@article {419, title = {Genome-scale analysis of anaerobic benzoate and phenol metabolism in the hyperthermophilic archaeon Ferroglobus placidus.}, journal = {ISME J}, volume = {6}, year = {2012}, month = {2012 Jan}, pages = {146-57}, abstract = {Insight into the mechanisms for the anaerobic metabolism of aromatic compounds by the hyperthermophilic archaeon Ferroglobus placidus is expected to improve understanding of the degradation of aromatics in hot (>80{\textdegree} C) environments and to identify enzymes that might have biotechnological applications. Analysis of the F. placidus genome revealed genes predicted to encode enzymes homologous to those previously identified as having a role in benzoate and phenol metabolism in mesophilic bacteria. Surprisingly, F. placidus lacks genes for an ATP-independent class II benzoyl-CoA (coenzyme A) reductase (BCR) found in all strictly anaerobic bacteria, but has instead genes coding for a bzd-type ATP-consuming class I BCR, similar to those found in facultative bacteria. The lower portion of the benzoate degradation pathway appears to be more similar to that found in the phototroph Rhodopseudomonas palustris, than the pathway reported for all heterotrophic anaerobic benzoate degraders. Many of the genes predicted to be involved in benzoate metabolism were found in one of two gene clusters. Genes for phenol carboxylation proceeding through a phenylphosphate intermediate were identified in a single gene cluster. Analysis of transcript abundance with a whole-genome microarray and quantitative reverse transcriptase polymerase chain reaction demonstrated that most of the genes predicted to be involved in benzoate or phenol metabolism had higher transcript abundance during growth on those substrates vs growth on acetate. These results suggest that the general strategies for benzoate and phenol metabolism are highly conserved between microorganisms living in moderate and hot environments, and that anaerobic metabolism of aromatic compounds might be analyzed in a wide range of environments with similar molecular targets.}, keywords = {Acetates, Archaea, Bacteria, Anaerobic, Benzoates, Metabolic Networks and Pathways, Phenol, Rhodopseudomonas}, issn = {1751-7370}, doi = {10.1038/ismej.2011.88}, author = {Holmes, Dawn E and Risso, Carla and Smith, Jessica A and Lovley, Derek R} } @article {421, title = {Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus.}, journal = {Appl Environ Microbiol}, volume = {77}, year = {2011}, month = {2011 Sep}, pages = {5926-33}, abstract = {Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85{\textdegree}C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [(14)C]benzene to [(14)C]carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism, and [(14)C]benzoate was produced from [(14)C]benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not upregulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- than in benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much-needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms.}, keywords = {Anaerobiosis, Archaeoglobales, Benzene, Carbon Radioisotopes, Ferric Compounds, Gene Expression Profiling, Hot Temperature, Isotope Labeling, Oxidation-Reduction}, issn = {1098-5336}, doi = {10.1128/AEM.05452-11}, author = {Holmes, Dawn E and Risso, Carla and Smith, Jessica A and Lovley, Derek R} } @article {438, title = {Genome-scale dynamic modeling of the competition between Rhodoferax and Geobacter in anoxic subsurface environments.}, journal = {ISME J}, volume = {5}, year = {2011}, month = {2011 Feb}, pages = {305-16}, abstract = {The advent of rapid complete genome sequencing, and the potential to capture this information in genome-scale metabolic models, provide the possibility of comprehensively modeling microbial community interactions. For example, Rhodoferax and Geobacter species are acetate-oxidizing Fe(III)-reducers that compete in anoxic subsurface environments and this competition may have an influence on the in situ bioremediation of uranium-contaminated groundwater. Therefore, genome-scale models of Geobacter sulfurreducens and Rhodoferax ferrireducens were used to evaluate how Geobacter and Rhodoferax species might compete under diverse conditions found in a uranium-contaminated aquifer in Rifle, CO. The model predicted that at the low rates of acetate flux expected under natural conditions at the site, Rhodoferax will outcompete Geobacter as long as sufficient ammonium is available. The model also predicted that when high concentrations of acetate are added during in situ bioremediation, Geobacter species would predominate, consistent with field-scale observations. This can be attributed to the higher expected growth yields of Rhodoferax and the ability of Geobacter to fix nitrogen. The modeling predicted relative proportions of Geobacter and Rhodoferax in geochemically distinct zones of the Rifle site that were comparable to those that were previously documented with molecular techniques. The model also predicted that under nitrogen fixation, higher carbon and electron fluxes would be diverted toward respiration rather than biomass formation in Geobacter, providing a potential explanation for enhanced in situ U(VI) reduction in low-ammonium zones. These results show that genome-scale modeling can be a useful tool for predicting microbial interactions in subsurface environments and shows promise for designing bioremediation strategies.