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Surface Loop Motion in FepA. Daniel C. Scott, 2002.Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops of Escherichia coli FepA during its transport of the siderophore ferric enterobactin . Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein . The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2 . Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332 . These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 Å, from a ligand-free open state to a ligand-bound closed state . The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide . Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length. Koichi Tamano, 2002.The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion . The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ . In S . flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated . Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity . When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm . Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length . The spa32 mutant overexpressing MxiH produced extremely long (>5 µm) needles . Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system . The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells . Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells . These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp . and is involved in the control of needle length . Furthermore, its function is interchangeable with that of Salmonella InvJ . Efflux of Cytoplasmically Acting Antibiotics from Gram-Negative Bacteria: Periplasmic Substrate Capture by Multicomponent Efflux Pumps Inferred from Their Cooperative Action with Single-Component Transporters. Michael Palmer, 2003.In gram-negative bacteria, coexpression of single- and multicomponent efflux pumps may result in multiplicative enhancement of the level of resistance against cytoplasmically acting antibiotics . Here, a simple model is presented to show that this cooperative effect can be accounted for only if substrate capture by the multicomponent efflux transporter occurs in the periplasm but not the cytosol .
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