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Natural Selection and Evolution of Streptococcal Virulence Genes Involved in Tissue-Specific Adaptations. Awdhesh Kalia, 2004.The molecular mechanisms underlying niche adaptation in bacteria are not fully understood . Primary infection by the pathogen group A streptococcus (GAS) takes place at either the throat or the skin of its human host, and GAS strains differ in tissue site preference . Many skin-tropic strains bind host plasminogen via the plasminogen-binding group A streptococcal M protein (PAM) present on the cell surface; inactivation of genes encoding either PAM or streptokinase (a plasminogen activator) leads to loss of virulence at the skin . Unlike PAM, which is present in only a subset of GAS strains, the gene encoding streptokinase (ska) is present in all GAS isolates . In this study, the evolution of the virulence genes known to be involved in skin infection was examined . Most genetic diversity within ska genes was localized to a region encoding the plasminogen-docking domain (ß-domain) . The gene encoding PAM displayed strong linkage disequilibrium (P << 0.01) with a distinct phylogenetic cluster of the ska ß-domain-encoding region . Yet, ska alleles of distant taxa showed a history of intragenic recombination, and high intrinsic levels of recombination were found among GAS strains having different tissue tropisms . The data suggest that tissue-specific adaptations arise from epistatic coselection of bacterial virulence genes . Additional analysis of ska genes showed that  4%
of the codons underwent strong diversifying selection . Horizontal
acquisition of one ska lineage from a commensal Streptococcus
donor species was also evident . Together, the data suggest that
new phenotypes can be acquired through interspecies recombination
between orthologous genes, while constrained functions can be
preserved; in this way, orthologous genes may provide a rich and
ready source for new phenotypes and thereby play a facilitating role
in the emergence of new niche adaptations in bacteria .
Lysostaphin-Coated Catheters Eradicate Staphylococccus aureus Challenge and Block Surface Colonization. Anjali Shah, 2004.Lysostaphin is an endopeptidase that kills Staphylococcus aureus, a predominant organism in catheter-related infections . Lysostaphin-coated catheters prevented catheter colonization by several strains of S . aureus, and activity was maintained for at least 4 days . Prophylactic use of lysostaphin in catheters may help prevent the occurrence of catheter-related staphylococcal infections . Molecular Profiling of Rhizosphere Microbial Communities Associated with Healthy and Diseased Black Spruce (Picea mariana) Seedlings Grown in a Nursery. M. Filion, 2004.Bacterial and fungal populations associated with the rhizosphere of healthy black spruce (Picea mariana) seedlings and seedlings with symptoms of root rot were characterized by cloned rRNA gene sequence analysis . Triplicate bacterial and fungal rRNA gene libraries were constructed, and 600 clones were analyzed by amplified ribosomal DNA restriction analysis and grouped into operational taxonomical units (OTUs) . A total of 84 different bacterial and 31 different fungal OTUs were obtained and sequenced . Phylogenetic analyses indicated that the different OTUs belonged to a wide range of bacterial and fungal taxa . For both groups, pairwise comparisons revealed that there was greater similarity between replicate libraries from each treatment than between libraries from different treatments . Significant differences between pooled triplicate samples from libraries of genes from healthy seedlings and pooled triplicate samples from libraries of genes from diseased seedlings were also obtained for both bacteria and fungi, clearly indicating that the rhizosphere-associated bacterial and fungal communities of healthy and diseased P . mariana seedlings were different . The communities associated with healthy and diseased seedlings also showed distinct ecological parameters as indicated by the calculated diversity, dominance, and evenness indices . Among the main differences observed at the community level, there was a higher proportion of Acidobacteria, Gammaproteobacteria, and Homobasidiomycetes clones associated with healthy seedlings, while the diseased-seedling rhizosphere harbored a higher proportion of Actinobacteria, Sordariomycetes, and environmental clones . The methodological approach described in this study appears promising for targeting potential rhizosphere-competent biological control agents against root rot diseases occurring in conifer nurseries . The SKHR Motif Is Required for Biological Function of the VirR Response Regulator from Clostridium perfringens. Sheena McGowan, 2003.The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens . The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins . VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain . Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein . Random mutagenesis identified a unique residue, Met-172, that was required for biological function . Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity . Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein . The Bacillus thuringiensis PlcR-Regulated Gene inhA2 Is Necessary, but Not Sufficient, for Virulence. Sinda Fedhila, 2003.We previously reported that Bacillus thuringiensis strain 407 Cry 32- secretes a zinc-requiring metalloprotease, InhA2, that is essential for virulence in orally infected insects . Analysis of the inhA2-lacZ transcriptional fusion showed that inhA2 expression is repressed in a PlcR- background . Using DNase I footprinting experiments, we demonstrated that PlcR activates inhA2 transcription directly by binding to a DNA sequence showing a one-residue mismatch with the previously reported PlcR box . It was previously reported that PlcR is essential for B . thuringiensis virulence in oral infection by contributing to the synergistic properties of the spores on the insecticidal activity of the Cry1C protein . We used complementation experiments to investigate whether the PlcR- phenotype was due to the absence of InhA2 . The results indicated that overexpression of inhA2 in the  plcR strain did not restore the wild-type phenotype . However, virulence was fully restored in the
inhA2 complemented mutant . Thus, inhA2 is the first example of a PlcR-regulated gene found to be directly involved in virulence . However, it is not sufficient for pathogenicity when the other members of the PlcR regulon are lacking . This suggests that InhA2 may act in concert with other PlcR-regulated gene products to provide virulence .
Bacterioplankton Community Shifts in an Arctic Lake Correlate with Seasonal Changes in Organic Matter Source. Byron C. Crump, 2003.Seasonal shifts in bacterioplankton community composition in Toolik Lake, a tundra lake on the North Slope of Alaska, were related to shifts in the source (terrestrial versus phytoplankton) and lability of dissolved organic matter (DOM) . A shift in community composition, measured by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes, occurred at 4°C in near-surface waters beneath seasonal ice and snow cover in spring . This shift was associated with an annual peak in bacterial productivity ([14C]leucine incorporation) driven by the large influx of labile terrestrial DOM associated with snow meltwater . A second shift occurred after the flux of terrestrial DOM had ended in early summer as ice left the lake and as the phytoplankton community developed . Bacterioplankton communities were composed of persistent populations present throughout the year and transient populations that appeared and disappeared . Most of the transient populations could be divided into those that were advected into the lake with terrestrial DOM in spring and those that grew up from low concentrations during the development of the phytoplankton community in early summer . Sequencing of DNA in DGGE bands demonstrated that most bands represented single ribotypes and that matching bands from different samples represented identical ribotypes . Bacteria were identified as members of globally distributed freshwater phylogenetic clusters within the  - and ß-Proteobacteria, the Cytophaga-Flavobacteria-Bacteroides group, and the Actinobacteria .
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