Marolda C L, Welsh J, Dafoe L, Valvano M A
Department of Microbiology and Immunology, Health Sciences Centre, University of Western Ontario, London, Canada.
J Bacteriol. 1990 Jul;172(7):3590-9. doi: 10.1128/jb.172.7.3590-3599.1990.
We recently cloned biosynthesis genes for the O7-lipopolysaccharide (O7-LPS) side chain from the Escherichia coli K-1 strain VW187 (M. A. Valvano, and J. H. Crosa, Infect. Immun. 57:937-943, 1989). To characterize the O7-LPS region, the recombinant cosmids pJHCV31 and pJHCV32 were mutagenized by transposon mutagenesis with Tn3HoHo1, which carries a promoterless lac operon and can therefore generate lacZ transcriptional fusions with target DNA sequences. Cells containing mutated plasmids were examined for their ability to react by coagglutination with O7 antiserum. The LPS pattern profiles of the insertion mutants were also investigated by electrophoresis of cell envelope fractions, followed by silver staining and immunoblotting analysis. These experiments identified three phenotypic classes of mutants and defined a region in the cloned DNA of about 14 kilobase pairs that is essential for O7-LPS expression. Analysis of beta-galactosidase production by cells carrying plasmids with transposon insertions indicated that transcription occurs in only one direction along the O7-LPS region. In vitro transcription-translation experiments revealed that the O7-LPS region encodes at least 16 polypeptides with molecular masses ranging from 20 to 48 kilodaltons. Also, the O7-LPS region in VW187 was mutagenized by homologous recombination with subsets of the cloned O7-LPS genes subcloned into a suicide plasmid vector. O7-LPS-deficient mutants of VW187 were complemented with pJHCV31 and pJHCV32, confirming that these cosmids contain genetic information that is essential for the expression of the O7 polysaccharide.
我们最近从大肠杆菌K-1菌株VW187中克隆了O7-脂多糖（O7-LPS）侧链的生物合成基因（M. A. 瓦尔瓦诺和J. H. 克罗萨，《感染与免疫》57:937 - 943，1989年）。为了表征O7-LPS区域，用携带无启动子乳糖操纵子的Tn3HoHo1通过转座子诱变对重组粘粒pJHCV31和pJHCV32进行诱变，因此它可以与靶DNA序列产生lacZ转录融合。检查含有突变质粒的细胞与O7抗血清通过凝聚反应的能力。还通过对细胞膜组分进行电泳，随后进行银染和免疫印迹分析来研究插入突变体的LPS模式图谱。这些实验确定了三类表型突变体，并在克隆的DNA中定义了一个约14千碱基对的区域，该区域对于O7-LPS表达至关重要。对携带带有转座子插入的质粒的细胞中β-半乳糖苷酶产生的分析表明，转录仅沿O7-LPS区域的一个方向发生。体外转录-翻译实验表明，O7-LPS区域编码至少16种分子量在20至48千道尔顿之间的多肽。此外，通过与亚克隆到自杀质粒载体中的克隆O7-LPS基因子集进行同源重组，对VW187中的O7-LPS区域进行诱变。VW187的O7-LPS缺陷型突变体用pJHCV31和pJHCV32进行互补，证实这些粘粒包含对O7多糖表达至关重要的遗传信息。
