@article {356, title = {Analysis of the genome of the sexually transmitted insect virus Helicoverpa zea nudivirus 2.}, journal = {Viruses}, volume = {4}, year = {2012}, month = {2012 Jan}, pages = {28-61}, abstract = {The sexually transmitted insect virus Helicoverpa zea nudivirus 2 (HzNV-2) was determined to have a circular double-stranded DNA genome of 231,621 bp coding for an estimated 113 open reading frames (ORFs). HzNV-2 is most closely related to the nudiviruses, a sister group of the insect baculoviruses. Several putative ORFs that share homology with the baculovirus core genes were identified in the viral genome. However, HzNV-2 lacks several key genetic features of baculoviruses including the late transcriptional regulation factor, LEF-1 and the palindromic hrs, which serve as origins of replication. The HzNV-2 genome was found to code for three ORFs that had significant sequence homology to cellular genes which are not generally found in viral genomes. These included a presumed juvenile hormone esterase gene, a gene coding for a putative zinc-dependent matrix metalloprotease, and a major facilitator superfamily protein gene; all of which are believed to play a role in the cellular proliferation and the tissue hypertrophy observed in the malformation of reproductive organs observed in HzNV-2 infected corn earworm moths, Helicoverpa zea.}, keywords = {Amino Acid Sequence, Animals, Baculoviridae, Base Sequence, Biological Evolution, Consensus Sequence, DNA Viruses, Female, Genome, Viral, Insect Viruses, Molecular Sequence Data, Moths, Open Reading Frames, Phylogeny, Sequence Alignment, Sequence Homology, Species Specificity, Viral Proteins}, issn = {1999-4915}, doi = {10.3390/v4010028}, author = {Burand, John P and Kim, Woojin and Afonso, Claudio L and Tulman, Edan R and Kutish, Gerald F and Lu, Zhiqiang and Rock, Daniel L} } @article {1205, title = {Complete genome sequence of the hyperthermophilic archaeon Thermococcus sp. strain CL1, isolated from a Paralvinella sp. polychaete worm collected from a hydrothermal vent.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 Sep}, pages = {4769-70}, abstract = {

Thermococcus sp. strain CL1 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a Paralvinella sp. polychaete worm living on an active deep-sea hydrothermal sulfide chimney on the Cleft Segment of the Juan de Fuca Ridge. To further understand the distinct characteristics of this archaeon at the genome level, its genome was completely sequenced and analyzed. Here, we announce the complete genome sequence (1,950,313 bp) of Thermococcus sp. strain CL1, with a focus on H(2)- and energy-producing capabilities and its amino acid biosynthesis and acquisition in an extreme habitat.

}, keywords = {Animals, Base Sequence, Chromosome Mapping, DNA, Archaeal, DNA, Ribosomal, Genome, Bacterial, Hydrothermal Vents, Molecular Sequence Data, Phylogeny, Polychaeta, Sequence Analysis, DNA, Thermococcus}, issn = {1098-5530}, doi = {10.1128/JB.01016-12}, author = {Jung, Jong-Hyun and Holden, James F and Seo, Dong-Ho and Park, Kwan-Hwa and Shin, Hakdong and Ryu, Sangryeol and Lee, Ju-Hoon and Park, Cheon-Seok} } @article {3149, title = {The genome of Pelobacter carbinolicus reveals surprising metabolic capabilities and physiological features.}, journal = {BMC Genomics}, volume = {13}, year = {2012}, month = {2012 Dec 10}, pages = {690}, abstract = {

BACKGROUND: The bacterium Pelobacter carbinolicus is able to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols, hydrogen or formate; it does not oxidize acetate and is not known to ferment any sugars or grow autotrophically. The genome of P. carbinolicus was sequenced in order to understand its metabolic capabilities and physiological features in comparison with its relatives, acetate-oxidizing Geobacter species.

RESULTS: Pathways were predicted for catabolism of known substrates: 2,3-butanediol, acetoin, glycerol, 1,2-ethanediol, ethanolamine, choline and ethanol. Multiple isozymes of 2,3-butanediol dehydrogenase, ATP synthase and [FeFe]-hydrogenase were differentiated and assigned roles according to their structural properties and genomic contexts. The absence of asparagine synthetase and the presence of a mutant tRNA for asparagine encoded among RNA-active enzymes suggest that P. carbinolicus may make asparaginyl-tRNA in a novel way. Catabolic glutamate dehydrogenases were discovered, implying that the tricarboxylic acid (TCA) cycle can function catabolically. A phosphotransferase system for uptake of sugars was discovered, along with enzymes that function in 2,3-butanediol production. Pyruvate:ferredoxin/flavodoxin oxidoreductase was identified as a potential bottleneck in both the supply of oxaloacetate for oxidation of acetate by the TCA cycle and the connection of glycolysis to production of ethanol. The P. carbinolicus genome was found to encode autotransporters and various appendages, including three proteins with similarity to the geopilin of electroconductive nanowires.

CONCLUSIONS: Several surprising metabolic capabilities and physiological features were predicted from the genome of P. carbinolicus, suggesting that it is more versatile than anticipated.

}, keywords = {Base Pairing, Base Sequence, Butylene Glycols, Choline, Deltaproteobacteria, Ethanolamine, Ethylene Glycol, Genome, Bacterial, Glycerol, Metabolic Networks and Pathways, Molecular Sequence Annotation, Molecular Sequence Data, Mutation, Oxidation-Reduction, Oxidoreductases, Propylene Glycols, RNA, Transfer, Asn, Sequence Analysis, DNA}, issn = {1471-2164}, doi = {10.1186/1471-2164-13-690}, author = {Aklujkar, Muktak and Haveman, Shelley A and DiDonato, Raymond and Chertkov, Olga and Han, Cliff S and Land, Miriam L and Brown, Peter and Lovley, Derek R} } @article {804, title = {Two isoforms of Geobacter sulfurreducens PilA have distinct roles in pilus biogenesis, cytochrome localization, extracellular electron transfer, and biofilm formation.}, journal = {J Bacteriol}, volume = {194}, year = {2012}, month = {2012 May}, pages = {2551-63}, abstract = {Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein. The short PilA isoform is found predominantly in an intracellular fraction. It seems to stabilize the long isoform and to influence the secretion of several outer-surface c-type cytochromes. The long PilA isoform is required for secretion of PilA to the outer cell surface, a process that requires coexpression of pilA with nine downstream genes. The long isoform was determined to be essential for biofilm formation on certain surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxides.}, keywords = {Amino Acid Sequence, Bacterial Adhesion, Base Sequence, Biofilms, Cytochromes, Electron Transport, Fimbriae Proteins, Fimbriae, Bacterial, Gene Expression Regulation, Bacterial, Geobacter, Molecular Sequence Data, Protein Isoforms, Protein Transport, Reverse Transcriptase Polymerase Chain Reaction, Surface Properties}, issn = {1098-5530}, doi = {10.1128/JB.06366-11}, author = {Richter, Lubna V and Sandler, Steven J and Weis, Robert M} } @article {357, title = {Identification of a nucleopolyhedrovirus in winter moth populations from Massachusetts.}, journal = {J Invertebr Pathol}, volume = {108}, year = {2011}, month = {2011 Nov}, pages = {217-9}, abstract = {Winter moth, Operophtera brumata, originally from Europe, has invaded eastern Massachusetts causing widespread defoliation and damage to many deciduous tree species and a variety of crop plants in the infested area. We identified O. brumata nucleopolyhedrovirus (OpbuNPV) in winter moth larvae collected from field sites in Massachusetts by using PCR to amplify a 482 bp region of the baculovirus polyhedrin gene. Viral sequences were also detected in winter moth pupae that failed to emerge, suggesting that these insects may have died as a result of viral infection. This represents the first report of OpbuNPV in winter moth populations in the US.}, keywords = {Animals, Base Sequence, Environmental Monitoring, Larva, Massachusetts, Molecular Sequence Data, Moths, Nucleopolyhedrovirus, Viral Proteins, Virus Diseases}, issn = {1096-0805}, doi = {10.1016/j.jip.2011.08.005}, author = {Burand, John P and Kim, Woojin and Welch, Anna and Elkinton, Joseph S} } @article {805, title = {Structure of the SSB-DNA polymerase III interface and its role in DNA replication.}, journal = {EMBO J}, volume = {30}, year = {2011}, month = {2011 Oct 19}, pages = {4236-47}, abstract = {Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB{\textquoteright}s association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, DNA Polymerase III, DNA Replication, DNA, Single-Stranded, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Holoenzymes, Molecular Sequence Data}, issn = {1460-2075}, doi = {10.1038/emboj.2011.305}, author = {Marceau, Aimee H and Bahng, Soon and Massoni, Shawn C and George, Nicholas P and Sandler, Steven J and Marians, Kenneth J and Keck, James L} } @article {677, title = {ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids.}, journal = {Nucleic Acids Res}, volume = {38}, year = {2010}, month = {2010 Jul}, pages = {W529-33}, abstract = {It is informative to detect highly conserved positions in proteins and nucleic acid sequence/structure since they are often indicative of structural and/or functional importance. ConSurf (http://consurf.tau.ac.il) and ConSeq (http://conseq.tau.ac.il) are two well-established web servers for calculating the evolutionary conservation of amino acid positions in proteins using an empirical Bayesian inference, starting from protein structure and sequence, respectively. Here, we present the new version of the ConSurf web server that combines the two independent servers, providing an easier and more intuitive step-by-step interface, while offering the user more flexibility during the process. In addition, the new version of ConSurf calculates the evolutionary rates for nucleic acid sequences. The new version is freely available at: http://consurf.tau.ac.il/.}, keywords = {Amino Acid Sequence, Base Sequence, Conserved Sequence, Evolution, Molecular, Internet, Nucleic Acid Conformation, Protein Conformation, Sequence Analysis, Sequence Homology, Amino Acid, Software}, issn = {1362-4962}, doi = {10.1093/nar/gkq399}, author = {Ashkenazy, Haim and Erez, Elana and Martz, Eric and Pupko, Tal and Ben-Tal, Nir} } @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 {439, title = {Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus.}, journal = {BMC Evol Biol}, volume = {10}, year = {2010}, month = {2010}, pages = {230}, abstract = {BACKGROUND: Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. RESULTS: CRISPR spacer $\#$1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer $\#$1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. CONCLUSIONS: The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer $\#$1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of CRISPR on genome-scale evolution by interference with an essential gene.}, keywords = {Base Sequence, Comparative Genomic Hybridization, Computational Biology, Deltaproteobacteria, DNA, Bacterial, DNA, Intergenic, Evolution, Molecular, Genes, Bacterial, Genome, Bacterial, Geobacillus, Histidine-tRNA Ligase, Inverted Repeat Sequences, Molecular Sequence Data, Phylogeny, Sequence Alignment, Sequence Analysis, DNA}, issn = {1471-2148}, doi = {10.1186/1471-2148-10-230}, author = {Aklujkar, Muktak and Lovley, Derek R} } @article {1570, title = {Effect of nutrient and selective inhibitor amendments on methane oxidation, nitrous oxide production, and key gene presence and expression in landfill cover soils: characterization of the role of methanotrophs, nitrifiers, and denitrifiers.}, journal = {Appl Microbiol Biotechnol}, volume = {85}, year = {2009}, month = {2009 Nov}, pages = {389-403}, abstract = {

Methane and nitrous oxide are both potent greenhouse gasses, with global warming potentials approximately 25 and 298 times that of carbon dioxide. A matrix of soil microcosms was constructed with landfill cover soils collected from the King Highway Landfill in Kalamazoo, Michigan and exposed to geochemical parameters known to affect methane consumption by methanotrophs while also examining their impact on biogenic nitrous oxide production. It was found that relatively dry soils (5\% moisture content) along with 15 mg NH (4) (+) (kg soil)(-1) and 0.1 mg phenylacetylene(kg soil)(-1) provided the greatest stimulation of methane oxidation while minimizing nitrous oxide production. Microarray analyses of pmoA showed that the methanotrophic community structure was dominated by Type II organisms, but Type I genera were more evident with the addition of ammonia. When phenylacetylene was added in conjunction with ammonia, the methanotrophic community structure was more similar to that observed in the presence of no amendments. PCR analyses showed the presence of amoA from both ammonia-oxidizing bacteria and archaea, and that the presence of key genes associated with these cells was reduced with the addition of phenylacetylene. Messenger RNA analyses found transcripts of pmoA, but not of mmoX, nirK, norB, or amoA from either ammonia-oxidizing bacteria or archaea. Pure culture analyses showed that methanotrophs could produce significant amounts of nitrous oxide, particularly when expressing the particulate methane monooxygenase (pMMO). Collectively, these data suggest that methanotrophs expressing pMMO played a role in nitrous oxide production in these microcosms.

