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Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49. Carine Desplats, 2002.RB49 is a virulent bacteriophage that infects Escherichia coli . Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage . To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the  170-kb genome . Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis . However, when translated, about 70% of them encoded proteins with homology to T4 proteins . Among these sequences were the numerous components of the virion and the phage DNA replication apparatus . Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome . The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication . This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues . Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences . The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies . For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters . These results indicate that RB49 and T4 have diverged substantially from their last common ancestor . The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes .
Mechanism of ToxT-Dependent Transcriptional Activation at the Vibrio cholerae tcpA Promoter. Robin R. Hulbert, 2002.The AraC homolog ToxT coordinately regulates virulence gene expression in Vibrio cholerae . ToxT is required for transcriptional activation of the genes encoding cholera toxin and the toxin coregulated pilus, among others . In this work we focused on the interaction of ToxT with the tcpA promoter and investigated the mechanism of ToxT-dependent transcriptional activation at tcpA. Deletion analysis showed that a region from -95 to +2 was sufficient for ToxT binding and activation, both of which were simultaneously lost when the deletion was extended to -63 . A collection of point mutations generated by error-prone PCR revealed two small regions required for ToxT-dependent transactivation . Binding studies performed with representative mutations showed that the two regions define sites at which ToxT binds to the tcpA promoter region, most likely as a dimer . Results obtained by using a rpoA truncation mutation showed that ToxT-dependent activation at tcpA involves the C-terminal domain of the RNA polymerase alpha subunit . A model of ToxT-dependent transcriptional activation at tcpA is proposed, in which ToxT interacts with two A-rich regions of tcpA centered at -72 and -51 and requires the alpha C-terminal domain of RNA polymerase . Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27. Trevor D. Lawley, 2002.The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay . Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein . Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype . Conjugative transfer was restored for each transfer mutant by genetic complementation . An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT) . The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay . Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids . Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production . TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases . TraJ contains both an Arc repressor motif and a leucine zipper motif . A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer . This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system . Osmotic Adaptation of Thermus thermophilus RQ-1: Lesson from a Mutant Deficient in Synthesis of Trehalose. Zélia Silva, 2003.Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O . C . Nunes, C . M . Manaia, M . S . da Costa, and H . Santos, Appl . Environ . Microbiol . 61:2351-2357, 1995) . A 2.4-kbp DNA fragment from T . thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library . The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked . To understand the role of trehalose during salt stress in T . thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement . Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl . However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose . The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6 . The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media . In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities . The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T . thermophilus RQ-1 . Genomic Diversity and Relatedness of Bifidobacteria Isolated from a Porcine Cecum. P. J. Simpson, 2003.This study initially involved the isolation of a number of bifidobacteria from either the lumen or the epithelium of a porcine cecum . A total of 160 isolates were selected at random on MRS plates containing cysteine hydrochloride (0.5 g/liter) and mupirocin (50 mg/liter) . All were identified as bifidobacteria based on fructose-6-phosphate phosphoketolase activity . Following genomic digestion with the restriction enzyme XbaI and pulsed-field gel electrophoresis (PFGE), the isolates produced 15 distinct macro-restriction patterns . Several of the PFGE patterns differed by only 1, 2, or 3 DNA fragments and were grouped as related patterns into seven PFGE types, termed A through G . The related patterns appeared to show genomic plasticity within the isolates arising from chromosomal mutations or possibly horizontal transfer of plasmids . The relative frequency of each PFGE type was maintained within each cecal sample, with PFGE type E representing approximately 50% of the isolates . Randomly amplified polymorphic DNA PCR, cell morphology, whole-cell protein profiling, 16S ribosomal DNA sequencing, and DNA-DNA hybridization were used to determine if the seven apparently unrelated PFGE types represented genetically distinct isolates . Four groups were identified: PFGE types A, C/D/G, B/E, and F, and these appeared to represent Bifidobacterium minimum, Bifidobacterium pseudolongum subsp . pseudolongum, and Bifidobacterium pseudolongum subsp . globosum and two new species, respectively . The data demonstrate the presence of considerable genomic diversity within a relatively simple bifidobacteria population, consisting of 15 distinct strains representing four groups, which was maintained throughout the porcine cecal contents and epithelial layer . Identification of Lactobacillus reuteri Genes Specifically Induced in the Mouse Gastrointestinal Tract. Jens Walter, 2003. Microbial Ecology of an Extreme Acidic Environment, the Tinto River. E. González-Toril, 2003.The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals . The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt . Molecular ecology techniques were used to analyze the diversity of this microbial community . The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification . A good correlation between the two approaches was found . Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., "Ferrimicrobium acidiphilum," Ferroplasma acidiphilum, and Thermoplasma acidophilum . The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes . More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to Archaea . Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected . Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined . Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested .
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