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Bacterial Outer Membrane Ushers Contain Distinct Targeting and Assembly Domains for Pilus Biogenesis. David G. Thanassi, 2002.Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway . In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface . We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a ß-barrel with short extracellular loops and extensive periplasmic domains . Several periplasmic regions were localized, including two domains containing conserved cysteine pairs . Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding . The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo . Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo . These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus . Identification and Molecular Characterization of the Mg2+ Stimulon of Escherichia coli. Shu Minagawa, 2003.Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg2+ stimulon that respond to the availability of external Mg2+ in a PhoP/PhoQ two-component system-dependent manner . The mRNA levels of W3110 in the presence of 30 mM MgCl2, WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl2 . The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes . Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon . Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg2+ stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously . In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP . Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg2+ stimulon in E . coli .
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