}, keywords = {Archaea, Bacteria, Base Sequence, DNA, DNA Primers, DNA, Archaeal, DNA, Bacterial, Global Warming, Greenhouse Effect, Inorganic Chemicals, Methane, Nitrites, Nitrogen, Nitrogen Oxides, Nitrous Oxide, Oxidation-Reduction, Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, RNA, Bacterial, RNA, Messenger, Soil, Water Pollutants, Chemical}, issn = {1432-0614}, doi = {10.1007/s00253-009-2238-7}, author = {Lee, Sung-Woo and Im, Jeongdae and Dispirito, Alan A and Bodrossy, Levente and Barcelona, Michael J and Semrau, Jeremy D} } @article {455, title = {GSEL version 2, an online genome-wide query system of operon organization and regulatory sequence elements of Geobacter sulfurreducens.}, journal = {OMICS}, volume = {13}, year = {2009}, month = {2009 Oct}, pages = {439-49}, abstract = {Geobacter sulfurreducens is a model organism within the delta-Proteobacterial family Geobacteraceae, members of which can participate in environmental bioremediation of metal and organic waste contaminants and in production of bioenergy. In this report, we describe a new, significantly expanded and updated, version 2 of the GSEL (Geobacter Sequence Elements) database ( http://geobacter.org/research/gsel2/ and http://geobacter.org/refs/gsel2/ ) and its accompanying online query system, which compiles information on operon organization and regulatory sequence elements in the genome of G. sulfurreducens. It incorporates a new online graphical browser, provides novel search capabilities, and includes updated operon predictions along with new information on predicted and experimentally validated genome regulatory sites. The GSEL database and online search system provides a unique and comprehensive tool cataloging information about gene regulation in G. sulfurreducens, aiding in investigation of mechanisms that regulate its ability to generate electric power, bioremediate environmental waste, and adapt to environmental changes.}, keywords = {Base Sequence, Databases, Genetic, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Humans, Internet, Online Systems, Operon, Regulatory Sequences, Nucleic Acid, Software, User-Computer Interface}, issn = {1557-8100}, doi = {10.1089/omi.2009.0081}, author = {Qu, Yanhua and Brown, Peter and Barbe, Jose F and Puljic, Marko and Merino, Enrique and Adkins, Ronald M and Lovley, Derek R and Krushkal, Julia} } @article {489, title = {Computational and experimental analysis of redundancy in the central metabolism of Geobacter sulfurreducens.}, journal = {PLoS Comput Biol}, volume = {4}, year = {2008}, month = {2008 Feb}, pages = {e36}, abstract = {Previous model-based analysis of the metabolic network of Geobacter sulfurreducens suggested the existence of several redundant pathways. Here, we identified eight sets of redundant pathways that included redundancy for the assimilation of acetate, and for the conversion of pyruvate into acetyl-CoA. These equivalent pathways and two other sub-optimal pathways were studied using 5 single-gene deletion mutants in those pathways for the evaluation of the predictive capacity of the model. The growth phenotypes of these mutants were studied under 12 different conditions of electron donor and acceptor availability. The comparison of the model predictions with the resulting experimental phenotypes indicated that pyruvate ferredoxin oxidoreductase is the only activity able to convert pyruvate into acetyl-CoA. However, the results and the modeling showed that the two acetate activation pathways present are not only active, but needed due to the additional role of the acetyl-CoA transferase in the TCA cycle, probably reflecting the adaptation of these bacteria to acetate utilization. In other cases, the data reconciliation suggested additional capacity constraints that were confirmed with biochemical assays. The results demonstrate the need to experimentally verify the activity of key enzymes when developing in silico models of microbial physiology based on sequence-based reconstruction of metabolic networks.}, keywords = {Bacterial Proteins, Base Sequence, Computer Simulation, Gene Expression Regulation, Bacterial, Geobacter, Models, Biological, Molecular Sequence Data, Multienzyme Complexes, Signal Transduction}, issn = {1553-7358}, doi = {10.1371/journal.pcbi.0040036}, author = {Segura, Daniel and Mahadevan, Radhakrishnan and Ju{\'a}rez, Katy and Lovley, Derek R} } @article {395, title = {A degenerate tripartite DNA-binding site required for activation of ComA-dependent quorum response gene expression in Bacillus subtilis.}, journal = {J Mol Biol}, volume = {381}, year = {2008}, month = {2008 Aug 29}, pages = {261-75}, abstract = {In Bacillus subtilis, the transcription factor ComA activates several biological processes in response to increasing population density. Extracellular peptide signaling is used to coordinate the activity of ComA with population density. At low culture densities, when the concentration of signaling peptides is lowest, ComA is largely inactive. At higher densities, when the concentration of signaling peptides is higher, ComA is active and activates the transcription of at least nine operons involved in the development of competence and in the production of degradative enzymes and antibiotics. We found that ComA binds a degenerate tripartite sequence consisting of three DNA-binding determinants or "recognition elements." Mutational analyses showed that all three recognition elements are required for transcription activation in vivo and for specific DNA binding by ComA in vitro. Degeneracy of the recognition elements in the ComA-binding site is an important regulatory feature for coordinating transcription with population density (i.e., promoters containing an optimized binding site have high activity at low culture density and are no longer regulated in the normal-density-dependent manner). We found that purified ComA forms a dimer in solution, and we propose a model for how two dimers of ComA bind to an odd number of DNA-binding determinants to activate transcription of target genes. This DNA-protein architecture for transcription activation appears to be conserved for ComA homologs in other Bacillus species.}, keywords = {Bacillus subtilis, Bacterial Proteins, Base Sequence, Binding Sites, Dimerization, DNA, Bacterial, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Gene Expression Regulation, Bacterial, Peptide Synthases, Phosphoprotein Phosphatases, Promoter Regions, Genetic, Protein Binding, Quorum Sensing, Sequence Analysis, DNA, Transcription, Genetic, Transcriptional Activation}, issn = {1089-8638}, doi = {10.1016/j.jmb.2008.06.035}, author = {Griffith, Kevin L and Grossman, Alan D} } @article {503, title = {Genome-wide expression profiling in Geobacter sulfurreducens: identification of Fur and RpoS transcription regulatory sites in a relGsu mutant.}, journal = {Funct Integr Genomics}, volume = {7}, year = {2007}, month = {2007 Jul}, pages = {229-55}, abstract = {Rel(Gsu) is the single Geobacter sulfurreducens homolog of RelA and SpoT proteins found in many organisms. These proteins are involved in the regulation of levels of guanosine 3{\textquoteright}, 5{\textquoteright} bispyrophosphate, ppGpp, a molecule that signals slow growth and stress response under nutrient limitation in bacteria. We used information obtained from genome-wide expression profiling of the rel(Gsu) deletion mutant to identify putative regulatory sites involved in transcription networks modulated by Rel(Gsu) or ppGpp. Differential gene expression in the rel(Gsu) deletion mutant, as compared to the wild type, was available from two growth conditions, steady state chemostat cultures and stationary phase batch cultures. Hierarchical clustering analysis of these two datasets identified several groups of operons that are likely co-regulated. Using a search for conserved motifs in the upstream regions of these co-regulated operons, we identified sequences similar to Fur- and RpoS-regulated sites. These findings suggest that Fur- and RpoS-dependent gene expression in G. sulfurreducens is affected by Rel(Gsu)-mediated signaling.}, keywords = {Bacterial Proteins, Base Sequence, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genome, Bacterial, Geobacter, Ligases, Mutation, Operon, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid, Repressor Proteins, Sigma Factor, Transcription, Genetic}, issn = {1438-793X}, doi = {10.1007/s10142-007-0048-5}, author = {Krushkal, Julia and Yan, Bin and DiDonato, Laurie N and Puljic, Marko and Nevin, Kelly P and Woodard, Trevor L and Adkins, Ronald M and Meth{\'e}, Barbara A and Lovley, Derek R} } @article {507, title = {Heat-shock sigma factor RpoH from Geobacter sulfurreducens.}, journal = {Microbiology}, volume = {153}, year = {2007}, month = {2007 Mar}, pages = {838-46}, abstract = {Recent studies with Myxococcus xanthus have suggested that homologues of the Escherichia coli heat-shock sigma factor, RpoH, may not be involved in the heat-shock response in this delta-proteobacterium. The genome of another delta-proteobacterium, Geobacter sulfurreducens, which is considered to be a representative of the Fe(III)-reducing Geobacteraceae that predominate in a diversity of subsurface environments, contains an rpoH homologue. Characterization of the G. sulfurreducens rpoH homologue revealed that it was induced by a temperature shift from 30 degrees C to 42 degrees C and that an rpoH-deficient mutant was unable to grow at 42 degrees C. The predicted heat-shock genes, hrcA, grpE, dnaK, groES and htpG, were heat-shock inducible in an rpoH-dependent manner, and comparison of promoter regions of these genes identified the consensus sequences for the -10 and -35 promoter elements. In addition, DNA elements identical to the CIRCE consensus sequence were found in promoters of rpoH, hrcA and groES, suggesting that these genes are regulated by a homologue of the repressor HrcA, which is known to bind the CIRCE element. These results suggest that the G. sulfurreducens RpoH homologue is the heat-shock sigma factor and that heat-shock response in G. sulfurreducens is regulated positively by RpoH as well as negatively by the HrcA/CIRCE system.}, keywords = {Adaptation, Physiological, Base Sequence, Binding Sites, Consensus Sequence, DNA, Bacterial, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Heat-Shock Proteins, Hot Temperature, Molecular Sequence Data, Promoter Regions, Genetic, RNA, Bacterial, RNA, Messenger, Sigma Factor, Transcription, Genetic}, issn = {1350-0872}, doi = {10.1099/mic.0.2006/000638-0}, author = {Ueki, Toshiyuki and Lovley, Derek R} } @article {744, title = {Salinity constraints on subsurface archaeal diversity and methanogenesis in sedimentary rock rich in organic matter.}, journal = {Appl Environ Microbiol}, volume = {73}, year = {2007}, month = {2007 Jul}, pages = {4171-9}, abstract = {The diversity of microorganisms active within sedimentary rocks provides important controls on the geochemistry of many subsurface environments. In particular, biodegradation of organic matter in sedimentary rocks contributes to the biogeochemical cycling of carbon and other elements and strongly impacts the recovery and quality of fossil fuel resources. In this study, archaeal diversity was investigated along a salinity gradient spanning 8 to 3,490 mM Cl(-) in a subsurface shale rich in CH(4) derived from biodegradation of sedimentary hydrocarbons. Shale pore waters collected from wells in the main CH(4)-producing zone lacked electron acceptors such as O(2), NO(3)(-), Fe(3+), or SO(4)(2-). Acetate was detected only in high-salinity waters, suggesting that acetoclastic methanogenesis is inhibited at Cl(-) concentrations above approximately 1,000 mM. Most-probable-number series revealed differences in methanogen substrate utilization (acetate, trimethylamine, or H(2)/CO(2)) associated with chlorinity. The greatest methane production in enrichment cultures was observed for incubations with salinity at or close to the native pore water salinity of the inoculum. Restriction fragment length polymorphism analyses of archaeal 16S rRNA genes from seven wells indicated that there were links between archaeal communities and pore water salinity. Archaeal clone libraries constructed from sequences from 16S rRNA genes isolated from two wells revealed phylotypes similar to a halophilic methylotrophic Methanohalophilus species and a hydrogenotrophic Methanoplanus species at high salinity and a single phylotype closely related to Methanocorpusculum bavaricum at low salinity. These results show that several distinct communities of methanogens persist in this subsurface, CH(4)-producing environment and that each community is adapted to particular conditions of salinity and preferential substrate use and each community induces distinct geochemical signatures in shale formation waters.}, keywords = {Archaea, Base Sequence, Cloning, Molecular, DNA Primers, Genes, Archaeal, Genetic Variation, Geologic Sediments, Methane, Michigan, Molecular Sequence Data, Organic Chemicals, Phylogeny, RNA, Archaeal, RNA, Ribosomal, 16S, Sodium Chloride, Soil, Soil Microbiology, Water Microbiology}, issn = {0099-2240}, doi = {10.1128/AEM.02810-06}, author = {Waldron, Patricia J and Petsch, Steven T and Martini, Anna M and N{\"u}sslein, Klaus and N{\"u}slein, Klaus} } @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} } @article {748, title = {Distribution of extensive nifH gene diversity across physical soil microenvironments.