with arsenic concentrations in the groundwater. In contrast, Geobacter arrA transcript numbers lagged behind the major arsenic release and remained high even after arsenic concentrations declined. This suggested that factors other than As(V) availability regulated the transcription of arrA in situ, even though the presence of As(V) increased the transcription of arrA in cultures of Geobacter lovleyi, which was capable of As(V) reduction. These results demonstrate that subsurface Geobacter species can tightly regulate their physiological response to changes in groundwater arsenic concentrations. The transcriptomic approach developed here should be useful for the study of a diversity of other environments in which Geobacter species are considered to have an important influence on arsenic biogeochemistry.

}, keywords = {Acetates, Arsenate Reductases, Arsenic, Biodegradation, Environmental, Colorado, Gene Expression Regulation, Bacterial, Genes, Bacterial, Geobacter, Groundwater, Transcriptome, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2012.109}, author = {Giloteaux, Ludovic and Holmes, Dawn E and Williams, Kenneth H and Wrighton, Kelly C and Wilkins, Michael J and Montgomery, Alison P and Smith, Jessica A and Orellana, Roberto and Thompson, Courtney A and Roper, Thomas J and Long, Philip E and Lovley, Derek R} } @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 {444, title = {Expression of acetate permease-like (apl ) genes in subsurface communities of Geobacter species under fluctuating acetate concentrations.}, journal = {FEMS Microbiol Ecol}, volume = {73}, year = {2010}, month = {2010 Sep}, pages = {441-9}, abstract = {The addition of acetate to uranium-contaminated aquifers in order to stimulate the growth and activity of Geobacter species that reduce uranium is a promising in situ bioremediation option. Optimizing this bioremediation strategy requires that sufficient acetate be added to promote Geobacter species growth. We hypothesized that under acetate-limiting conditions, subsurface Geobacter species would increase the expression of either putative acetate symporters genes (aplI and aplII). Acetate was added to a uranium-contaminated aquifer (Rifle, CO) in two continuous amendments separated by 5 days of groundwater flush to create changing acetate concentrations. While the expression of aplI in monitoring well D04 (high acetate) weakly correlated with the acetate concentration over time, the transcript levels for this gene were relatively constant in well D08 (low acetate). At the lowest acetate concentrations during the groundwater flush, the transcript levels of aplII were the highest. The expression of aplII decreased 2-10-fold upon acetate reintroduction. However, the overall instability of acetate concentrations throughout the experiment could not support a robust conclusion regarding the role of apl genes in response to acetate limitation under field conditions, in contrast to previous chemostat studies, suggesting that the function of a microbial community cannot be inferred based on lab experiments alone.}, keywords = {Acetates, Bacterial Proteins, Biodegradation, Environmental, Fresh Water, Gene Expression Regulation, Bacterial, Gene Library, Geobacter, Membrane Transport Proteins, Multigene Family, RNA, Bacterial, Uranium, Water Pollutants, Radioactive}, issn = {1574-6941}, doi = {10.1111/j.1574-6941.2010.00907.x}, author = {Elifantz, Hila and N{\textquoteright}guessan, Lucie A and Mouser, Paula J and Williams, Kenneth H and Wilkins, Michael J and Risso, Carla and Holmes, Dawn E and Long, Philip E and Lovley, Derek R} } @article {459, title = {Influence of heterogeneous ammonium availability on bacterial community structure and the expression of nitrogen fixation and ammonium transporter genes during in situ bioremediation of uranium-contaminated groundwater.}, journal = {Environ Sci Technol}, volume = {43}, year = {2009}, month = {2009 Jun 15}, pages = {4386-92}, abstract = {The influence of ammonium availability on bacterial community structure and the physiological status of Geobacter species during in situ bioremediation of uranium-contaminated groundwater was evaluated. Ammonium concentrations varied by 2 orders of magnitude (< 4 to 400 microM) across th study site. Analysis of 16S rRNA sequences suggested that ammonium may have been one factor influencing the community composition prior to acetate amendment with Rhodoferax species predominating over Geobacter species with higher ammonium and Dechloromonas species dominating at the site with lowest ammonium. However, once acetate was added and dissimilatory metal reduction was stimulated, Geobacter species became the predominant organisms at all locations. Rates of U(VI) reduction appeared to be more related to acetate concentrations rather than ammonium levels. In situ mRNA transcript abundance of the nitrogen fixation gene, nifD, and the ammonium transporter gene, amtB, in Geobacter species indicated that ammonium was the primary source of nitrogen during uranium reduction. The abundance of amtB was inversely correlated to ammonium levels, whereas nifD transcript levels were similar across all sites examined. These results suggest that nifD and amtB expression are closely regulated in response to ammonium availability to ensure an adequate supply of nitrogen while conserving cell resources. Thus, quantifying nifD and amtB transcript expression appears to be a useful approach for monitoring the nitrogen-related physiological status of subsurface Geobacter species. This study also emphasizes the need for more detailed analysis of geochemical and physiological interactions at the field scale in order to adequately model subsurface microbial processes during bioremediation.