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Remarkable Increase in Central Japan in 2001-2002 of Neisseria gonorrhoeae Isolates with Decreased Susceptibility to Penicillin, Tetracycline, Oral Cephalosporins, and Fluoroquinolones. Masayasu Ito, 2004.Four hundred sixty-two clinical isolates of Neisseria gonorrhoeae recovered from 1999 through 2002 in central Japan were examined for MICs of antimicrobial agents . The majority was sensitive to ceftriaxone and spectinomycin, but a remarkable increase in isolates with decreased susceptibility to penicillin, tetracycline, oral cephalosporins, and fluoroquinolones was observed from 2001 through 2002 . Genetic Variation at the O-Antigen Biosynthetic Locus in Pseudomonas aeruginosa. Christopher K. Raymond, 2002.The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described . Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus . We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains . DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region . Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis . The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome . The overall pattern underscores the plasticity of the P . aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences . Biochemical Properties of Neisseria gonorrhoeae LgtE. Andrzej Piekarowicz, 2002.A fragment of chromosomal DNA encoding the lgtE gene of Neisseria gonorrhoeae strain F62 was amplified by PCR and cloned into the expression vector pET15b . Functional LgtE was purified and its biochemical properties were determined . The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations . LgtE was only able to mediate the addition of UDP-galactose into neisserial lipooligosaccharides (LOSs) . We used a variety of genetically defined and chemically verified LOS structures to determine acceptor specificity . LgtE was able to mediate the addition of galactose into a variety of LOS structures, indicating the this enzyme possesses broad acceptor specificity . Furthermore, it was able to add multiple galactose residues onto LOS . We also determined that this enzyme was capable of adding galactose onto both the  and ß chains of neisserial LOS .
Striking Complexity of Lipopolysaccharide Defects in a Collection of Sinorhizobium meliloti Mutants. Gordon R. O. Campbell, 2003.Although the role that lipopolysaccharide (LPS) plays in the symbiosis between Sinorhizobium meliloti and alfalfa has been studied for over a decade, its function in this process remains controversial and poorly understood . This is largely due to a lack of mutants affected by its synthesis . In one of the definitive studies concerning this issue, Clover et al . (R . H . Clover, J . Kieber, and E . R . Signer, J . Bacteriol . 171:3961-3967, 1989) identified a series of mutants with putative LPS defects, judged them to be symbiotically proficient on Medicago sativa, and concluded that LPS might not have a symbiotic function in S . meliloti . The mutations in these strains were never characterized at the molecular level nor was the LPS from most of them analyzed . We have transduced these mutations from the Rm2011 background from which they were originally isolated into the sequenced strain Rm1021 and have characterized the resulting strains in greater detail . We found the LPS from these mutants to display a striking complexity of phenotypes on polyacrylamide electrophoresis gels, including additional rough LPS bands and alterations in the molecular weight distribution of the smooth LPS . We found that some of the mutants contain insertions in genes that are predicted to be involved in the synthesis of carbohydrate components of LPS, including ddhB, lpsB, lpsC, and lpsE . The majority, however, code for proteins predicted to be involved in a wide variety of functions not previously recognized to play a role in LPS synthesis, including a possible transcription elongation factor (GreA), a possible queuine synthesis protein, and a possible chemotaxis protein . Furthermore, using more extensive assays, we have found that most of these strains have symbiotic deficiencies . These results support more recent findings that alterations in LPS structure can affect the ability of S . meliloti to form an effective symbiosis . N-Linked Protein Glycosylation Is Required for Full Competence in Campylobacter jejuni 81-176. Joseph C. Larsen, 2004.The recent sequencing of the virulence plasmid of Campylobacter jejuni 81-176 revealed the presence of genes homologous to type IV secretion systems (TFSS) that have subsequently been found in Helicobacter pylori and Wolinella succinogenes . Mutational analyses of some of these genes have implicated their involvement in intestinal epithelial cell invasion and natural competence . In this report, we demonstrate that one of these type IV secretion homologs, Cjp3/VirB10, is a glycoprotein . Treatment with various glycosidases and binding to soybean agglutinin indicated that the structure of the glycan present on VirB10 contains a terminal GalNAc, consistent with previous reports of N-linked glycans in C . jejuni . Site-directed mutagenesis of five putative N-linked glycosylation sites indicated that VirB10 is glycosylated at two sites, N32 and N97 . Mutants in the N-linked general protein glycosylation (pgl) system of C . jejuni are significantly reduced in natural transformation, which is likely due, in part, to lack of glycosylation of VirB10 . The natural transformation defect in a virB10 mutant can be complemented in trans by using a plasmid expressing wild-type VirB10 or an N32A substitution but not by using a mutant expressing VirB10 with an N97A substitution . Taken together, these results suggest that glycosylation of VirB10 specifically at N97 is required for the function of the TFSS and for full competence in C . jejuni 81-176 . In Vivo Tracking of Campylobacter jejuni by Using a Novel Recombinant Expressing Green Fluorescent Protein. Philip F. Mixter, 2003.Campylobacter jejuni is a leading cause of food-borne disease in developed countries . The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C . jejuni in a variety of niches . C . jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry . Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C . jejuni harboring the gfp-containing vector . Four hours after injection of the mice, flow cytometry analyses determined that C . jejuni synthesizing GFP were predominantly associated with granulocytes . More specifically, the proportion of CD11b+ Gr-1+ lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b+ Gr-1- lavage macrophages ranged from 77.0 to 80.0% . In contrast, few CD11b- CD45R+ B lymphocytes from the lavage of the C . jejuni-injected mice were associated with green-fluorescent C . jejuni (proportions ranged from 0.75 to 0.77%) . Cell-free C . jejuni was recovered from tissue homogenates after intraperitoneal injection . Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C . jejuni F38011 wild-type isolate . In vivo this variant displayed a phenotype identical to that of the wild-type isolate . In summary, we demonstrate that C . jejuni associates with marker-defined cellular subsets in vivo with a novel gfp reporter system and that C . jejuni genotypic variants can be isolated from both in vitro and in vivo systems .
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