}, journal = {Microb Ecol}, volume = {51}, year = {2006}, month = {2006 May}, pages = {441-52}, abstract = {The diversity of nitrogen-fixing bacteria is well described for aquatic environments; however, terrestrial analyses remain mostly biased to rhizobial plant-microbe associations. We maximized the level of resolution for this study through the use of nucleotide sequence information extracted from a series of soil microenvironments, ranging from macroaggregates at 2000 microm to the clay fraction at < 75 microm in diameter. In addition, we attempted to create an overview of the distribution of terrestrial nitrogen fixers across such microenvironments by combining culture-independent techniques with a suite of natural soil environments from uniquely different origins. Soil diazotroph diversity was analyzed phylogenetically for 600 terrestrial nifH sequences from 12 midsized clone libraries based on microenvironments of three separate soils across a global scale. Statistical analyses of nifH gene clone libraries were used to estimate coverage, establish degrees of sequence overlap, and compare cluster distributions. These analyses revealed an extensive diversity in a tropical (19 phylotypes) and an arctic soil (17 phylotypes), and moderate diversity in a temperate soil (11 phylotypes). Within each soil, comparisons across aggregate size fractions delineated nifH gene cluster shifts within populations and degrees of sequence overlap that ranged from significantly different (arctic, tropical) to significantly similar (temperate). We suggest that this is due to population separation across aggregates of different size classes, which results from differences in the temporal stability of aggregates as niches for microbial communities. This study not only provides new knowledge of the arrangement of diazotrophic communities at the soil microscale, but it also contributes to the underrepresented knowledge of soil nifH sequences in the public databases.}, keywords = {Bacteria, Base Sequence, DNA Primers, Genes, Bacterial, Multigene Family, Oxidoreductases, Phylogeny, Polymerase Chain Reaction, Soil Microbiology}, issn = {0095-3628}, doi = {10.1007/s00248-006-9044-x}, author = {Izquierdo, Javier A and N{\"u}sslein, Klaus} } @article {531, title = {Adaptation to disruption of the electron transfer pathway for Fe(III) reduction in Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {187}, year = {2005}, month = {2005 Sep}, pages = {5918-26}, abstract = {Previous studies demonstrated that an outer membrane c-type cytochrome, OmcB, was involved in Fe(III) reduction in Geobacter sulfurreducens. An OmcB-deficient mutant was greatly impaired in its ability to reduce both soluble and insoluble Fe(III). Reintroducing omcB restored the capacity for Fe(III) reduction at a level proportional to the level of OmcB production. Here, we report that the OmcB-deficient mutant gradually adapted to grow on soluble Fe(III) but not insoluble Fe(III). The adapted OmcB-deficient mutant reduced soluble Fe(III) at a rate comparable to that of the wild type, but the cell yield of the mutant was only ca. 60\% of that of the wild type under steady-state culturing conditions. Analysis of proteins and transcript levels demonstrated that expression of several membrane-associated cytochromes was higher in the adapted mutant than in the wild type. Further comparison of transcript levels during steady-state growth on Fe(III) citrate with a whole-genome DNA microarray revealed a significant shift in gene expression in an apparent attempt to adapt metabolism to the impaired electron transport to Fe(III). These results demonstrate that, although there are many other membrane-bound c-type cytochromes in G. sulfurreducens, increased expression of these cytochromes cannot completely compensate for the loss of OmcB. The concept that outer membrane cytochromes are promiscuous reductases that are interchangeable in function appears to be incorrect. Furthermore, the results indicate that there may be different mechanisms for electron transfer to soluble Fe(III) and insoluble Fe(III) oxides in G. sulfurreducens, which emphasizes the importance of studying electron transport to the environmentally relevant Fe(III) oxides.}, keywords = {Adaptation, Physiological, Bacterial Outer Membrane Proteins, Base Sequence, Cytochromes c, DNA Primers, Electron Transport, Ferric Compounds, Gene Expression Regulation, Bacterial, Geobacter, Kinetics, Oxidation-Reduction, Reverse Transcriptase Polymerase Chain Reaction}, issn = {0021-9193}, doi = {10.1128/JB.187.17.5918-5926.2005}, author = {Leang, Ching and Adams, L A and Chin, K-J and Nevin, K P and Meth{\'e}, B A and Webster, J and Sharma, M L and Lovley, D R} } @article {534, title = {A novel Geobacteraceae-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe(III) and Mn(IV) oxides in Geobacter sulfurreducens.}, journal = {BMC Microbiol}, volume = {5}, year = {2005}, month = {2005}, pages = {41}, abstract = {BACKGROUND: Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. RESULTS: When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe(III) and insoluble Mn(IV) oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50\% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal reduction. CONCLUSION: OmpJ is a putative porin found in the outer membrane of the model metal reducer G. sulfurreducens that is required for respiration of extracellular electron acceptors such as soluble and insoluble metals. The effect of OmpJ in extracellular electron transfer is indirect, as OmpJ is required to keep the integrity of the periplasmic space necessary for proper folding and functioning of periplasmic and outer membrane electron transport components. The exclusive presence of ompJ in members of the Geobacteraceae family as well as its role in metal reduction suggest that the ompJ sequence may be useful in tracking the growth or activity of Geobacteraceae in sedimentary environments.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Base Sequence, Biological Transport, Deltaproteobacteria, DNA Primers, Ferric Compounds, Gene Deletion, Genome, Bacterial, Geobacter, Manganese Compounds, Molecular Sequence Data, Oxides, Peptide Fragments, Phylogeny, Protein Structure, Secondary, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, issn = {1471-2180}, doi = {10.1186/1471-2180-5-41}, author = {Afkar, Eman and Reguera, Gemma and Schiffer, Marianne and Lovley, Derek R} } @article {527, title = {Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 Dec}, pages = {8634-41}, abstract = {The potential role of outer membrane proteins in electron transfer to insoluble Fe(III) oxides by Geobacter sulfurreducens was investigated because this organism is closely related to the Fe(III) oxide-reducing organisms that are predominant in many Fe(III)-reducing environments. Two of the most abundant proteins that were easily sheared from the outer surfaces of intact cells were c-type cytochromes. One, designated OmcS, has a molecular mass of ca. 50 kDa and is predicted to be an outer membrane hexaheme c-type cytochrome. Transcripts for omcS could be detected during growth on Fe(III) oxide, but not on soluble Fe(III) citrate. The omcS mRNA consisted primarily of a monocistronic transcript, and to a lesser extent, a longer transcript that also contained the downstream gene omcT, which is predicted to encode a second hexaheme outer membrane cytochrome with 62.6\% amino acid sequence identity to OmcS. The other abundant c-type cytochrome sheared from the outer surface of G. sulfurreducens, designated OmcE, has a molecular mass of ca. 30 kDa and is predicted to be an outer membrane tetraheme c-type cytochrome. When either omcS or omcE was deleted, G. sulfurreducens could no longer reduce Fe(III) oxide but could still reduce soluble electron acceptors, including Fe(III) citrate. The mutants could reduce Fe(III) in Fe(III) oxide medium only if the Fe(III) chelator, nitrilotriacetic acid, or the electron shuttle, anthraquinone 2,6-disulfonate, was added. Expressing omcS or omcE in trans restored the capacity for Fe(III) oxide reduction. OmcT was not detected among the sheared proteins, and genetic studies indicated that G. sulfurreducens could not reduce Fe(III) oxide when omcT was expressed but OmcS was absent. In contrast, Fe(III) oxide was reduced when omcS was expressed in the absence of OmcT. These results suggest that OmcS and OmcE are involved in electron transfer to Fe(III) oxides in G. sulfurreducens. They also emphasize the importance of evaluating mechanisms for Fe(III) reduction with environmentally relevant Fe(III) oxide, rather than the more commonly utilized Fe(III) citrate, because additional electron transfer components are required for Fe(III) oxide reduction that are not required for Fe(III) citrate reduction.}, keywords = {Amino Acid Sequence, Bacterial Outer Membrane Proteins, Bacterial Proteins, Base Sequence, Cytochromes c, DNA Primers, Ferric Compounds, Geobacter, Kinetics, Manganese Compounds, Molecular Sequence Data, Oxidation-Reduction, Oxides, Peptide Fragments}, issn = {0099-2240}, doi = {10.1128/AEM.71.12.8634-8641.2005}, author = {Mehta, T and Coppi, M V and Childers, S E and Lovley, D R} } @article {751, title = {Shifts in rhizoplane communities of aquatic plants after cadmium exposure.}, journal = {Appl Environ Microbiol}, volume = {71}, year = {2005}, month = {2005 May}, pages = {2484-92}, abstract = {In this study we present the comparative molecular analysis of bacterial communities of the aquatic plant Lemna minor from a contaminated site (RCP) and from a laboratory culture (EPA), as well as each of these with the addition of cadmium. Plants were identified as L. minor by analysis of the rpl16 chloroplast region. Comparative bacterial community studies were based on the analyses of 16S rRNA clone libraries, each containing about 100 clones from the root surfaces of plants. Bacterial communities were compared at three phylogenetic levels of resolution. At the level of bacterial divisions, differences in diversity index scores between treatments, with and without cadmium within the same plant type (EPA or RCP), were small, indicating that cadmium had little effect. When we compared genera within the most dominant group, the beta-proteobacteria, differences between unamended and cadmium-amended libraries were much larger. Bacterial diversity increased upon cadmium addition for both EPA and RCP libraries. Analyses of diversity at the phylotype level showed parallel shifts to more even communities upon cadmium addition; that is, percentage changes in diversity indices due to cadmium addition were the same for either plant type, indicating that contamination history might be independent of disturbance-induced diversity shifts. At finer phylogenetic levels of resolution, the effects of cadmium addition on bacterial communities were very noticeable. This study is a first step in understanding the role of aquatic plant-associated microbial communities in phytoremediation of heavy metals.}, keywords = {Araceae, Bacteria, Base Sequence, Cadmium, Molecular Sequence Data, Phylogeny, Plant Roots, RNA, Ribosomal, 16S}, issn = {0099-2240}, doi = {10.1128/AEM.71.5.2484-2492.2005}, author = {Stout, Lisa M and N{\"u}sslein, Klaus} } @article {548, title = {Direct correlation between rates of anaerobic respiration and levels of mRNA for key respiratory genes in Geobacter sulfurreducens.}, journal = {Appl Environ Microbiol}, volume = {70}, year = {2004}, month = {2004 Sep}, pages = {5183-9}, abstract = {The predominance of Geobacter species in environments in which Fe(III) reduction is important has suggested that Fe(III) reduction rates might be estimated in Geobacter-dominated environments by assessing in situ activity with molecular techniques. To determine whether mRNA levels of key respiratory genes might be correlated with respiration rates in Geobacter sulfurreducens, studies were conducted with fumarate as the electron acceptor and acetate as the limiting electron donor in anaerobic continuous cultures. Levels of mRNA for a fumarate reductase gene, frdA, quantified by real-time reverse transcription-PCR were directly correlated with fumarate reduction rates. In similar studies with Fe(III) as the electron acceptor, mRNA levels for omcB, a gene for an outer membrane c-type cytochrome involved in Fe(III) reduction, were positively correlated with Fe(III) reduction rates. Levels of mRNA for frdA and omcB were also positively correlated with fumarate and Fe(III) reduction rates, respectively, when growth was limited by the availability of fumarate or Fe(III), but mRNA levels were higher than in acetate-limited cultures. Levels of mRNA for omcC, which encodes a c-type cytochrome highly similar to OmcB but not necessary for Fe(III) reduction, followed patterns different than those of omcB. This agrees with the previous finding that OmcC is not involved in Fe(III) reduction and suggests that changes in mRNA levels of omcB are related to its role in Fe(III) reduction. These results demonstrate that mRNA levels for respiratory genes might be used to estimate in situ Fe(III) reduction rates in Geobacter-dominated environments but suggest that information on environmental conditions and/or the metabolic state of Geobacter species is also required for accurate rate estimates.