}, keywords = {Carrier Proteins, DNA, Bacterial, Environmental Remediation, Gene Expression Regulation, Bacterial, Gene Library, Geobacter, Nitrogen Fixation, Quaternary Ammonium Compounds, Time Factors, Uranium, Water, Water Pollutants, Radioactive}, issn = {0013-936X}, author = {Mouser, Paula J and N{\textquoteright}guessan, Lucie A and Elifantz, Hila and Holmes, Dawn E and Williams, Kenneth H and Wilkins, Michael J and Long, Philip E and Lovley, Derek R} } @article {467, title = {Quantifying expression of Geobacter spp. oxidative stress genes in pure culture and during in situ uranium bioremediation.}, journal = {ISME J}, volume = {3}, year = {2009}, month = {2009 Apr}, pages = {454-65}, abstract = {As part of an effort to diagnose the physiological status of Geobacter species during in situ bioremediation of uranium-contaminated groundwater, transcript levels for two genes potentially associated with oxidative stress, cydA and sodA, were quantified throughout a bioremediation field study in Rifle, CO, USA. Despite the accumulation of Fe(II) in the groundwater, which is inconsistent with the presence of dissolved oxygen, both genes were highly expressed during the bioremediation process. Therefore, the response to oxidative stress was further evaluated with Geobacter uraniireducens, an isolate from the Rifle site. When G. uraniireducens cultured with fumarate as the electron acceptor was exposed to 5\% oxygen for 8 h, there was a significant increase in cydA and sodA transcripts as well as other genes associated with oxygen respiration or oxidative stress. Oxygen-exposed cells had lower transcript abundance for genes associated with anaerobic respiration, metabolism and motility. Short-term oxygen exposure had little impact on cydA transcript levels, as more than 1 h was required for increases to levels comparable to the subsurface. Abundance of cydA and sodA transcripts for the isolate G. sulfurreducens were always higher in cells cultured with Fe(III) compared with fumarate as an electron acceptor, even when fumarate-grown cells were exposed to oxygen, and Fe(III)-grown cells were grown anaerobically. These results suggest that the apparently high Geobacter cydA and sodA expression during bioremediation cannot necessarily be attributed to oxidative stress and demonstrate that diagnosis of the metabolic status of subsurface microorganisms through transcript analysis should be coupled with appropriate geochemical analyses.}, keywords = {Anaerobiosis, Bacterial Proteins, Biodegradation, Environmental, Colorado, Ferric Compounds, Fumarates, Gene Expression Profiling, Geobacter, Oxidative Stress, Soil Microbiology, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2008.126}, author = {Mouser, Paula J and Holmes, Dawn E and Perpetua, Lorrie A and DiDonato, Raymond and Postier, Brad and Liu, Anna and Lovley, Derek R} } @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 {488, title = {Gene transcript analysis of assimilatory iron limitation in Geobacteraceae during groundwater bioremediation.}, journal = {Environ Microbiol}, volume = {10}, year = {2008}, month = {2008 May}, pages = {1218-30}, abstract = {Limitations on the availability of Fe(III) as an electron acceptor are thought to play an important role in restricting the growth and activity of Geobacter species during bioremediation of contaminated subsurface environments, but the possibility that these organisms might also be limited in the subsurface by the availability of iron for assimilatory purposes was not previously considered because copious quantities of Fe(II) are produced as the result of Fe(III) reduction. Analysis of multiple Geobacteraceae genomes revealed the presence of a three-gene cluster consisting of homologues of two iron-dependent regulators, fur and dtxR (ideR), separated by a homologue of feoB, which encodes an Fe(II) uptake protein. This cluster appears to be conserved among members of the Geobacteraceae and was detected in several environments. Expression of the fur-feoB-ideR cluster decreased as Fe(II) concentrations increased in chemostat cultures. The number of Geobacteraceae feoB transcripts in groundwater samples from a site undergoing in situ uranium bioremediation was relatively high until the concentration of dissolved Fe(II) increased near the end of the field experiment. These results suggest that, because much of the Fe(II) is sequestered in solid phases, Geobacter species, which have a high requirement for iron for iron-sulfur proteins, may be limited by the amount of iron available for assimilatory purposes. These results demonstrate the ability of transcript analysis to reveal previously unsuspected aspects of the in situ physiology of microorganisms in subsurface environments.}, keywords = {Bacterial Proteins, Biodegradation, Environmental, Culture Media, Ferric Compounds, Ferrous Compounds, Fresh Water, Gene Expression Regulation, Bacterial, Geobacter, Iron, Multigene Family, Phylogeny, Polymerase Chain Reaction, Repressor Proteins, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Uranium, Water Pollution, Radioactive}, issn = {1462-2920}, doi = {10.1111/j.1462-2920.2007.01537.x}, author = {O{\textquoteright}Neil, Regina A and Holmes, Dawn E and Coppi, Maddalena V and Adams, Lorrie A and Larrahondo, M Juliana and Ward, Joy E and Nevin, Kelly P and Woodard, Trevor L and Vrionis, Helen A and N{\textquoteright}guessan, Lucie A 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 {472, title = {Highly conserved genes in Geobacter species with expression patterns indicative of acetate limitation.