@article {3057, title = {Generation of High Current Densities in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor.}, journal = {Microbiol Spectr}, volume = {9}, year = {2021}, month = {2021 Oct 31}, pages = {e0087721}, abstract = {

Geobacter sulfurreducens is commonly employed as a model for the study of extracellular electron transport mechanisms in the species. Deletion of , which is known to encode the pilus assembly motor protein for type IV pili in other bacteria, has been proposed as an effective strategy for evaluating the role of electrically conductive pili (e-pili) in G. sulfurreducens extracellular electron transfer. In those studies, the inhibition of e-pili expression associated with deletion was not demonstrated directly but was inferred from the observation that deletion mutants produced lower current densities than wild-type cells. Here, we report that deleting did not diminish current production. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-type G. sulfurreducens or when e-pili are expressed heterologously from the G. sulfurreducens pilin gene in Escherichia coli. Immunogold labeling demonstrated that a G. sulfurreducens strain expressing a pilin monomer with a His tag continued to express His tag-labeled filaments when was deleted. These results suggest that a reinterpretation of the results of previous studies on G. sulfurreducens deletion strains may be necessary. Geobacter sulfurreducens is a model microbe for the study of biogeochemically and technologically significant processes, such as the reduction of Fe(III) oxides in soils and sediments, bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity, direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments, and metal corrosion. Elucidating the phenotypes associated with gene deletions is an important strategy for determining the mechanisms for extracellular electron transfer in G. sulfurreducens. The results reported here demonstrate that we cannot replicate the key phenotype reported for a gene deletion that has been central to the development of models for long-range electron transport in G. sulfurreducens.

}, keywords = {Bacterial Proteins, Electric Conductivity, Electron Transport, Fimbriae Proteins, Fimbriae, Bacterial, Gene Deletion, Geobacter, Geologic Sediments, Microscopy, Atomic Force, Oxidoreductases}, issn = {2165-0497}, doi = {10.1128/Spectrum.00877-21}, author = {Ueki, Toshiyuki and Walker, David J F and Nevin, Kelly P and Ward, Joy E and Woodard, Trevor L and Nonnenmann, Stephen S and Lovley, Derek R} } @article {3090, title = {Toward establishing minimum requirements for extracellular electron transfer in Geobacter sulfurreducens.}, journal = {FEMS Microbiol Lett}, volume = {364}, year = {2017}, month = {2017 May 01}, abstract = {

The highly redundant pathways for extracellular electron transfer in Geobacter sulfurreducens must be simplified for this microorganism to serve as an effective chassis for applications such as the development of sensors and biocomputing. Five homologs of the periplasmic c-type cytochromes, PpcA-E, offer the possibility of multiple routes of electron transfer across the periplasm. The presence of a large number of outer membrane c-type cytochromes allows G. sulfurreducens to adapt to disruption of an electron transfer pathway in the outer membrane. A strain in which genes for all five periplasmic cytochromes, PpcA-E, were deleted did not reduce Fe(III). Introducing ppcA under the control of an IPTG-inducible system in the quintuple deletion strain yielded a strain that reduced Fe(III) only in the presence of IPTG. A strain lacking known major outer membrane cytochromes, OmcB, OmcE, OmcS and OmcT, and putative functional homologs of OmcB, did not reduce Fe(III). Introduction of omcB in this septuple deletion strain restored the ability to reduce Fe(III). These results demonstrate that it is possible to trim redundancy from the extracellular electron transfer pathways in G. sulfurreducens in order to construct strains with defined extracellular electron transfer routes.