}, keywords = {Anaerobiosis, Base Sequence, DNA Primers, Geobacter, Kinetics, Oxygen Consumption, Polymerase Chain Reaction, RNA, Messenger}, issn = {0099-2240}, doi = {10.1128/AEM.70.9.5183-5189.2004}, author = {Chin, Kuk-Jeong and Esteve-N{\'u}{\~n}ez, Abraham and Leang, Ching and Lovley, Derek R} } @article {824, title = {A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12.}, journal = {Genetics}, volume = {167}, year = {2004}, month = {2004 Jun}, pages = {569-78}, abstract = {The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.}, keywords = {Base Sequence, Codon, DNA Primers, DNA Repair, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli K12, Escherichia coli Proteins, Molecular Sequence Data, Mutation, Plasmids, Polymerase Chain Reaction, Sequence Deletion}, issn = {0016-6731}, doi = {10.1534/genetics.103.025296}, author = {McCool, Jesse D and Ford, Christopher C and Sandler, Steven J} } @article {397, title = {Genetic evidence for pre-recruitment as the mechanism of transcription activation by SoxS of Escherichia coli: the dominance of DNA binding mutations of SoxS.}, journal = {J Mol Biol}, volume = {344}, year = {2004}, month = {2004 Nov 12}, pages = {1-10}, abstract = {SoxS, the direct transcriptional activator of the Escherichia coli superoxide (SoxRS) regulon, displays several unusual characteristics which suggest that it is unlikely to activate transcription by the ususal recruitment mechanism. Thus, agents that generate superoxide endogenously and thereby provoke the defense response elicit the de novo synthesis of SoxS, and with the SoxS binding site being highly degenerate, the number of SoxS binding sites per cell far exceeds the number of SoxS molecules per cell. To account for these distinctive features of the SoxRS system, we proposed "pre-recruitment" as the mechanism by which SoxS activates transcription of the regulon{\textquoteright}s genes. In pre-recruitment, newly synthesized SoxS molecules form binary complexes with RNA polymerase in solution. These complexes provide the information content to allow the 2500 molecules of SoxS per cell to scan the 65,000 SoxS binding sites per cell for the 200 binding sites per cell that reside within SoxS-dependent promoters. As a test of whether SoxS activates transcription by recruitment or pre-recruitment, we determined the dominance relationships of SoxS mutations conferring defective DNA binding. We found that soxS DNA binding mutations are dominant to the wild-type allele, a result consistent with the pre-recruitment hypothesis, but opposite to that expected for an activator that functions by recruitment. Moreover, whereas positive control mutations of activators functioning by recruitment are usually dominant, a soxS positive control mutation was not. Lastly, with the SoxRS system as an example, we discuss the physiological requirement for stringent regulation of transcriptional activators that function by pre-recruitment.}, keywords = {Base Sequence, Binding Sites, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genes, Dominant, Mutation, Regulon, Trans-Activators, Transcriptional Activation}, issn = {0022-2836}, doi = {10.1016/j.jmb.2004.09.007}, author = {Griffith, Kevin L and Wolf, Richard E} } @article {544, title = {Microbial communities associated with electrodes harvesting electricity from a variety of aquatic sediments.}, journal = {Microb Ecol}, volume = {48}, year = {2004}, month = {2004 Aug}, pages = {178-90}, abstract = {The microbial communities associated with electrodes from underwater fuel cells harvesting electricity from five different aquatic sediments were investigated. Three fuel cells were constructed with marine, salt-marsh, or freshwater sediments incubated in the laboratory. Fuel cells were also deployed in the field in salt marsh sediments in New Jersey and estuarine sediments in Oregon, USA. All of the sediments produced comparable amounts of power. Analysis of 16S rRNA gene sequences after 3-7 months of incubation demonstrated that all of the energy-harvesting anodes were highly enriched in microorganisms in the delta-Proteobacteria when compared with control electrodes not connected to a cathode. Geobacteraceae accounted for the majority of delta-Proteobacterial sequences or all of the energy-harvesting anodes, except the one deployed at the Oregon estuarine site. Quantitative PCR analysis of 16S rRNA genes and culturing studies indicated that Geobacteraceae were 100-fold more abundant on the marine-deployed anodes versus controls. Sequences most similar to microorganisms in the family Desulfobulbaceae predominated on the anode deployed in the estuarine sediments, and a significant proportion of the sequences recovered from the freshwater anodes were closely related to the Fe(III)-reducing isolate, Geothrix fermentans. There was also a specific enrichment of microorganisms on energy harvesting cathodes, but the enriched populations varied with the sediment/water source. Thus, future studies designed to help optimize the harvesting of electricity from aquatic sediments or waste organic matter should focus on the electrode interactions of these microorganisms which are most competitive in colonizing anodes and cathodes.}, keywords = {Base Sequence, Biodiversity, Bioelectric Energy Sources, Cloning, Molecular, DNA Primers, Electrodes, Gammaproteobacteria, Geologic Sediments, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Restriction Mapping, RNA, Ribosomal, 16S, Sequence Analysis, DNA}, issn = {0095-3628}, doi = {10.1007/s00248-003-0004-4}, author = {Holmes, D E and Bond, D R and O{\textquoteright}Neil, R A and Reimers, C E and Tender, L R and Lovley, D R} } @article {398, title = {Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons.}, journal = {Mol Microbiol}, volume = {51}, year = {2004}, month = {2004 Mar}, pages = {1801-16}, abstract = {In Escherichia coli, the SoxRS regulon confers resistance to redox-cycling compounds, and the Mar regulon provides a defence against multiple antibiotics. The response regulators, SoxS and MarA, are synthesized de novo in response to their inducing signals and directly activate transcription of a common set of target genes. Although the mechanisms of transcription activation by SoxS and MarA have been well studied, little is known about how the systems are shut-off once the inducing stress has subsided, except that de novo synthesis of the regulators is known to cease almost immediately. Here, we induced the SoxRS regulon and determined that, upon removal of the inducer, expression of the regulon{\textquoteright}s genes quickly returns to the preinduced level. This rapid shut-off indicates that the system is reset by an active process. We found that SoxS is unstable and infer that SoxS degradation is responsible for the rapid return of the system to the ground state upon removal of the inducing signal. We also found that MarA is unstable and that the instability of both proteins is intrinsic and unregulated. We used null mutations of protease genes to identify the proteases involved in the degradation of SoxS and MarA. Among single protease mutations, only lon mutations increased the half-life of SoxS and MarA. In addition, SoxS appeared to be nearly completely stable in a lon ftsH double mutant. Using hexahistidine tags placed at the respective ends of the activators, we found that access to the amino-terminus is essential for the proteolytic degradation.}, keywords = {Base Sequence, Blotting, Western, DNA Primers, DNA-Binding Proteins, Drug Resistance, Bacterial, Drug Resistance, Multiple, Bacterial, Endopeptidases, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Half-Life, Hydrolysis, Kinetics, Mutation, Oxidation-Reduction, Promoter Regions, Genetic, Regulon, Trans-Activators, Transcription, Genetic}, issn = {0950-382X}, author = {Griffith, Kevin L and Shah, Ishita M and Wolf, Richard E} } @article {549, title = {The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens.}, journal = {J Bacteriol}, volume = {186}, year = {2004}, month = {2004 Aug}, pages = {5543-6}, abstract = {Geobacter sulfurreducens RpoS sigma factor was shown to contribute to survival in stationary phase and upon oxygen exposure. Furthermore, a mutation in rpoS decreased the rate of reduction of insoluble Fe(III) but not of soluble forms of iron. This study suggests that RpoS plays a role in regulating metabolism of Geobacter under suboptimal conditions in subsurface environments.}, keywords = {Adaptation, Physiological, Bacterial Proteins, Base Sequence, Ferric Compounds, Gene Expression Regulation, Bacterial, Genes, Bacterial, Geobacter, Molecular Sequence Data, Mutation, Oxidation-Reduction, Oxidative Stress, Sigma Factor, Transcription Initiation Site}, issn = {0021-9193}, doi = {10.1128/JB.186.16.5543-5546.2004}, author = {N{\'u}{\~n}ez, Cinthia and Adams, Lorrie and Childers, Susan and Lovley, Derek R} } @article {576, title = {Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens.}, journal = {Biochem J}, volume = {369}, year = {2003}, month = {2003 Jan 1}, pages = {153-61}, abstract = {A 9.6 kDa periplasmic c -type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfurreducens and characterized. The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c (7) cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans. The gene encoding PpcA was identified from the G. sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58 kDa) with 49\% identity to the c (7) cytochrome of D. acetoxidans. In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method. Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant. When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored. The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered. The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor. When taken together with previous studies on other electron transport proteins in G. sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.}, keywords = {Amino Acid Sequence, Base Sequence, Cytochrome c Group, DNA, Bacterial, Molecular Sequence Data, Periplasm, Proteobacteria, Sequence Homology, Amino Acid}, issn = {0264-6021}, doi = {10.1042/BJ20020597}, author = {Lloyd, Jon R and Leang, Ching and Hodges Myerson, Allison L and Coppi, Maddalena V and Cuifo, Stacey and Methe, Barb and Sandler, Steven J and Lovley, Derek R} } @article {42, title = {Expression of the yeast PIS1 gene requires multiple regulatory elements including a Rox1p binding site.}, journal = {J Biol Chem}, volume = {278}, year = {2003}, month = {2003 Oct 3}, pages = {38646-52}, abstract = {The PIS1 gene is required for de novo synthesis of phosphatidylinositol (PI), an essential phospholipid in Saccharomyces cerevisiae. PIS1 gene expression is unusual because it is uncoupled from the other phospholipid biosynthetic genes, which are regulated in response to inositol and choline. Relatively little is known about regulation of transcription of the PIS1 gene. We reported previously that PIS1 transcription is sensitive to carbon source. To further our understanding of the regulation of PIS1 transcription, we carried out a promoter deletion analysis that identified three regions required for PIS1 gene expression (upstream activating sequence (UAS) elements 1-3). Deletion of either UAS1 or UAS2 resulted in an approximately 45\% reduction in expression, whereas removal of UAS3 yielded an 84\% decrease in expression. A comparison of promoters among several Saccharomyces species shows that these sequences are highly conserved. Curiously, the UAS3 element region (-149 to -138) includes a Rox1p binding site. Rox1p is a repressor of hypoxic genes under aerobic growth conditions. Consistent with this, we have found that expression of a PIS1-cat reporter was repressed under aerobic conditions, and this repression was dependent on both Rox1p and its binding site. Furthermore, PI levels were elevated under anaerobic conditions. This is the first evidence that PI levels are affected by regulation of PIS1 transcription.}, keywords = {Anoxia, Base Sequence, Binding Sites, Carbon, Chloramphenicol O-Acetyltransferase, Choline, Chromatography, Thin Layer, Conserved Sequence, DNA, DNA, Complementary, DNA-Binding Proteins, Gene Deletion, Gene Expression Regulation, Fungal, Genes, Reporter, Inositol, Lipid Metabolism, Models, Biological, Models, Genetic, Molecular Sequence Data, Oxygen, Phospholipids, Plasmids, Promoter Regions, Genetic, Protein Binding, Repressor Proteins, RNA, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Transferases (Other Substituted Phosphate Groups)}, issn = {0021-9258}, doi = {10.1074/jbc.M305251200}, author = {Gardocki, Mary Elizabeth and Lopes, John M} } @article {386, title = {Trypanosoma brucei has two distinct mitochondrial DNA polymerase beta enzymes.