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The Sorbitol Phosphotransferase System Is Responsible for Transport of 2-C-Methyl-D-Erythritol into Salmonella enterica Serovar Typhimurium. Charles A. Testa, 2004.2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate . Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate . In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated . We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter . Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell . RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-D-erythritol . Ubiquinone-8 was isolated from these cells and analyzed for deuterium content . Efficient incorporation of deuterium was observed for RMC26 . However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants . Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936). Takahiro Hirata, 2004.The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined . Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain . In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 µM did not cause proton translocation . This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity . These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant . Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor . The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold . Tigecycline inhibited active ethidium bromide efflux from intact E . coli cells overproducing AcrAB . Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters . A Novel Outer Membrane Protein, Wzi, Is Involved in Surface Assembly of the Escherichia coli K30 Group 1 Capsule. Andrea Rahn, 2003.Escherichia coli group 1 K antigens form a tightly associated capsule structure on the cell surface . Although the general features of the early steps in capsular polysaccharide biosynthesis have been described, little is known about the later stages that culminate in assembly of a capsular structure on the cell surface . Group 1 capsule biosynthesis gene clusters (cps) in E . coli and Klebsiella pneumoniae include a conserved open reading frame, wzi . The wzi gene is the first of a block of four conserved genes (wzi-wza-wzb-wzc) found in all group 1 K-antigen serotypes . Unlike wza, wzb, and wzc homologs that are found in gene clusters responsible for production of exopolysaccharides (i.e., predominantly cell-free polymer) in a range of bacteria, wzi is found only in systems that assemble capsular polysaccharides . The predicted Wzi protein shows no similarity to any other known proteins in the databases, but computer analysis of Wzi predicted a cleavable signal sequence . Wzi was expressed with a C-terminal hexahistidine tag, purified, and used for the production of specific antibodies that facilitated localization of Wzi to the outer membrane . Circular dichroism spectroscopy indicates that Wzi consists primarily of a ß-barrel structure, and dynamic light scattering studies established that the protein behaves as a monomer in solution . A nonpolar wzi chromosomal mutant retained a mucoid phenotype and remained sensitive to lysis by a K30-specific bacteriophage . However, the mutant showed a significant reduction in cell-bound polymer, with a corresponding increase in cell-free material . Furthermore, examination of the mutant by electron microscopy showed that it lacked a coherent capsule structure . It is proposed that the Wzi protein plays a late role in capsule assembly, perhaps in the process that links high-molecular-weight capsule to the cell surface .
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