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Analysis of Differences in the Functional Properties of the Substrate Binding Proteins of the Borrelia burgdorferi Oligopeptide Permease (opp) Operon. Xing-Guo Wang, 2004.The Borrelia burgdorferi genome encodes five orthologues of the substrate binding protein oligopeptide permease A (OppA) . It was previously shown that these genes are under the control of separate promoters and are differentially expressed under various environmental conditions . We were interested in determining whether there are also differences in substrate specificities among the proteins . The substrate specificities of recombinant proteins were determined by screening for high-affinity peptides by use of a combinatorial phage display heptapeptide library . Different heptapeptides with high affinities for OppA-1, OppA-2, and OppA-3 were identified . No heptapeptide binding OppA-4 or OppA-5 could be identified . Competitive binding assays were performed under various conditions to determine the substrate preferences of the OppA proteins . OppA-1 retained maximal activity over a broad range of pHs (5.5 to 7.5), whereas OppA-2 and OppA-3 showed peak activities at pHs below 5.5 . OppA-1 and OppA-2 showed preferences for tripeptides over dipeptides and longer-chain peptides . Although a wide variety of amino acyl side chains were tolerated by all three OppA proteins, OppA-1 showed the broadest substrate specificity and was able to accommodate peptides composed of bulky hydrophobic residues; OppA-2 and OppA-3 showed preferences for peptides composed of small nonpolar amino acids . All three OppA proteins showed preferences for peptides composed of L- rather than D-amino acids . OppA-3 showed the greatest tolerance for changes in stereochemistry . Substantial differences in the substrate specificities of the OppA proteins of B . burgdorferi suggest that they may have distinct functions in the organism . Endotoxin of Neisseria meningitidis Composed Only of Intact Lipid A: Inactivation of the Meningococcal 3-Deoxy-D-Manno-Octulosonic Acid Transferase. Yih-Ling Tzeng, 2002.Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria . A necessary step in endotoxin biosynthesis is 3-deoxy-D-manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA) . In enteric gram-negative bacteria, this step is essential for survival . A nonpolar kdtA::aphA-3 mutation was created in Neisseria meningitidis via allelic exchange, and the mutant was viable . Detailed structural analysis demonstrated that the endotoxin of the kdtA::aphA-3 mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation . In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IVA did not accumulate . The LOS structure of the kdtA::aphA-3 mutant was restored to the wild-type structure by complementation with kdtA from N . meningitidis or Escherichia coli . The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in N . meningitidis can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus .
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