}, journal = {J Biol Chem}, volume = {278}, year = {2003}, month = {2003 Dec 5}, pages = {49095-101}, abstract = {In higher eukaryotes, DNA polymerase (pol) beta resides in the nucleus and participates primarily in DNA repair. The DNA polymerase beta from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol beta-like proteins. One is approximately 70\% identical to the C. fasciculata pol beta and is likely the homolog of this enzyme. The other, although approximately 30\% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different.}, keywords = {Animals, Base Sequence, Cell Line, DNA Polymerase beta, DNA Primers, Isoenzymes, Mitochondria, Recombinant Proteins, Trypanosoma brucei brucei}, issn = {0021-9258}, doi = {10.1074/jbc.M308565200}, author = {Saxowsky, Tina T and Choudhary, Gunjan and Klingbeil, Michele M and Englund, Paul T} } @article {780, title = {Ancient lineages of non-LTR retrotransposons in the primitive eukaryote, Giardia lamblia.}, journal = {Mol Biol Evol}, volume = {19}, year = {2002}, month = {2002 May}, pages = {619-30}, abstract = {Mobile elements that use reverse transcriptase to make new copies of themselves are found in all major lineages of eukaryotes. The non-long terminal repeat (non-LTR) retrotransposons have been suggested to be the oldest of these eukaryotic elements. Phylogenetic analysis of non-LTR elements suggests that they have predominantly undergone vertical transmission, as opposed to the frequent horizontal transmissions found for other mobile elements. One prediction of this vertical model of inheritance is that the oldest lineages of eukaryotes should exclusively harbor the oldest lineages of non-LTR retrotransposons. Here we characterize the non-LTR retrotransposons present in one of the most primitive eukaryotes, the diplomonad Giardia lamblia. Two families of elements were detected in the WB isolate of G. lamblia currently being used for the genome sequencing project. These elements are clearly distinct from all other previously described non-LTR lineages. Phylogenetic analysis indicates that these Genie elements (for Giardia early non-LTR insertion element) are among the oldest known lineages of non-LTR elements consistent with strict vertical descent. Genie elements encode a single open reading frame with a carboxyl terminal endonuclease domain. Genie 1 is site specific, as seven to eight copies are present in a single tandem array of a 771-bp repeat near the telomere of one chromosome. The function of this repeat is not known. One additional, highly divergent, element within the Genie 1 lineage is not located in this tandem array but is near a second telomere. Four different telomere addition sites could be identified within or near the Genie elements on each of these chromosomes. The second lineage of non-LTR elements, Genie 2, is composed of about 10 degenerate copies. Genie 2 elements do not appear to be site specific in their insertion. An unusual aspect of Genie 2 is that all copies contain inverted repeats up to 172 bp in length.}, keywords = {Animals, Base Sequence, DNA, Protozoan, Evolution, Molecular, Giardia lamblia, Long Interspersed Nucleotide Elements, Molecular Sequence Data, Open Reading Frames, Phylogeny, Repetitive Sequences, Nucleic Acid, Sequence Homology, Nucleic Acid, Telomere}, issn = {0737-4038}, author = {Burke, William D and Malik, Harmit S and Rich, Stephen M and Eickbush, Thomas H} } @article {399, title = {A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation.}, journal = {J Mol Biol}, volume = {322}, year = {2002}, month = {2002 Sep 13}, pages = {237-57}, abstract = {SoxS is the direct transcriptional activator of the superoxide regulon. SoxS recognizes a highly degenerate "soxbox" DNA sequence, and activates transcription from class I and class II promoters. SoxS is the smallest member of the AraC/XylS family of transcription regulators whose hallmark is dual helix-turn-helix (HTH) DNA-binding motifs. Evidence suggests that the N-terminal HTH motif of SoxS interacts with a highly conserved region of the soxbox termed recognition element 1 (RE1), while the C-terminal HTH motif interacts with the less conserved recognition element 2 (RE2). In the work described here, we prepared a complete library of 101 SoxS mutants containing single alanine substitutions of SoxS, and we characterized the mutant proteins in vivo and in vitro. With SoxS being closely related to MarA, we analyzed the effects of the SoxS mutations in the context of the MarA-mar crystal structure and with respect to the NMR study of MarA-DNA complexes in solution. From the properties of the alanine substitutions, we conclude the following. (1) Surface-exposed residues of helix 3 and helix 6, the recognition helices of the dual HTH motifs, are important to DNA binding and transcription activation; however, substitutions of residues predicted from the MarA-mar crystal structure to make contact with the sugar-phosphate backbone are more detrimental to DNA binding than mutations predicted to make base-specific contacts. (2) Substitution of several residues within the recognition helix predicted to make base-specific contacts with RE2 have relatively little effect on DNA-binding, suggesting the possibility of alternative protein-DNA interactions than those inferred from the MarA-mar crystal structure. (3) DNA binding and transcription activation were reduced by substitution of conserved amino acid residues comprising the hydrophobic core, presumably because they disrupt the structural integrity of SoxS. (4) Mutant K30A appears to be a positive control mutant defective in a protein-protein interaction with RNA polymerase that is required for transcription activation at all SoxS-dependent promoters because it binds and bends DNA normally but fails to activate transcription from both classes of promoters. Alanine substitutions of surface-exposed residues H3, K5, D9, S31, and V45 confer a similar phenotype. Since these residues are near K30 on the surface of the protein, the surface formed by the six residues may be used to make protein-protein interactions with RNA polymerase that are required for transcription activation at both class I and class II SoxS-dependent promoters. (5) Mutants F74A, D75A, M78A, D79A and Q85A appear to define a surface required for protein-protein interaction with RNA polymerase specifically at class II promoters because these positive control mutants bind and bend DNA normally but are defective in activation of class II promoters but not class I promoters. These SoxS mutants that bind and bend DNA normally but are defective in transcription activation represent the first positive control mutants with putative defects in protein-protein interactions with RNA polymerase among the SoxS/MarA/Rob subset of the AraC/XylS family of transcription regulators.}, keywords = {Alanine, Amino Acid Substitution, Base Sequence, Binding Sites, DNA, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Lethal, Hydrophobic and Hydrophilic Interactions, Lac Operon, Models, Molecular, Mutagenesis, Nucleic Acid Conformation, Phenotype, Phosphates, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Structure-Activity Relationship, Trans-Activators, Transcriptional Activation}, issn = {0022-2836}, author = {Griffith, Kevin L and Wolf, Richard E} } @article {783, title = {Antigen polymorphism in Borrelia hermsii, a clonal pathogenic bacterium.}, journal = {Proc Natl Acad Sci U S A}, volume = {98}, year = {2001}, month = {2001 Dec 18}, pages = {15038-43}, abstract = {The relapsing fever spirochete, Borrelia hermsii, escapes immune selection by alternating expression of surface lipoprotein alleles. The switch results from a duplicative transposition of one of several surface lipoprotein-encoding nucleotide sequences into the singular expression site. These nucleotide sequences constitute a large gene family whose diversity originated, in some cases, before the major divergences of Borrelia species. We have examined the B. hermsii vsp subfamily of alleles, which are carried on linear plasmids within each cell and maintained in several diverse copies as an antigenic archive. Each encodes a distinct serotype-specific protein. We sequenced more than 90\% of the alleles within a single strain-B. hermsii strain HS1. A preponderance of allelic mosaicism suggests that intragenic recombination, coupled with selection imposed by host immune response, has driven diversification of the archived ensemble of vsp alleles. The recombinational diversification of vsp alleles generates change in the associated serotypes of the magnitude (30-40\% amino acid differentiation) necessary for overcoming cross-reactivity of neutralizing antibodies. We conclude that evolution of vsp has occurred by punctuated occurrence of allelic differentiation, rather than by gradual selection of incremental point mutations that do not meet the threshold for antigenic diversity.}, keywords = {Antigens, Bacterial, Base Sequence, Borrelia, DNA, Bacterial, Polymorphism, Genetic, Repetitive Sequences, Nucleic Acid}, issn = {0027-8424}, doi = {10.1073/pnas.071042098}, author = {Rich, S M and Sawyer, S A and Barbour, A G} } @article {402, title = {Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation.}, journal = {Mol Microbiol}, volume = {40}, year = {2001}, month = {2001 Jun}, pages = {1141-54}, abstract = {SoxS is the direct transcriptional activator of at least 15 genes of the Escherichia coli superoxide regulon. SoxS is small (107 amino acids), binds DNA as a monomer and recognizes a highly degenerate DNA binding site, termed {\textquoteright}soxbox{\textquoteright}. Like other members of the AraC/XylS family, SoxS has two putative helix-turn-helix (HTH) DNA-binding motifs, and it has been proposed that each HTH motif recognizes a highly conserved recognition element of the soxbox. To determine which nucleotides are important for SoxS binding, we conducted a systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters and determined the effect of the soxbox mutations on SoxS DNA binding and transcription activation in vivo by measuring beta-galactosidase activity in strains with fusions to lacZ. We found that the sequences GCAC and CAAA, termed recognition elements 1 and 2 (RE 1 and RE 2), respectively, are critical for SoxS binding, as mutations within these elements severely hinder or eliminate SoxS-dependent transcription activation; substitutions within RE 2 (CAAA), however, are tolerated better than changes within RE 1 (GCAC). Although substitutions at the seven positions separating the two REs had only a modest effect on SoxS binding, AT basepairs were favoured within this {\textquoteright}spacer{\textquoteright} region, presumably because, by facilitating DNA bending, they help bring the two recognition elements into proper juxtaposition. We also found that the {\textquoteright}invariant A{\textquoteright} present at position 1 of 14/15 functional soxboxes identified thus far is important for SoxS binding, as a change to any other nucleotide at this position reduced SoxS-dependent transcription by approximately 50\%. In addition, positions surrounding the REs seem to show a context effect, in that certain substitutions there have little or no effect when the RE has the optimal binding sequence, but produce a pronounced effect when the RE has a suboptimal sequence. We propose that these nucleotides play an important role in effecting differential expression from the various promoters. Lastly, we used gel retardation assays to show that alterations in transcription activation in vivo are caused by effects on DNA binding. Based on this exhaustive mutagenesis, we propose the following optimal sequence for SoxS binding: AnVGCACWWWnKRHCAAAHn (n = A, C, G, T; V = A, C, G; W = A, T; K = G, T; R = A, G; H = A, C, T).}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, Binding Sites, Carrier Proteins, DNA, Bacterial, Electrophoresis, Escherichia coli, Escherichia coli Proteins, Molecular Sequence Data, Mutagenesis, Promoter Regions, Genetic, Trans-Activators, Transcription Factors, Transcriptional Activation}, issn = {0950-382X}, author = {Griffith, K L and Wolf, R E} } @article {787, title = {Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.}, journal = {Appl Environ Microbiol}, volume = {66}, year = {2000}, month = {2000 Aug}, pages = {3344-9}, abstract = {Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.}, keywords = {Animals, Base Sequence, Cattle, Cryptosporidiosis, Cryptosporidium parvum, DNA, Protozoan, Humans, Karyotyping, Mice, Mice, Knockout, Microsatellite Repeats, Molecular Sequence Data, Polymerase Chain Reaction, Polymorphism, Genetic, Sequence Analysis, DNA}, issn = {0099-2240}, author = {Feng, X and Rich, S M and Akiyoshi, D and Tumwine, J K and Kekitiinwa, A and Nabukeera, N and Tzipori, S and Widmer, G} } @article {784, title = {Genetic variation and the recent worldwide expansion of Plasmodium falciparum.