Mutations in the genes for synthesis of the outer core region of the lipopolysaccharide of Yersinia enterocolitica O:3

被引:3
|
作者
Sirisena, DM [1 ]
Skurnik, M [1 ]
机构
[1] Univ Turku, Inst Biomed, Dept Med Biochem & Mol Biol, Turku, Finland
关键词
allelic exchange; frame-shift mutations; lipopolysaccharide; outer core; Yersinia enterocolitica O : 3;
D O I
10.1046/j.1365-2672.2003.01897.x
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Aims: The aim of this study was to construct non-polar frame-shift mutations in some of the individual genes responsible for the biosynthesis of the branching outer core (OC) hexasaccharide of the lipopolysaccharide (LPS) in Yersinia enterocolitica O:3 (YeO:3). Methods and Results: Chromosomal segments of YeO:3 containing wbcN, wbcO and wbcQ genes were cloned into a suicide vector. A frame-shift mutation was introduced into each gene by modifying a unique restriction enzyme recognition site. Each recombinant plasmid with a modified OC gene was mobilized into YeO:3 to allow for allelic exchange between the modified gene and the wild type chromosomal gene. The exchange was confirmed by demonstrating the absence of the particular restriction site in the chromosome of each mutant strain. Analysis of LPS by gel electrophoresis showed that the LPS of the mutants was lacking the OC. Therefore, the constructed wbcN, wbcO and wbcQ strains are true mutants with frame-shifts in the corresponding genes. Conclusions: The products of the wbcN, wbcO and wbcQ genes are putative glycosyltransferases and, based on the present analysis, essential for the biosynthesis of the OC hexasaccharide. The absence of OC in the LPS of these mutants further supports the hypothesis that the OC hexasaccharide is a single O-antigen O-unit that is not polymerized in YeO:3. Significance and Impact of the Study: These mutants provide information on the unique nature of the synthesis of OC of YeO:3 LPS. They are valuable for future biochemical studies to establish the roles of the products of individual OC genes.
引用
收藏
页码:686 / 692
页数:7
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