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Spa32 Regulates a Switch in Substrate Specificity of the Type III Secreton of Shigella flexneri from Needle Components to Ipa Proteins. Juana Magdalena, 2002.Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes . The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid . By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb . In the present study, we generated a Shigella spa32 mutant and studied its phenotype . The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK . The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium . However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro . Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction . Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type . In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH . Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH . Chloride, a New Environmental Signal Molecule Involved in Gene Regulation in a Moderately Halophilic Bacterium, Halobacillus halophilus. Markus Roeßler, 2002.The gram-positive, aerobic, moderately halophilic bacterium Halobacillus halophilus is challenged in its environment by frequently changing salt (NaCl) concentrations . Recently, H . halophilus was shown to be the first prokaryote that is dependent on Cl- for growth . In a search for the biological function of Cl- in this prokaryote, we identified different Cl--dependent processes, which suggests a more general role for Cl- in the metabolism of H . halophilus . To analyze the effect of Cl- in more detail, we concentrated on one model system, the Cl--dependent production of flagella, and aimed to identify the molecular basis for the Cl- dependence of flagellum production . Here, we report that synthesis of the major subunit of the flagellum, FliC, is dependent on the Cl- concentration of the medium, as determined by Western blot analyses . The gene encoding FliC was cloned and sequenced, and Northern blot as well as reverse transcriptase PCR analyses revealed that expression of fliC is Cl- dependent . FliC is the first protein of known function demonstrated to be synthesized in a Cl--dependent manner in a prokaryote . Two-dimensional gel electrophoresis of cells grown under different conditions revealed five more Cl--induced proteins; these were identified by N-terminal sequencing and database searches to be orthologs of proteins involved in stress response in Bacillus subtilis . The data indicate that Cl- is an important environmental signal in this moderate halophile and regulates protein synthesis and gene expression . Furthermore, the data may suggest that Cl- plays a role in the signal transduction involved in salt perception by this bacterium .
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