}, journal = {Gene}, volume = {261}, year = {2000}, month = {2000 Dec 30}, pages = {161-70}, abstract = {Plasmodium falciparum, the agent of human malignant malaria, diverged from Plasmodium reichenowi, the chimpanzee parasite, about the time the human and chimpanzee lineages diverged from each other. The absence of synonymous nucleotide variation at ten loci indicates that the world populations of P. falciparum derive most recently from one single strain, or {\textquoteright}cenancestor,{\textquoteright} which lived a few thousand years ago. Antigenic genes of P. falciparum (such as Csp, Msp-1, and Msp-2) exhibit numerous polymorphisms that have been estimated to be millions of years old. We have discovered in these antigenic genes short repetitive sequences that distort the alignment of alleles and account for the apparent old age of the polymorphisms. The processes of intragenic recombination that generate the repeats occur at rates about 10(-3) to 10(-2), several orders of magnitude greater than the typical mutational process of nucleotide substitutions. We conclude that the antigenic polymorphisms of P. falciparum are consistent with a recent expansion of the world populations of the parasite from a cenancestor that lived in tropical Africa a few thousand years ago.}, keywords = {Animals, Antigens, Protozoan, Base Sequence, Genetic Variation, Merozoite Surface Protein 1, Molecular Sequence Data, Phylogeny, Plasmodium, Plasmodium falciparum, Polymorphism, Genetic, Protozoan Proteins, Repetitive Sequences, Nucleic Acid, RNA, Ribosomal, Sequence Homology, Nucleic Acid, Species Specificity}, issn = {0378-1119}, author = {Ayala, F J and Rich, S M} } @article {827, title = {Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12.}, journal = {Genetics}, volume = {155}, year = {2000}, month = {2000 Jun}, pages = {487-97}, abstract = {In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3{\textquoteright}-5{\textquoteright} helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.}, keywords = {Base Sequence, DNA Primers, DNA Replication, Escherichia coli, Genes, Lethal, Mutation, Polymerase Chain Reaction}, issn = {0016-6731}, author = {Sandler, S J} } @article {786, title = {The origin of antigenic diversity in Plasmodium falciparum.}, journal = {Parasitol Today}, volume = {16}, year = {2000}, month = {2000 Sep}, pages = {390-6}, abstract = {Most studies of genetic variability of Plasmodium falciparum have focused on protein antigens and the genes that encode them. The consensus is that populations exhibit high levels of genetic polymorphism, most notably the genes encoding surface proteins of the merozoite (Msp1, Msp2) and the sporozoite (Csp). The age and derivation of this variation is a subject that warrants further careful consideration, as discussed here by Stephen Rich, Marcelo Ferreira and Francisco Ayala.}, keywords = {Amino Acid Sequence, Animals, Antigens, Protozoan, Base Sequence, Genes, Protozoan, Genetic Variation, Merozoite Surface Protein 1, Molecular Sequence Data, Plasmodium falciparum, Protozoan Proteins, Sequence Homology}, issn = {0169-4758}, author = {Rich, S M and Ferreira, M U and Ayala, F J} } @article {788, title = {Population structure and recent evolution of Plasmodium falciparum.}, journal = {Proc Natl Acad Sci U S A}, volume = {97}, year = {2000}, month = {2000 Jun 20}, pages = {6994-7001}, abstract = {Plasmodium falciparum is the agent of malignant malaria, one of mankind{\textquoteright}s most severe maladies. The parasite exhibits antigenic polymorphisms that have been postulated to be ancient. We have proposed that the extant world populations of P. falciparum have derived from one single parasite, a cenancestor, within the last 5, 000-50,000 years. This inference derives from the virtual or complete absence of synonymous nucleotide polymorphisms at genes not involved in immune or drug responses. Seeking to conciliate this claim with extensive antigenic polymorphism, we first note that allele substitutions or polymorphisms can arise very rapidly, even in a single generation, in large populations subject to strong natural selection. Second, new alleles can arise not only by single-nucleotide mutations, but also by duplication/deletion of short simple-repeat DNA sequences, a process several orders of magnitude faster than single-nucleotide mutation. We analyze three antigenic genes known to be extremely polymorphic: Csp, Msp-1, and Msp-2. We identify regions consisting of tandem or proximally repetitive short DNA sequences, including some previously unnoticed. We conclude that the antigenic polymorphisms are consistent with the recent origin of the world populations of P. falciparum inferred from the analysis of nonantigenic genes.}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Biological Evolution, Genes, Protozoan, Genetics, Population, Molecular Sequence Data, Plasmodium falciparum}, issn = {0027-8424}, author = {Rich, S M and Ayala, F J} } @article {790, title = {Evolution of Plasmodium and the recent origin of the world populations of Plasmodium falciparum.}, journal = {Parassitologia}, volume = {41}, year = {1999}, month = {1999 Sep}, pages = {55-68}, abstract = {We have investigated the evolution of Plasmodium parasites by analyzing DNA sequences of several genes. We reach the following conclusions: (1) The four human parasites, P. falciparum, P. malariae, P. ovale, and P. vivax are very remotely related to each other, so that their evolutionary divergence predates the origin of the hominids; several of these parasites became associated with the human lineage by lateral transfer from other hosts. (2) P. falciparum diverged from P. reichenowi about 8 million years ago, consistently with the time of divergence of the human lineage from the apes; a parsimonious inference is that falciparum has been associated with humans since the origin of the hominids. (3) P. malariae is genetically indistinguishable from P. brasilianum, a parasite of New World monkeys; and, similarly. (4) P. vivax is genetically indistinguishable from the New World monkey parasite P. simium. We infer in each of these two cases a very recent lateral transfer between the human and monkey hosts, and explore alternative hypotheses about the direction of the transfer. We have also investigated the population structure of P. falciparum by analyzing 10 genes and conclude that the extant world populations of this parasite have evolved from a single strain within the last several thousand years. The extensive polymorphisms observed in the highly repetitive central region of the Csp gene, as well as the apparently very divergent two classes of alleles at the Msa-1 gene, are consistent with this conclusion.}, keywords = {Amino Acid Sequence, Animals, Base Sequence, DNA, Protozoan, Evolution, Molecular, Hominidae, Humans, Merozoite Surface Protein 1, Molecular Sequence Data, Plasmodium, Plasmodium falciparum, Polymorphism, Genetic, Sequence Alignment}, issn = {0048-2951}, author = {Ayala, F J and Escalante, A A and Rich, S M} } @article {403, title = {Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters.}, journal = {Mol Microbiol}, volume = {34}, year = {1999}, month = {1999 Nov}, pages = {414-30}, abstract = {SoxS is the direct transcriptional activator of the member genes of the Escherichia coli superoxide regulon. At class I SoxS-dependent promoters, e.g. zwf and fpr, whose SoxS binding sites ({\textquoteright}soxbox{\textquoteright}) lie upstream of the -35 region of the promoter, activation requires the C-terminal domain of the RNA polymerase alpha-subunit, while at class II SoxS-dependent promoters, e.g. fumC and micF, whose binding sites overlap the -35 region, activation is independent of the alpha-CTD. To determine whether SoxS activation of its class I promoters shows the same helical phase-dependent spacing requirement as class I promoters activated by catabolite gene activator protein, we increased the 7 bp distance between the 20 bp zwf soxbox and the zwf -35 promoter hexamer by 5 bp and 11 bp, and we decreased the 15 bp distance between the 20 bp fpr soxbox and the fpr -35 promoter hexamer by the same amounts. In both cases, displacement of the binding site by a half or full turn of the DNA helix prevented transcriptional activation. With constructs containing the binding site of one gene fused to the promoter of the other, we demonstrated that the positional requirements are a function of the specific binding site, not the promoter. Supposing that opposite orientation of the SoxS binding site at the two promoters might account for the positional requirements, we placed the zwf and fpr soxboxes in the reverse orientation at the various positions upstream of the promoters and determined the effect of orientation on transcription activation. We found that reversing the orientation of the zwf binding site converts its positional requirement to that of the fpr binding site in its normal orientation, and vice versa. Analysis by molecular information theory of DNA sequences known to bind SoxS in vitro is consistent with the opposite orientation of the zwf and fpr soxboxes.}, keywords = {Bacterial Proteins, Base Sequence, Binding Sites, Escherichia coli, Escherichia coli Proteins, Molecular Sequence Data, Plasmids, Promoter Regions, Genetic, Sequence Analysis, DNA, Superoxides, Trans-Activators, Transcription Factors, Transcription, Genetic, Transcriptional Activation}, issn = {0950-382X}, author = {Wood, T I and Griffith, K L and Fawcett, W P and Jair, K W and Schneider, T D and Wolf, R E} } @article {831, title = {Replication fork assembly at recombination intermediates is required for bacterial growth.}, journal = {Proc Natl Acad Sci U S A}, volume = {96}, year = {1999}, month = {1999 Mar 30}, pages = {3552-5}, abstract = {PriA, a 3{\textquoteright} --> 5{\textquoteright} DNA helicase, directs assembly of a primosome on some bacteriophage and plasmid DNAs. Primosomes are multienzyme replication machines that contribute both the DNA-unwinding and Okazaki fragment-priming functions at the replication fork. The role of PriA in chromosomal replication is unclear. The phenotypes of priA null mutations suggest that the protein participates in replication restart at recombination intermediates. We show here that PriA promotes replication fork assembly at a D loop, an intermediate formed during initiation of homologous recombination. We also show that DnaC810, encoded by a naturally arising intergenic suppressor allele of the priA2::kan mutation, bypasses the need for PriA during replication fork assembly at D loops in vitro. These findings underscore the essentiality of replication fork restart at recombination intermediates under normal growth conditions in bacteria.}, keywords = {Bacteria, Bacteriophage phi X 174, Base Sequence, DNA Polymerase III, DNA Replication, DNA-Binding Proteins, Escherichia coli, Molecular Sequence Data, Oligodeoxyribonucleotides, Open Reading Frames, Recombination, Genetic, Replication Protein A, Templates, Genetic}, issn = {0027-8424}, author = {Liu, J and Xu, L and Sandler, S J and Marians, K J} } @article {625, title = {Recovery of humic-reducing bacteria from a diversity of environments.}, journal = {Appl Environ Microbiol}, volume = {64}, year = {1998}, month = {1998 Apr}, pages = {1504-9}, abstract = {To evaluate which microorganisms might be responsible for microbial reduction of humic substances in sedimentary environments, humic-reducing bacteria were isolated from a variety of sediment types. These included lake sediments, pristine and contaminated wetland sediments, and marine sediments. In each of the sediment types, all of the humic reducers recovered with acetate as the electron donor and the humic substance analog, 2,6-anthraquinone disulfonate (AQDS), as the electron acceptor were members of the family Geobacteraceae. This was true whether the AQDS-reducing bacteria were enriched prior to isolation on solid media or were recovered from the highest positive dilutions of sediments in liquid media. All of the isolates tested not only conserved energy to support growth from acetate oxidation coupled to AQDS reduction but also could oxidize acetate with highly purified soil humic acids as the sole electron acceptor. All of the isolates tested were also able to grow with Fe(III) serving as the sole electron acceptor. This is consistent with previous studies that have suggested that the capacity for Fe(III) reduction is a common feature of all members of the Geobacteraceae. These studies demonstrate that the potential for microbial humic substance reduction can be found in a wide variety of sediment types and suggest that Geobacteraceae species might be important humic-reducing organisms in sediments.}, keywords = {Acetic Acid, Anthraquinones, Base Sequence, DNA Primers, Electron Transport, Fresh Water, Gram-Negative Anaerobic Bacteria, Humic Substances, Iron, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, RNA, Bacterial, RNA, Ribosomal, 16S, Seawater, Sulfur-Reducing Bacteria, Water Microbiology}, issn = {0099-2240}, author = {Coates, J D and Ellis, D J and Blunt-Harris, E L and Gaw, C V and Roden, E E and Lovley, D R} } @article {795, title = {Heterogeneity of the internal transcribed spacer (ITS-2) region within individual deer ticks.