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Characterization of Heme Uptake Cluster Genes in the Fish Pathogen Vibrio anguillarum. Susana Mouriño, 2004.Vibrio anguillarum can utilize hemin and hemoglobin as sole iron sources . In previous work we identified HuvA, the V . anguillarum outer membrane heme receptor by complementation of a heme utilization mutant with a cosmid clone [pML1] isolated from a genomic library of V . anguillarum . In the present study, we describe a gene cluster contained in cosmid pML1, coding for nine potential heme uptake and utilization proteins: HuvA, the heme receptor;HuvZ and HuvX; TonB, ExbB, and ExbD; HuvB, the putative periplasmic binding protein; HuvC, the putative inner membrane permease;and HuvD, the putative ABC transporter ATPase . A V . anguillarum strain with an in-frame chromosomal deletion of the nine-genecluster was impaired for growth with heme or hemoglobin as thesole iron source . Single-gene in-frame deletions were constructed, demonstrating that each of the huvAZBCD genes are essential for utilization of heme as an iron source in V . anguillarum, whereas huvX is not . When expressed in Escherichia coli hemA [strain EB53], a plasmid carrying the gene for the heme receptor, HuvA, was sufficient to allow the use of heme as the porphyrin source . For utilization of heme as an iron source in E . colient [strain 101ESD], the tonB exbBD and huvBCD genes were required in addition to huvA . The V . anguillarum heme uptake cluster shows some differences in gene arrangement when compared to homologous clusters described for other Vibrio species. Genetic Locus Encoding Functions Involved in Biosynthesis and Outer Membrane Localization of Xanthomonadin in Xanthomonas oryzae pv . oryzae. Ajay Kumar Goel, 2002.Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus Xanthomonas . We have characterized a genetic locus (pig) from Xanthomonas oryzae pv . oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production . Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis . The fourth ORF has no homologue in the database . For the first time, we report that a putative cytoplasmic membrane protein encoded in the pig locus is required for outer membrane localization of xanthomonadin in X . oryzae pv . oryzae . We also report the identification of a novel 145-bp palindromic Xanthomonas repetitive intergenic consensus element that is present in two places in the pig locus . We estimate that more than 100 copies of this element might be present in the genome of X . oryzae pv . oryzae and other xanthomonads . Functional Analysis of TraA, the Sex Pheromone Receptor Encoded by pPD1, in a Promoter Region Essential for the Mating Response in Enterococcus faecalis. Takaaki Horii, 2002.Conjugative transfer of a bacteriocin plasmid, pPD1, of Enterococcus faecalis is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells . cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA . Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells . In this study, we discuss how TraA transduces the signal of cPD1 to the mating response . Gel mobility shift assays indicated that TraA is bound to a traA-ipd intergenic region, which is essential for cPD1 response . DNase I footprinting analysis suggested the presence of one strong (tab1) and two weak (tab2 and tab3) TraA-binding sites in the intergenic region . Primer extension analysis implied that the transcriptional initiation sites of traA and ipd were located in the intergenic region . Northern analysis showed that cPD1 upregulated and downregulated transcription of ipd and traA, respectively . The circular permutation assay showed that TraA bent a DNA fragment corresponding to the tab1 region, and its angle was changed in the presence of cPD1 or iPD1 . From these data, we propose a model that TraA changes the conformation of the tab1 region in response to cPD1 and upregulates the transcription of ipd, which may lead to expression of genes required for the mating response . Membrane Interaction of the Glycosyltransferase MurG: a Special Role for Cardiolipin. Els van den Brink-van der Laan, 2003.MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis . The crystal structure of Escherichia coli MurG (S . Ha, D . Walker, Y . Shi, and S . Walker, Protein Sci . 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site . To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from E . coli in the absence of detergent . Surprisingly, we found that lipid vesicles copurify with MurG . Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression . This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell . The cardiolipin content of cells overexpressing MurG was increased from 1 ± 1 to 7 ± 1 mol% compared to nonoverexpressing cells . The lipids that copurify with MurG were even further enriched in cardiolipin (13 ± 4 mol%) . MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol . These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane . In addition, the results show a special role for cardiolipin in the MurG-membrane interaction . Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His  P) and HPr(Ser-P)(HisP) and Effects on Growth.Christian Lessard, 2003.The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism . S . thermophilus releases galactose into the medium during growth on lactose . Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS) . Unlike S . thermophilus, S . salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars . Analysis of the C-terminal end of S . salivarius LacS revealed a IIA-like domain (IIALacS) almost identical to the IIA domain of S . thermophilus LacS . Experiments performed with purified proteins showed that S . salivarius IIALacS was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(HisP) but also by HPr(Ser-P)(HisP), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S . salivarius cells . Two other major S . salivarius PTS proteins, IIABLMan and IIABHMan, were unable to phosphorylate IIALacS . The effect of LacS phosphorylation on growth was studied with strain G71, an S . salivarius enzyme I-negative mutant that cannot synthesize HPr(HisP) or HPr(Ser-P)(HisP) . These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose .
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