}, keywords = {Bacterial Outer Membrane Proteins, Bacterial Proteins, Cytochromes c, Electron Transport, Ferric Compounds, Gene Expression Regulation, Bacterial, Geobacter, Oxidation-Reduction, Periplasm}, issn = {1574-6968}, doi = {10.1093/femsle/fnx093}, author = {Ueki, Toshiyuki and DiDonato, Laurie N and Lovley, Derek R} } @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 {423, title = {Monitoring the metabolic status of geobacter species in contaminated groundwater by quantifying key metabolic proteins with Geobacter-specific antibodies.}, journal = {Appl Environ Microbiol}, volume = {77}, year = {2011}, month = {2011 Jul}, pages = {4597-602}, abstract = {Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the bioremediation community as a routine practice for guiding the development of bioremediation strategies. Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation but is technically challenging and does not account for the impact of translational modifications on protein abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species, increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of microbial communities responsible for bioremediation, aiding in the rational design of bioremediation strategies.}, keywords = {Acetates, Antibodies, Bacterial, Bacterial Proteins, Geobacter, Petroleum, Quaternary Ammonium Compounds, Soil Microbiology, Water Microbiology}, issn = {1098-5336}, doi = {10.1128/AEM.00114-11}, author = {Yun, Jiae and Ueki, Toshiyuki and Miletto, Marzia and Lovley, Derek R} } @article {452, title = {Genome-wide gene regulation of biosynthesis and energy generation by a novel transcriptional repressor in Geobacter species.}, journal = {Nucleic Acids Res}, volume = {38}, year = {2010}, month = {2010 Jan}, pages = {810-21}, abstract = {Geobacter species play important roles in bioremediation of contaminated environments and in electricity production from waste organic matter in microbial fuel cells. To better understand physiology of Geobacter species, expression and function of citrate synthase, a key enzyme in the TCA cycle that is important for organic acid oxidation in Geobacter species, was investigated. Geobacter sulfurreducens did not require citrate synthase for growth with hydrogen as the electron donor and fumarate as the electron acceptor. Expression of the citrate synthase gene, gltA, was repressed by a transcription factor under this growth condition. Functional and comparative genomics approaches, coupled with genetic and biochemical assays, identified a novel transcription factor termed HgtR that acts as a repressor for gltA. Further analysis revealed that HgtR is a global regulator for genes involved in biosynthesis and energy generation in Geobacter species. The hgtR gene was essential for growth with hydrogen, during which hgtR expression was induced. These findings provide important new insights into the mechanisms by which Geobacter species regulate their central metabolism under different environmental conditions.}, keywords = {Bacterial Proteins, Base Sequence, Citrate (si)-Synthase, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Molecular Sequence Data, Promoter Regions, Genetic, Repressor Proteins, Transcription, Genetic}, issn = {1362-4962}, doi = {10.1093/nar/gkp1085}, author = {Ueki, Toshiyuki and Lovley, Derek R} } @article {436, title = {Genome-wide survey for PilR recognition sites of the metal-reducing prokaryote Geobacter sulfurreducens.}, journal = {Gene}, volume = {469}, year = {2010}, month = {2010 Dec 1}, pages = {31-44}, abstract = {Geobacter sulfurreducens is a species from the bacterial family Geobacteraceae, members of which participate in bioenergy production and in environmental bioremediation. G. sulfurreducens pili are electrically conductive and are required for Fe(III) oxide reduction and for optimal current production in microbial fuel cells. PilR is an enhancer binding protein, which is an activator acting together with the alternative sigma factor, RpoN, in transcriptional regulation. Both RpoN and PilR are involved in regulation of expression of the pilA gene, whose product is pilin, a structural component of a pilus. Using bioinformatic approaches, we predicted G. sulfurreducens sequence elements that are likely to be regulated by PilR. The functional importance of the genome region containing a PilR binding site predicted upstream of the pilA gene was experimentally validated. The predicted G. sulfurreducens PilR binding sites are similar to PilR binding sites of Pseudomonas and Moraxella. While the number of predicted PilR-regulated sites did not deviate from that expected by chance, multiple sites were predicted upstream of genes with roles in biosynthesis and function of pili and flagella, in secretory pathways, and in cell wall biogenesis, suggesting the possible involvement of G. sulfurreducens PilR in regulation of production and assembly of pili and flagella.}, keywords = {Bacterial Proteins, Base Sequence, Binding Sites, Conserved Sequence, Ferric Compounds, Fimbriae Proteins, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Molecular Sequence Data, Promoter Regions, Genetic, Transcription Factors, Transcription, Genetic}, issn = {1879-0038}, doi = {10.1016/j.gene.2010.08.005}, author = {Krushkal, Julia and Ju{\'a}rez, Katy and Barbe, Jose F and Qu, Yanhua and Andrade, Angel and Puljic, Marko and Adkins, Ronald M and Lovley, Derek R and Ueki, Toshiyuki} } @article {440, title = {Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens.