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Complete Sequence and Evolutionary Genomic Analysis of the Pseudomonas aeruginosa Transposable Bacteriophage D3112. Pauline W. Wang, 2004.Bacteriophage D3112 represents one of two distinct groups of transposable phage found in the clinically relevant, opportunistic pathogen Pseudomonas aeruginosa . To further our understanding of transposable phage in P . aeruginosa, we have sequenced the complete genome of D3112 . The genome is 37,611 bp, with an overall G+C content of 65% . We have identified 53 potential open reading frames, including three genes (the c repressor gene and early genes A and B) that have been previously characterized and sequenced . The organization of the putative coding regions corresponds to published genetic and transcriptional maps and is very similar to that of enterobacteriophage Mu . In contrast, the International Committee on Taxonomy of Viruses has classified D3112 as a  -like
phage on the basis of its morphology . Similarity-based analyses
identified 27 open reading frames with significant matches to
proteins in the NCBI databases . Forty-eight percent of these were
similar to Mu-like phage and prophage sequences, including proteins
responsible for transposition, transcriptional regulation, virion
morphogenesis, and capsid formation . The tail proteins were highly
similar to prophage sequences in Escherichia coli and phage
Phi12 from Staphylococcus aureus, while proteins at the right
end were highly similar to proteins in Xylella fastidiosa . We
performed phylogenetic analyses to understand the evolutionary
relationships of D3112 with respect to Mu-like versus
-like
bacteriophages . Different results were obtained from similarity-based
versus phylogenetic analyses in some instances . Overall, our
findings reveal a highly mosaic structure and suggest that extensive
horizontal exchange of genetic material played an important role in
the evolution of D3112 .
PCR-Based Identification of Hyperthermophilic Archaea of the Family Thermococcaceae. Galina B. Slobodkina, 2004. Temperature-Sensitive Growth and Decreased Thermotolerance Associated with relA Mutations in Escherichia coli. Xiaoming Yang, 2003.The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation . The stringent response is thought to be mediated by ppGpp . Mutations in relA are known to result in pleiotropic phenotypes . We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41°C . The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M . The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42°C, a temperature that is normally nonlethal . The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities . The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis . This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone . The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the ß subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations . Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance . Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters. Angelo DePaola, 2003.Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish . From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala . The presence and densities of V . parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V . parahaemolyticus, respectively . V . parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g-1 . Higher V . parahaemolyticus densities were associated with higher water temperatures . Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment . Forty-six of 6,018 and 31 of 6,992 V . parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe . There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains . Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene . The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected . The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh+ V . parahaemolyticus than previously reported .
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