}, journal = {Microbiology}, volume = {154}, year = {2008}, month = {2008 Sep}, pages = {2589-99}, abstract = {Analysis of the genome of Geobacter sulfurreducens revealed four genes encoding putative symporters with homology to ActP, an acetate transporter in Escherichia coli. Three of these genes, aplA, aplB and aplC, are highly similar (over 90 \% identical) and fell within a tight phylogenetic cluster (Group I) consisting entirely of Geobacter homologues. Transcript levels for all three genes increased in response to acetate limitation. The fourth gene, aplD, is phylogenetically distinct (Group II) and its expression was not influenced by acetate availability. Deletion of any one of the three genes in Group I did not significantly affect acetate-dependent growth, suggesting functional redundancy. Attempts to recover mutants in which various combinations of two of these genes were deleted were unsuccessful, suggesting that at least two of these three transporter genes are required to support growth. Closely related Group I apl genes were found in the genomes of other Geobacter species whose genome sequences are available. Furthermore, related genes could be detected in genomic DNA extracted from a subsurface environment undergoing in situ uranium bioremediation. The transporter genes recovered from the subsurface were most closely related to Group I apl genes found in the genomes of cultured Geobacter species that were isolated from contaminated subsurface environments. The increased expression of these genes in response to acetate limitation, their high degree of conservation among Geobacter species and the ease with which they can be detected in environmental samples suggest that Group I apl genes of the Geobacteraceae may be suitable biomarkers for acetate limitation. Monitoring the expression of these genes could aid in the design of strategies for acetate-mediated in situ bioremediation of uranium-contaminated groundwater.}, keywords = {Acetates, Biodegradation, Environmental, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Gene Deletion, Gene Expression, Genes, Bacterial, Genome, Bacterial, Geobacter, Membrane Transport Proteins, Phylogeny, Uranium}, issn = {1350-0872}, doi = {10.1099/mic.0.2008/017244-0}, author = {Risso, Carla and Meth{\'e}, Barbara A and Elifantz, Hila and Holmes, Dawn E and Lovley, Derek R} } @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 {529, title = {Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 Nov}, pages = {6870-7}, abstract = {The Geobacteraceae citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of Geobacteraceae. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of Geobacter metabolism was evaluated in pure culture studies and in four different Geobacteraceae-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of Geobacter sulfurreducens grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene, gltA, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes recA, rpoD, and proC remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of gltA. The expression of gltA was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect gltA transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of Geobacteraceae by tracking the expression of the citrate synthase gene.}, keywords = {Acetates, Citrate (si)-Synthase, Deltaproteobacteria, DNA, Ribosomal, Electrodes, Ferric Compounds, Fresh Water, Geobacter, Geologic Sediments, Petroleum, Phylogeny, RNA, Ribosomal, 16S, Uranium, Water Pollutants, Chemical, Water Pollutants, Radioactive}, issn = {0099-2240}, doi = {10.1128/AEM.71.11.6870-6877.2005}, author = {Holmes, Dawn E and Nevin, Kelly P and O{\textquoteright}Neil, Regina A and Ward, Joy E and Adams, Lorrie A and Woodard, Trevor L and Vrionis, Helen A and Lovley, Derek R} } @article {583, title = {Enrichment of members of the family Geobacteraceae associated with stimulation of dissimilatory metal reduction in uranium-contaminated aquifer sediments.}, journal = {Appl Environ Microbiol}, volume = {68}, year = {2002}, month = {2002 May}, pages = {2300-6}, abstract = {Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family Geobacteraceae, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion, Geobacteraceae accounted for ca. 40\% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas Geobacteraceae accounted for fewer than 5\% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65\% of these Geobacteraceae sequences were most similar to sequences from Desulfuromonas species, with the remainder being most closely related to Geobacter species. Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR and TaqMan analyses indicated that the number of Geobacteraceae sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in Geobacteraceae sequences was observed in control sediments. In contrast to the predominance of Geobacteraceae sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that Geobacteraceae are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that Geobacteraceae will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.}, keywords = {Gene Library, Geologic Sediments, Iron, Oxidation-Reduction, Proteobacteria, RNA, Ribosomal, 16S, Soil Pollutants, Uranium}, issn = {0099-2240}, author = {Holmes, Dawn E and Finneran, Kevin T and O{\textquoteright}Neil, Regina A and Lovley, Derek R} }