}, journal = {Nucleic Acids Res}, volume = {38}, year = {2010}, month = {2010 Nov}, pages = {7485-99}, abstract = {Geobacter species often play an important role in bioremediation of environments contaminated with metals or organics and show promise for harvesting electricity from waste organic matter in microbial fuel cells. The ability of Geobacter species to fix atmospheric nitrogen is an important metabolic feature for these applications. We identified novel regulatory cascades controlling nitrogen-fixation gene expression in Geobacter sulfurreducens. Unlike the regulatory mechanisms known in other nitrogen-fixing microorganisms, nitrogen-fixation gene regulation in G. sulfurreducens is controlled by two two-component His-Asp phosphorelay systems. One of these systems appears to be the master regulatory system that activates transcription of the majority of nitrogen-fixation genes and represses a gene encoding glutamate dehydrogenase during nitrogen fixation. The other system whose expression is directly activated by the master regulatory system appears to control by antitermination the expression of a subset of the nitrogen-fixation genes whose transcription is activated by the master regulatory system and whose promoter contains transcription termination signals. This study provides a new paradigm for nitrogen-fixation gene regulation.}, keywords = {Bacterial Proteins, Binding Sites, DNA-Binding Proteins, Gene Expression Regulation, Bacterial, Geobacter, Nitrogen Fixation, Promoter Regions, Genetic, Protein Kinases, RNA-Binding Proteins, Transcription, Genetic}, issn = {1362-4962}, doi = {10.1093/nar/gkq652}, author = {Ueki, Toshiyuki and Lovley, Derek R} } @article {458, title = {Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens.}, journal = {BMC Genomics}, volume = {10}, year = {2009}, month = {2009}, pages = {331}, abstract = {BACKGROUND: The role of the RNA polymerase sigma factor RpoN in regulation of gene expression in Geobacter sulfurreducens was investigated to better understand transcriptional regulatory networks as part of an effort to develop regulatory modules for genome-scale in silico models, which can predict the physiological responses of Geobacter species during groundwater bioremediation or electricity production. RESULTS: An rpoN deletion mutant could not be obtained under all conditions tested. In order to investigate the regulon of the G. sulfurreducens RpoN, an RpoN over-expression strain was made in which an extra copy of the rpoN gene was under the control of a taclac promoter. Combining both the microarray transcriptome analysis and the computational prediction revealed that the G. sulfurreducens RpoN controls genes involved in a wide range of cellular functions. Most importantly, RpoN controls the expression of the dcuB gene encoding the fumarate/succinate exchanger, which is essential for cell growth with fumarate as the terminal electron acceptor in G. sulfurreducens. RpoN also controls genes, which encode enzymes for both pathways of ammonia assimilation that is predicted to be essential under all growth conditions in G. sulfurreducens. Other genes that were identified as part of the RpoN regulon using either the computational prediction or the microarray transcriptome analysis included genes involved in flagella biosynthesis, pili biosynthesis and genes involved in central metabolism enzymes and cytochromes involved in extracellular electron transfer to Fe(III), which are known to be important for growth in subsurface environment or electricity production in microbial fuel cells. The consensus sequence for the predicted RpoN-regulated promoter elements is TTGGCACGGTTTTTGCT. CONCLUSION: The G. sulfurreducens RpoN is an essential sigma factor and a global regulator involved in a complex transcriptional network controlling a variety of cellular processes.}, keywords = {Bacterial Proteins, DNA, Bacterial, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genome-Wide Association Study, Geobacter, Multigene Family, Oligonucleotide Array Sequence Analysis, Promoter Regions, Genetic, Regulon, RNA Polymerase Sigma 54}, issn = {1471-2164}, doi = {10.1186/1471-2164-10-331}, author = {Leang, Ching and Krushkal, Julia and Ueki, Toshiyuki and Puljic, Marko and Sun, Jun and Ju{\'a}rez, Katy and N{\'u}{\~n}ez, Cinthia and Reguera, Gemma and DiDonato, Raymond and Postier, Bradley and Adkins, Ronald M and Lovley, Derek R} } @article {512, title = {Computational prediction of RpoS and RpoD regulatory sites in Geobacter sulfurreducens using sequence and gene expression information.}, journal = {Gene}, volume = {384}, year = {2006}, month = {2006 Dec 15}, pages = {73-95}, abstract = {RpoS, the sigma S subunit of RNA polymerase, is vital during the growth and survival of Geobacter sulfurreducens under conditions typically encountered in its native subsurface environments. We investigated the conservation of sites that may be important for RpoS function in G. sulfurreducens. We also employed sequence information and expression microarray data to predict G. sulfurreducens genome sites that may be related to RpoS regulation. Hierarchical clustering identified three clusters of significantly downregulated genes in the rpoS deletion mutant. The search for conserved overrepresented motifs in co-regulated operons identified likely -35 and -10 promoter elements upstream of a number of functionally important G. sulfurreducens operons that were downregulated in the rpoS deletion mutant. Putative -35/-10 promoter elements were also identified in the G. sulfurreducens genome using sequence similarity searches to matrices of -35/-10 promoter elements found in G. sulfurreducens and in Escherichia coli. Due to a sufficient degree of sequence similarity between -35/-10 promoter elements for RpoS, RpoD, and other sigma factors, both the sequence similarity searches and the search for conserved overrepresented motifs using microarray data may identify promoter elements for both RpoS and other sigma factors.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, Citrates, Computational Biology, Conserved Sequence, DNA-Directed RNA Polymerases, Escherichia coli, Escherichia coli Proteins, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genome, Bacterial, Geobacter, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Operon, Promoter Regions, Genetic, Sigma Factor, Transcription, Genetic}, issn = {0378-1119}, doi = {10.1016/j.gene.2006.06.025}, author = {Yan, Bin and N{\'u}{\~n}ez, Cinthia and Ueki, Toshiyuki and Esteve-N{\'u}{\~n}ez, Abraham and Puljic, Marko and Adkins, Ronald M and Meth{\'e}, Barbara A and Lovley, Derek R and Krushkal, Julia} }