}, keywords = {Acetates, Anaerobiosis, Biodegradation, Environmental, Biomass, Comamonadaceae, Genome, Genome, Bacterial, Geobacter, Models, Biological, Nitrogen Fixation, Quaternary Ammonium Compounds, RNA, Ribosomal, 16S, Uranium, Water Microbiology, Water Pollutants, Radioactive}, issn = {1751-7370}, doi = {10.1038/ismej.2010.117}, author = {Zhuang, Kai and Izallalen, Mounir and Mouser, Paula and Richter, Hanno and Risso, Carla and Mahadevan, Radhakrishnan and Lovley, Derek R} } @article {415, title = {Geobacter: the microbe electric{\textquoteright}s physiology, ecology, and practical applications.}, journal = {Adv Microb Physiol}, volume = {59}, year = {2011}, month = {2011}, pages = {1-100}, abstract = {Geobacter species specialize in making electrical contacts with extracellular electron acceptors and other organisms. This permits Geobacter species to fill important niches in a diversity of anaerobic environments. Geobacter species appear to be the primary agents for coupling the oxidation of organic compounds to the reduction of insoluble Fe(III) and Mn(IV) oxides in many soils and sediments, a process of global biogeochemical significance. Some Geobacter species can anaerobically oxidize aromatic hydrocarbons and play an important role in aromatic hydrocarbon removal from contaminated aquifers. The ability of Geobacter species to reductively precipitate uranium and related contaminants has led to the development of bioremediation strategies for contaminated environments. Geobacter species produce higher current densities than any other known organism in microbial fuel cells and are common colonizers of electrodes harvesting electricity from organic wastes and aquatic sediments. Direct interspecies electron exchange between Geobacter species and syntrophic partners appears to be an important process in anaerobic wastewater digesters. Functional and comparative genomic studies have begun to reveal important aspects of Geobacter physiology and regulation, but much remains unexplored. Quantifying key gene transcripts and proteins of subsurface Geobacter communities has proven to be a powerful approach to diagnose the in situ physiological status of Geobacter species during groundwater bioremediation. The growth and activity of Geobacter species in the subsurface and their biogeochemical impact under different environmental conditions can be predicted with a systems biology approach in which genome-scale metabolic models are coupled with appropriate physical/chemical models. The proficiency of Geobacter species in transferring electrons to insoluble minerals, electrodes, and possibly other microorganisms can be attributed to their unique "microbial nanowires," pili that conduct electrons along their length with metallic-like conductivity. Surprisingly, the abundant c-type cytochromes of Geobacter species do not contribute to this long-range electron transport, but cytochromes are important for making the terminal electrical connections with Fe(III) oxides and electrodes and also function as capacitors, storing charge to permit continued respiration when extracellular electron acceptors are temporarily unavailable. The high conductivity of Geobacter pili and biofilms and the ability of biofilms to function as supercapacitors are novel properties that might contribute to the field of bioelectronics. The study of Geobacter species has revealed a remarkable number of microbial physiological properties that had not previously been described in any microorganism. Further investigation of these environmentally relevant and physiologically unique organisms is warranted.}, keywords = {Biotechnology, Ecology, Environmental Remediation, Ferric Compounds, Geobacter}, issn = {0065-2911}, doi = {10.1016/B978-0-12-387661-4.00004-5}, author = {Lovley, Derek R and Ueki, Toshiyuki and Zhang, Tian and Malvankar, Nikhil S and Shrestha, Pravin M and Flanagan, Kelly A and Aklujkar, Muktak and Butler, Jessica E and Giloteaux, Ludovic and Rotaru, Amelia-Elena and Holmes, Dawn E and Franks, Ashley E and Orellana, Roberto and Risso, Carla and Nevin, Kelly P} } @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 {434, title = {The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments.}, journal = {BMC Genomics}, volume = {11}, year = {2010}, month = {2010}, pages = {490}, abstract = {BACKGROUND: Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process. Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites. This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis compared to non-subsurface Geobacter species. RESULTS: Annotation of the genome sequence of G. bemidjiensis indicates several differences in metabolism compared to previously sequenced non-subsurface Geobacteraceae, which will be useful for in silico metabolic modeling of subsurface bioremediation processes involving Geobacter species. Pathways can now be predicted for the use of various carbon sources such as propionate by G. bemidjiensis. Additional metabolic capabilities such as carbon dioxide fixation and growth on glucose were predicted from the genome annotation. The presence of different dicarboxylic acid transporters and two oxaloacetate decarboxylases in G. bemidjiensis may explain its ability to grow by disproportionation of fumarate. Although benzoate is the only aromatic compound that G. bemidjiensis is known or predicted to utilize as an electron donor and carbon source, the genome suggests that this species may be able to detoxify other aromatic pollutants without degrading them. Furthermore, G. bemidjiensis is auxotrophic for 4-aminobenzoate, which makes it the first Geobacter species identified as having a vitamin requirement. Several features of the genome indicated that G. bemidjiensis has enhanced abilities to respire, detoxify and avoid oxygen. CONCLUSION: Overall, the genome sequence of G. bemidjiensis offers surprising insights into the metabolism and physiology of Geobacteraceae in subsurface environments, compared to non-subsurface Geobacter species, such as the ability to disproportionate fumarate, more efficient oxidation of propionate, enhanced responses to oxygen stress, and dependence on the environment for a vitamin requirement. Therefore, an understanding of the activity of Geobacter species in the subsurface is more likely to benefit from studies of subsurface isolates such as G. bemidjiensis than from the non-subsurface model species studied so far.}, keywords = {Aldehyde Oxidoreductases, Biodegradation, Environmental, Carbohydrate Metabolism, Carbon Dioxide, Cell Wall, Electrons, Environmental Microbiology, Fatty Acids, Frameshift Mutation, Fumarates, Genes, Bacterial, Genome, Bacterial, Geobacter, Glucose, Iron, Metabolic Networks and Pathways, Multienzyme Complexes, Multigene Family, Osmosis, Oxidation-Reduction, Oxo-Acid-Lyases, Propionic Acids, Pyruvic Acid, Species Specificity, Surface Properties}, issn = {1471-2164}, doi = {10.1186/1471-2164-11-490}, author = {Aklujkar, Muktak and Young, Nelson D and Holmes, Dawn and Chavan, Milind and Risso, Carla and Kiss, Hajnalka E and Han, Cliff S and Land, Miriam L and Lovley, Derek R} } @article {456, title = {Genome-scale comparison and constraint-based metabolic reconstruction of the facultative anaerobic Fe(III)-reducer Rhodoferax ferrireducens.}, journal = {BMC Genomics}, volume = {10}, year = {2009}, month = {2009}, pages = {447}, abstract = {BACKGROUND: Rhodoferax ferrireducens is a metabolically versatile, Fe(III)-reducing, subsurface microorganism that is likely to play an important role in the carbon and metal cycles in the subsurface. It also has the unique ability to convert sugars to electricity, oxidizing the sugars to carbon dioxide with quantitative electron transfer to graphite electrodes in microbial fuel cells. In order to expand our limited knowledge about R. ferrireducens, the complete genome sequence of this organism was further annotated and then the physiology of R. ferrireducens was investigated with a constraint-based, genome-scale in silico metabolic model and laboratory studies. RESULTS: The iterative modeling and experimental approach unveiled exciting, previously unknown physiological features, including an expanded range of substrates that support growth, such as cellobiose and citrate, and provided additional insights into important features such as the stoichiometry of the electron transport chain and the ability to grow via fumarate dismutation. Further analysis explained why R. ferrireducens is unable to grow via photosynthesis or fermentation of sugars like other members of this genus and uncovered novel genes for benzoate metabolism. The genome also revealed that R. ferrireducens is well-adapted for growth in the subsurface because it appears to be capable of dealing with a number of environmental insults, including heavy metals, aromatic compounds, nutrient limitation and oxidative stress. CONCLUSION: This study demonstrates that combining genome-scale modeling with the annotation of a new genome sequence can guide experimental studies and accelerate the understanding of the physiology of under-studied yet environmentally relevant microorganisms.}, keywords = {Comamonadaceae, Comparative Genomic Hybridization, DNA, Bacterial, Ferric Compounds, Genome, Bacterial, Genomics, Models, Biological, Oxidation-Reduction, Sequence Analysis, DNA}, issn = {1471-2164}, doi = {10.1186/1471-2164-10-447}, author = {Risso, Carla and Sun, Jun and Zhuang, Kai and Mahadevan, Radhakrishnan and DeBoy, Robert and Ismail, Wael and Shrivastava, Susmita and Huot, Heather and Kothari, Sagar and Daugherty, Sean and Bui, Olivia and Schilling, Christophe H and Lovley, Derek R and Meth{\'e}, Barbara A} } @article {491, title = {Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {190}, year = {2008}, month = {2008 Apr}, pages = {2266-74}, abstract = {The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coli, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. 13C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms.}, keywords = {Acetyl Coenzyme A, Bacterial Proteins, Biosynthetic Pathways, Butyric Acids, Carbon Isotopes, Geobacter, Isoleucine, Malates, Pyruvic Acid, Threonine, Threonine Dehydratase}, issn = {1098-5530}, doi = {10.1128/JB.01841-07}, author = {Risso, Carla and Van Dien, Stephen J and Orloff, Amber and Lovley, Derek R and Coppi, Maddalena V} } @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} }