}, journal = {Insect Mol Biol}, volume = {6}, year = {1997}, month = {1997 May}, pages = {123-9}, abstract = {To determine whether nuclear rDNA sequences provide a useful means for assessing the structure of populations of Ixodes ticks, we compared variability among copies of an internal transcribed spacer (ITS-2) sequence within individual ticks to the variability between ticks. At least 4\% of the nucleotides comprising this sequence vary among the copies present within individual ticks. ITS-2 diversity in each of two ticks is nearly half as great as that reported between ticks from geographically disparate populations. Because individual ticks retain ancestral polymorphism, ITS-2 variation does not accurately reflect descent relationships among these ticks. Sequencing single copies of PCR-amplified ITS-2 therefore does not permit assessment of the phylogenetic relationships among the I. ricinus-like ticks in eastern North America. We recommend caution in future analyses, and emphasize the importance of procedures designed to ensure that the many paralogous copies of the rDNA cistron have been sufficiently homogenized by concerted evolutionary processes. Such precautionary measures will make certain that phylogenetic trees based on these gene sequences reflect the phyletic relatedness of the biological species.}, keywords = {Animals, Base Sequence, Deer, DNA, DNA, Ribosomal, Genetic Heterogeneity, Ixodes, Molecular Sequence Data}, issn = {0962-1075}, author = {Rich, S M and Rosenthal, B M and Telford, S R and Spielman, A and Hartl, D L and Ayala, F J} } @article {633, title = {Isolation of Geobacter species from diverse sedimentary environments.}, journal = {Appl Environ Microbiol}, volume = {62}, year = {1996}, month = {1996 May}, pages = {1531-6}, abstract = {In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram-negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria. Unlike the type strain, Geobacter metallireducens, three of the five isolates could use H2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process.}, keywords = {Base Sequence, Ferric Compounds, Gram-Negative Anaerobic Bacteria, Molecular Sequence Data, Phylogeny, RNA, Bacterial, RNA, Ribosomal, 16S, Water Microbiology}, issn = {0099-2240}, author = {Coates, J D and Phillips, E J and Lonergan, D J and Jenter, H and Lovley, D R} } @article {796, title = {Mosaic structure of plasmids from natural populations of Escherichia coli.}, journal = {Genetics}, volume = {143}, year = {1996}, month = {1996 Jul}, pages = {1091-100}, abstract = {The distribution of plasmids related to the fertility factor F was examined in the ECOR reference collection of Escherichia coli. Probes specific for four F-related genes were isolated and used to survey the collection by DNA hybridization. To estimate the genetic diversity of genes in F-like plasmids, DNA sequences were obtained for four plasmid genes. The phylogenetic relationships among the plasmids in the ECOR strains is very different from that of the strains themselves. This finding supports the view that plasmid transfer has been frequent within and between the major groups of ECOR. Furthermore, the sequences indicate that recombination between genes in plasmids takes place at a considerably higher frequency than that observed for chromosomal genes. The plasmid genes, and by inference the plasmids themselves, are mosaic in structure with different regions acquired from different sources. Comparison of gene sequences from a variety of naturally occurring plasmids suggested a plausible donor of some of the recombinant regions as well as implicating a chi site in the mechanism of genetic exchange. The relatively high rate of recombination in F-plasmid genes suggests that conjugational gene transfer may play a greater role in bacterial population structure than previously appreciated.}, keywords = {Bacterial Proteins, Base Sequence, DNA Helicases, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Evolution, Molecular, Membrane Proteins, Molecular Sequence Data, Mosaicism, Nucleic Acid Hybridization, Phylogeny, Plasmids, Polymorphism, Genetic, Proteins, Recombination, Genetic, Repressor Proteins, RNA-Binding Proteins, Trans-Activators}, issn = {0016-6731}, author = {Boyd, E F and Hill, C W and Rich, S M and Hartl, D L} } @article {797, title = {A new Borrelia infecting Lone Star ticks.}, journal = {Lancet}, volume = {347}, year = {1996}, month = {1996 Jan 6}, pages = {67-8}, keywords = {Animals, Base Sequence, Borrelia, Borrelia Infections, Deer, Disease Vectors, DNA Primers, Maryland, Molecular Sequence Data, Ticks}, issn = {0140-6736}, author = {Armstrong, P M and Rich, S M and Smith, R D and Hartl, D L and Spielman, A and Telford, S R} } @article {634, title = {Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria.}, journal = {J Bacteriol}, volume = {178}, year = {1996}, month = {1996 Apr}, pages = {2402-8}, abstract = {Evolutionary relationships among strictly anaerobic dissimilatory Fe(III)-reducing bacteria obtained from a diversity of sedimentary environments were examined by phylogenetic analysis of 16S rRNA gene sequences. Members of the genera Geobacter, Desulfuromonas, Pelobacter, and Desulfuromusa formed a monophyletic group within the delta subdivision of the class Proteobacteria. On the basis of their common ancestry and the shared ability to reduce Fe(III) and/or S0, we propose that this group be considered a single family, Geobacteraceae. Bootstrap analysis, characteristic nucleotides, and higher-order secondary structures support the division of Geobacteraceae into two subgroups, designated the Geobacter and Desulfuromonas clusters. The genus Desulfuromusa and Pelobacter acidigallici make up a distinct branch within the Desulfuromonas cluster. Several members of the family Geobacteraceae, none of which reduce sulfate, were found to contain the target sequences of probes that have been previously used to define the distribution of sulfate-reducing bacteria and sulfate-reducing bacterium-like microorganisms. The recent isolations of Fe(III)-reducing microorganisms distributed throughout the domain Bacteria suggest that development of 16S rRNA probes that would specifically target all Fe(III) reducers may not be feasible. However, all of the evidence suggests that if a 16S rRNA sequence falls within the family Geobacteraceae, then the organism has the capacity for Fe(III) reduction. The suggestion, based on geological evidence, that Fe(III) reduction was the first globally significant process for oxidizing organic matter back to carbon dioxide is consistent with the finding that acetate-oxidizing Fe(III) reducers are phylogenetically diverse.}, keywords = {Bacteria, Anaerobic, Base Sequence, DNA, Ribosomal, Ferric Compounds, Iron, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Sequence Homology, Nucleic Acid}, issn = {0021-9193}, author = {Lonergan, D J and Jenter, H L and Coates, J D and Phillips, E J and Schmidt, T M and Lovley, D R} } @article {835, title = {recA-like genes from three archaean species with putative protein products similar to Rad51 and Dmc1 proteins of the yeast Saccharomyces cerevisiae.}, journal = {Nucleic Acids Res}, volume = {24}, year = {1996}, month = {1996 Jun 1}, pages = {2125-32}, abstract = {The process of homologous recombination has been documented in bacterial and eucaryotic organisms. The Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins are the archetypal members of two related families of proteins that play a central role in this process. Using the PCR process primed by degenerate oligonucleotides designed to encode regions of the proteins showing the greatest degree of identity, we examined DNA from three organisms of a third phylogenetically divergent group, Archaea, for sequences encoding proteins similar to RecA and Rad51. The archaeans examined were a hyperthermophilic acidophile, Sulfolobus sofataricus (Sso); a halophile, Haloferax volcanii (Hvo); and a hyperthermophilic piezophilic methanogen, Methanococcus jannaschii (Mja). The PCR generated DNA was used to clone a larger genomic DNA fragment containing an open reading frame (orf), that we refer to as the radA gene, for each of the three archaeans. As shown by amino acid sequence alignments, percent amino acid identities and phylogenetic analysis, the putative proteins encoded by all three are related to each other and to both the RecA and Rad51 families of proteins. The putative RadA proteins are more similar to the Rad51 family (approximately 40\% identity at the amino acid level) than to the RecA family (approximately 20\%). Conserved sequence motifs, putative tertiary structures and phylogenetic analysis implied by the alignment are discussed. The 5{\textquoteright} ends of mRNA transcripts to the Sso radA were mapped. The levels of radA mRNA do not increase after treatment with UV irradiation as do recA and RAD51 transcripts in E.coli and S.cerevisiae. Hence it is likely that radA in this organism is a constitutively expressed gene and we discuss possible implications of the lack of UV-inducibility.}, keywords = {Amino Acid Sequence, Archaea, Base Sequence, Cell Cycle Proteins, DNA-Binding Proteins, Escherichia coli, Halobacteriaceae, Methanococcus, Models, Molecular, Molecular Sequence Data, Phylogeny, Rad51 Recombinase, Rec A Recombinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Alignment, Sulfolobus}, issn = {0305-1048}, author = {Sandler, S J and Satin, L H and Samra, H S and Clark, A J} } @article {798, title = {Discriminating between Ixodes ticks by means of mitochondrial DNA sequences.}, journal = {Mol Phylogenet Evol}, volume = {4}, year = {1995}, month = {1995 Dec}, pages = {361-5}, abstract = {Ticks of the genus Ixodes have recently assumed prominence because they frequently serve as vectors of important zoonoses, including Lyme disease and babesiosis. The morphological characteristics that have been used in their identification often are ambiguous and are useful solely at a particular stage of development. Here we report the DNA sequence of the mitochondrially encoded 16S rRNA gene of nine different Ixodes ticks and an outgroup from another genus, Dermacentor. The sequences readily discriminate between these ticks. Samples of I. dammini from the northeastern and upper midwestern United States differ from southeastern I. scapularis at about 2\% of the nucleotides. This difference is about half that separating other members of the I. ricinus group of species, but exceeds typical levels of intraspecific variation. Two major clades exist within the I. ricinus complex. One includes I. cookei, I. hexagonus, and I. angustus. The other includes I. persulcatus, I. pacificus, I. muris, I. ricinus, I. scapularis, and I. dammini. We conclude that mtDNA sequences are useful for unravelling the systematics of these important vectors of human disease.}, keywords = {Animals, Arachnid Vectors, Base Sequence, Dermacentor, DNA Primers, DNA, Mitochondrial, Genes, Humans, Ixodes, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Species Specificity}, issn = {1055-7903}, doi = {10.1006/mpev.1995.1033}, author = {Caporale, D A and Rich, S M and Spielman, A and Telford, S R and Kocher, T D} } @article {799, title = {Distribution of the Ixodes ricinus-like ticks of eastern North America.}, journal = {Proc Natl Acad Sci U S A}, volume = {92}, year = {1995}, month = {1995 Jul 3}, pages = {6284-8}, abstract = {We analyzed the geographic distribution of the Ixodes ricinus-like ticks in eastern North America by comparing the mitochondrial 16S rDNA sequences of specimens sampled directly from the field during the 1990s. Two distinct lineages are evident. The southern clade includes ticks from the southeastern and middle-eastern regions of the United States. The range of the northern clade, which appears to have been restricted to the northeastern region until the mid-1900s, now extends throughout the northeastern and middle-eastern regions. These phyletic units correspond to northern and southern taxa that have previously been assigned specific status as Ixodes dammini and Ixodes scapularis, respectively. The expanding range of I. dammini appears to drive the present outbreaks of zoonotic disease in eastern North America that include Lyme disease and human babesiosis.}, keywords = {Animals, Base Sequence, Demography, DNA Primers, DNA, Mitochondrial, DNA, Ribosomal, Geography, Mitochondria, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Population, RNA, Ribosomal, 16S, Ticks, United States}, issn = {0027-8424}, author = {Rich, S M and Caporale, D A and Telford, S R and Kocher, T D and Hartl, D L and Spielman, A} } @article {639, title = {Fe(III) and S0 reduction by Pelobacter carbinolicus.}, journal = {Appl Environ Microbiol}, volume = {61}, year = {1995}, month = {1995 Jun}, pages = {2132-8}, abstract = {There is a close phylogenetic relationship between Pelobacter species and members of the genera Desulfuromonas and Geobacter, and yet there has been a perplexing lack of physiological similarities. Pelobacter species have been considered to have a fermentative metabolism. In contrast, Desulfuromonas and Geobacter species have a respiratory metabolism with Fe(III) serving as the common terminal electron acceptor in all species. However, the ability of Pelobacter species to reduce Fe(III) had not been previously evaluated. When a culture of Pelobacter carbinolicus that had grown by fermentation of 2,3-butanediol was inoculated into the same medium supplemented with Fe(III), the Fe(III) was reduced. There was less accumulation of ethanol and more production of acetate in the presence of Fe(III). P. carbinolicus grew with ethanol as the sole electron donor and Fe(III) as the sole electron acceptor. Ethanol was metabolized to acetate. Growth was also possible on Fe(III) with the oxidation of propanol to propionate or butanol to butyrate if acetate was provided as a carbon source. P. carbinolicus appears capable of conserving energy to support growth from Fe(III) respiration as it also grew with H2 or formate as the electron donor and Fe(III) as the electron acceptor. Once adapted to Fe(III) reduction, P. carbinolicus could also grow on ethanol or H2 with S0 as the electron acceptor. P. carbinolicus did not contain detectable concentrations of the c-type cytochromes that previous studies have suggested are involved in electron transport to Fe(III) in other organisms that conserve energy to support growth from Fe(III) reduction.