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Differences in Enzymatic Properties Allow SodCI but Not SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium Strain 14028. Radha Krishnakumar, 2004.Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII . While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background . Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth . By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter . In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter . Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes . In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome . We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment . Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added . The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is "tethered" within the periplasm by an apparently noncovalent interaction . We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium . Modified Serial Analysis of Gene Expression Method for Construction of Gene Expression Profiles of Microbial Eukaryotic Species. Kathryn J. Coyne, 2004.Serial analysis of gene expression (SAGE) is a powerful approach for the identification of differentially expressed genes, providing comprehensive and quantitative gene expression profiles in the form of short tag sequences . Each tag represents a unique transcript, and the relative frequencies of tags in the SAGE library are equal to the relative proportions of the transcripts they represent . One of the major obstacles in the preparation of SAGE libraries from microorganisms is the requirement for large amounts of starting material (i.e., mRNA) . Here, we present a novel approach for the construction of SAGE libraries from small quantities of total RNA by using Y linkers to selectively amplify 3' cDNA fragments . To validate this method, we constructed comprehensive gene expression profiles of the toxic dinoflagellate Pfiesteria shumwayae . SAGE libraries were constructed from an actively toxic fish-fed culture of P . shumwayae and from a recently toxic alga-fed culture . P . shumwayae-specific gene transcripts were identified by comparison of tag sequences in the two libraries . Representative tags with frequencies ranging from 0.026 to 3.3% of the total number of tags in the libraries were chosen for further analysis . Expression of each transcript was confirmed in separate control cultures of toxic P . shumwayae . The modified SAGE method described here produces gene expression profiles that appear to be both comprehensive and quantitative, and it is directly applicable to the study of gene expression in other environmentally relevant microbial species . Imbroglios of Viral Taxonomy: Genetic Exchange and Failings of Phenetic Approaches. Jeffrey G. Lawrence, 2002.The practice of classifying organisms into hierarchical groups originated with Aristotle and was codified into nearly immutable biological law by Linnaeus . The heart of taxonomy is the biological species, which forms the foundation for higher levels of classification . Whereas species have long been established among sexual eukaryotes, achieving a meaningful species concept for prokaryotes has been an onerous task and has proven exceedingly difficult for describing viruses and bacteriophages . Moreover, the assembly of viral "species" into higher-order taxonomic groupings has been even more tenuous, since these groupings were based initially on limited numbers of morphological features and more recently on overall genomic similarities . The wealth of nucleotide sequence information that catalyzed a revolution in the taxonomy of free-living organisms necessitates a reevaluation of the concept of viral species, genera, families, and higher levels of classification . Just as microbiologists discarded dubious morphological traits in favor of more accurate molecular yardsticks of evolutionary change, virologists can gain new insight into viral evolution through the rigorous analyses afforded by the molecular phylogenetics of viral genes . For bacteriophages, such dissections of genomic sequences reveal fundamental flaws in the Linnaean paradigm that necessitate a new view of viral evolution, classification, and taxonomy . Polynucleotide Phosphorylase-Deficient Mutants of Pseudomonas putida. Rebecca Favaro, 2003.In bacteria, polynucleotide phosphorylase (PNPase) is one of the main exonucleolytic activities involved in RNA turnover and is widely conserved . In spite of this, PNPase does not seem to be essential for growth if the organisms are not subjected to special conditions, such as low temperature . We identified the PNPase-encoding gene (pnp) of Pseudomonas putida and constructed deletion mutants that did not exhibit cold sensitivity . In addition, we found that the transcription pattern of pnp upon cold shock in P . putida was markedly different from that in Escherichia coli . It thus appears that pnp expression control and the physiological roles in the cold may be different in different bacterial species . Polymorphism in the Collagen-Like Region of the Bacillus anthracis BclA Protein Leads to Variation in Exosporium Filament Length. Patricia Sylvestre, 2003.We recently identified a Bacillus anthracis glycoprotein which is a structural constituent of the exosporium filaments (P . Sylvestre, E . Couture-Tosi, and M . Mock, Mol . Microbiol . 45:169-178, 2002) . This Bacillus collagen-like protein (BclA) contains an internal collagen-like region (CLR) of GXX repeats which includes a large proportion of GPT triplets . Here, we report that the polymorphic marker Ceb-Bams13, for which there are nine alleles (P . Le Flèche et al., BMC Microbiol . 1:2, 2001), maps within the open reading frame encoding BclA . The bclA gene in 11 B . anthracis strains representative of seven Ceb-Bams13 alleles was sequenced and compared to the Ames bclA gene sequence . The amino- and carboxy-terminal sequences surrounding the CLR are conserved . The CLR itself is highly polymorphic: it contains between 17 and 91 GXX repeats and one to eight copies of the 21-amino-acid sequence (GPT)5GDTGTT, named the BclA repeat . The length of the filament on the spore surface differed between the strains . We exchanged the bclA gene between strains with different CLRs and examined the spore surfaces by electron microscopy analysis . The length of the BclA CLR is responsible for the variation in filament length . Molecular, Serological, and Virulence Characteristics of Vibrio parahaemolyticus Isolated from Environmental, Food, and Clinical Sources in North America and Asia. Angelo DePaola, 2003.Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia . The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene . The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York . Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh . Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production . Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh . A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups . All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia . The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates . The combination of serotyping and ribotyping showed that the Pacific Coast V . parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast . The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease .
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