(ABSTRACT TRUNCATED AT 250 WORDS)}, keywords = {Bacteria, Anaerobic, Base Sequence, Butylene Glycols, Culture Media, Ethanol, Ferrous Compounds, Hydrogen, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Sulfur}, issn = {0099-2240}, author = {Lovley, D R and Phillips, E J and Lonergan, D J and Widman, P K} } @article {642, title = {Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism.}, journal = {Appl Environ Microbiol}, volume = {60}, year = {1994}, month = {1994 Oct}, pages = {3752-9}, abstract = {A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PPi, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutyrate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metallireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter sulfurreducens, is proposed.}, keywords = {Acetic Acid, Acetic Acids, Base Sequence, DNA Primers, DNA, Bacterial, DNA, Ribosomal, Electron Transport, Gram-Negative Anaerobic Bacteria, Hydrogen, Metals, Microscopy, Electron, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Bacterial, RNA, Ribosomal, 16S, Soil Microbiology}, issn = {0099-2240}, author = {Caccavo, F and Lonergan, D J and Lovley, D R and Davis, M and Stolz, J F and McInerney, M J} } @article {840, title = {Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression.}, journal = {J Bacteriol}, volume = {176}, year = {1994}, month = {1994 Jul}, pages = {4011-6}, abstract = {The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF. In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence. Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation. When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally. We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible. In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.}, keywords = {Bacterial Proteins, Base Sequence, DNA Mutational Analysis, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Half-Life, Lac Operon, Molecular Sequence Data, Protein Biosynthesis, Recombinant Fusion Proteins, Regulatory Sequences, Nucleic Acid, Ribosomes}, issn = {0021-9193}, author = {Sandler, S J and Clark, A J} } @article {841, title = {RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.}, journal = {J Bacteriol}, volume = {176}, year = {1994}, month = {1994 Jun}, pages = {3661-72}, abstract = {The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.}, keywords = {Bacterial Proteins, Base Sequence, DNA Damage, DNA Repair, DNA-Binding Proteins, Epistasis, Genetic, Escherichia coli, Escherichia coli Proteins, Models, Genetic, Molecular Sequence Data, Phenotype, Plasmids, SOS Response (Genetics), Suppression, Genetic, Ultraviolet Rays}, issn = {0021-9193}, author = {Sandler, S J and Clark, A J} } @article {839, title = {Studies on the mechanism of reduction of UV-inducible sulAp expression by recF overexpression in Escherichia coli K-12.}, journal = {Mol Gen Genet}, volume = {245}, year = {1994}, month = {1994 Dec 15}, pages = {741-9}, abstract = {UV-inducible sulAp expression, an indicator of the SOS response, is reduced by recF+ overexpression in vivo. Different DNA-damaging agents and amounts of RecO and RecR were tested for their effects on this phenotype. It was found that recF+ overexpression reduced sulAp expression after DNA damage by mitomycin C or nalidixic acid, recO+ and recR+ overexpression partially suppressed the reduction of UV-induced sulAp expression caused by recF+ overexpression. The requirement for ATP binding to RecF to produce the phenotype was tested by genetically altering the putative phosphate binding cleft of recF in a way that should prevent the mutant recF protein from binding ATP. It was found that a change of lysine to glutamine at codon 36 results in a mutant recF protein (RecF4115) that is unable to reduce UV-inducible sulAp expression when overproduced. It is inferred from these results that recF overexpression may reduce UV-inducible sulAp expression by a mechanism that is sensitive to the ability of RecF to bind ATP and to the levels of RecO and RecR (RecOR) in the cell, but not to the type of DNA damage per se. Models are explored that can explain how recF+ overexpression reduces UV induction of sulAp and how RecOR overproduction might suppress this phenotype.}, keywords = {Bacterial Proteins, Base Sequence, DNA Primers, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Mutagenesis, Site-Directed, Restriction Mapping, RNA, Messenger, SOS Response (Genetics), Structure-Activity Relationship, Ultraviolet Rays}, issn = {0026-8925}, author = {Sandler, S J} } @article {392, title = {Characterization of cDNA clones for the 2-methyl branched-chain enoyl-CoA reductase. An enzyme involved in branched-chain fatty acid synthesis in anaerobic mitochondria of the parasitic nematode Ascaris suum.}, journal = {J Biol Chem}, volume = {268}, year = {1993}, month = {1993 Oct 25}, pages = {22391-6}, abstract = {The 2-methyl branched-chain enoyl-CoA reductase plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode Ascaris suum. An affinity-purified polyclonal anti-serum against the reductase was used to screen a cDNA library constructed in lambda gt11 with poly(A)+ RNA from adult A. suum muscle. A 1.2-kilobase partial cDNA clone was isolated, subcloned, and sequenced in both directions. Additional sequence at the 5{\textquoteright} end of the mRNA was determined by the RACE (rapid amplification of cDNA ends) procedure. Nucleotide sequence analysis of the cDNAs revealed the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1236 nucleotides and a 3{\textquoteright}-untranslated sequence of 109 nucleotides including a short poly(A) tail 14 nucleotides from a polyadenylation signal (AATAAA). The open reading frame encoded a 396-amino acid sequence (M(r) 43,046) including a 16-amino acid leader peptide. Two-dimensional gel electrophoresis of the purified reductase yielded multiple spots with two distinct but overlapping amino-terminal amino acid sequences. Both sequences overlapped with the sequence predicted from the mRNA, and one of the sequences was identical to the predicted sequence. Comparison of the ascarid sequence with that of mammalian acyl-CoA dehydrogenases revealed a high degree of sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene even though the ascarid enzyme functions as a reductase, not as a dehydrogenase. Immunoblotting of A. suum larval stages and adult tissues with antisera that cross-reacted with each of the spots separated on two-dimensional gels suggested that the reductase was only found in adult muscle. Northern blotting using the partial cDNA revealed a hybridization band of about 1.5 kilobases and also suggested that the enzyme was tissue-specific and developmentally regulated in agreement with the results of the immunoblotting.}, keywords = {Amino Acid Sequence, Anaerobiosis, Animals, Ascaris suum, Base Sequence, Cloning, Molecular, Consensus Sequence, DNA, DNA Primers, DNA, Complementary, Fatty Acid Desaturases, Gene Library, Humans, Mitochondria, Molecular Sequence Data, Oligonucleotides, Antisense, Oxidoreductases, Oxidoreductases Acting on CH-CH Group Donors, Poly A, RNA, RNA, Messenger, Sequence Homology, Amino Acid}, issn = {0021-9258}, author = {Duran, E and Komuniecki, R W and Komuniecki, P R and Wheelock, M J and Klingbeil, M M and Ma, Y C and Johnson, K R} } @article {843, title = {Use of high and low level overexpression plasmids to test mutant alleles of the recF gene of Escherichia coli K-12 for partial activity.}, journal = {Genetics}, volume = {135}, year = {1993}, month = {1993 Nov}, pages = {643-54}, abstract = {We showed that sufficient overexpression of the wild-type recF gene interfered with three normal cell functions: (1) UV induction of transcription from the LexA-protein-repressed sulA promoter, (2) UV resistance and (3) cell viability at 42 degrees. To show this, we altered a low-level overexpressing recF+ plasmid with a set of structurally neutral mutations that increased the rate of expression of recF. The resulting high-level overexpressing plasmid interfered with UV induction of the sulA promoter, as did the low-level overexpressing plasmid. It also reduced UV resistance more than its low level progenitor and decreased viability at 42 degrees, an effect not seen with the low-level plasmid. We used the high-level plasmid to test four recF structural mutations for residual activity. The structural alleles consisted of an insertion mutation, two single amino acid substitution mutations and a double amino acid substitution mutation. On the Escherichia coli chromosome the three substitution mutations acted similarly to a recF deletion in reducing UV resistance in a recB21 recC22 sbcB15 sbcC201 genetic background. By this test, therefore, all three appeared to be null alleles. Measurements of conjugational recombination revealed, however, that the three substitution mutations may have residual activity. On the high-level overexpressing plasmid all three substitution mutations definitely showed partial activity. By contrast, the insertion mutation on the high-level overexpressing plasmid showed no partial activity and can be considered a true null mutation. One of the substitutions, recF143, showed a property attributable to a leaky mutation. Another substitution, recF4101, may block selectively two of the three interference phenotypes, thus allowing us to infer a mechanism for them.}, keywords = {Alleles, Bacterial Proteins, Base Sequence, Chromosomes, Bacterial, DNA Repair, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression, Genes, Bacterial, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Phenotype, Plasmids, Radiation Tolerance, Recombination, Genetic, Ultraviolet Rays}, issn = {0016-6731}, author = {Sandler, S J and Clark, A J} } @article {844, title = {Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.}, journal = {Nucleic Acids Res}, volume = {20}, year = {1992}, month = {1992 Feb 25}, pages = {839-45}, abstract = {We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92\% and 38\% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20\% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations.}, keywords = {Amino Acid Sequence, Bacillus subtilis, Bacterial Proteins, Base Sequence, Binding Sites, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Genetic Complementation Test, Molecular Sequence Data, Nucleic Acid Conformation, Phosphates, Pseudomonas putida, Salmonella typhimurium, Sequence Alignment}, issn = {0305-1048}, author = {Sandler, S J and Chackerian, B and Li, J T and Clark, A J} } @article {845, title = {Factors affecting expression of the recF gene of Escherichia coli K-12.}, journal = {Gene}, volume = {86}, year = {1990}, month = {1990 Jan 31}, pages = {35-43}, abstract = {This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control. The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95\% of the dnaA gene and lambda bet, N (double amber) and 40\% of the exo gene. When most of this DNA was deleted, RecF became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.}, keywords = {Bacterial Proteins, Base Sequence, Cloning, Molecular, Escherichia coli, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Peptide Chain Initiation, Translational, Recombinant Fusion Proteins, Recombination, Genetic, Transcription, Genetic}, issn = {0378-1119}, author = {Sandler, S J and Clark, A J} } @article {846, title = {Molecular analysis of the recF gene of Escherichia coli.}, journal = {Proc Natl Acad Sci U S A}, volume = {81}, year = {1984}, month = {1984 Aug}, pages = {4622-6}, abstract = {We analyzed the nucleotide sequence of a 1.325-kilobase region of wild-type Escherichia coli containing a functional recF gene and six Tn3 mutations that inactivate recF. The analysis shows a potentially translatable reading frame of 1071 nucleotides, which is interrupted by all six insertions. A protein of 40.5 kilodaltons would result from translation of the open reading frame, and a radioactive band of protein of an apparent molecular weight of approximately 40 kilodaltons was seen by the maxicell method using a recF+ plasmid. Putative truncated peptides were seen when two recF::Tn3 mutant plasmids were used. Differential expression of dnaN and recF from a common promoter was noted. recF332::Tn3 was transferred to the chromosome where, in hemizygous condition, it produced UV sensitivity indistinguishable from that produced by two presumed recF point mutations.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, DNA Repair, DNA Replication, Escherichia coli, Genes, Bacterial, Molecular Weight, Mutation, Plasmids, Recombination, Genetic}, issn = {0027-8424}, author = {Blanar, M A and Sandler, S J and Armengod, M E and Ream, L W and Clark, A J} }