Cloning, complementation, and characterization of an rfaE homolog from Neisseria gonorrhoeae.
Cloning, complementation, and characterization of an rfaE homolog from Neisseria gonorrhoeae.
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淋病奈瑟氏菌 rfaE 同源物的克隆、互补和表征。
DOI：
10.1128/jb.178.15.4571-4575.1996
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发表时间：
1996
影响因子：
3.2
通讯作者：
Stein,DC
中科院分区：
文献类型：
--
作者：
Levin,JC;Stein,DC
淋病奈瑟菌WS1是淋病奈瑟菌FA19的一种自发产脓毒素（一种由铜绿假单胞菌产生的细菌素）抗性突变体，它产生一种截断的低脂糖（LOS），并且是不可转化的。通过将WS1的染色体DNA转化为FA19（产生菌株JWS-1）， WS1的los特异性突变转移到可转换的背景中。通过获得与单克隆抗体的反应性以及互补的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱，分离出一个能够将JWS-1恢复到野生型LOS表达的克隆（pJCL2）。pJCL2的序列单向缺失和DNA序列分析发现了一个开放阅读框，命名为lsi-7，可以弥补JWS-1的缺陷。对多个数据库的同源性检索表明，lsi-7与多个参与糖磷酸化的大肠杆菌基因具有同源性。lsi-7与另一个参与LOS生物合成的基因lsi-6相邻。利用鼠伤寒沙门菌脂多糖突变体进行的互补研究表明，lsi-6和lsi-7分别是鼠伤寒沙门菌rfaD和rfaE的淋球菌同源物。逆转录酶PCR分析表明，lsi-6和lsi-7是同一转录单元的一部分。
Neisseria gonorrhoeae WS1 is a spontaneous pyocin (a bacteriocin produced by Pseudomonas aeruginosa)-resistant mutant of N. gonorrhoeae FA19 that produces a truncated lipooligosaccharide (LOS) and is non-transformable. The LOS-specific mutation in WS1 was moved into a transformable background by transforming FA19 with chromosomal DNA from WS1 (generating strain JWS-1). A clone (pJCL2) capable of restoring JWS-1 to wild-type LOS expression, as detected by its acquisition of reactivity with monoclonal antibodies and by its complemented sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile, was isolated. Sequential unidirectional deletion and DNA sequence analysis of pJCL2 identified an open reading frame, designated lsi-7, that could complement the defect in JWS-1. Homology searches against various databases indicated that lsi-7 bad homology with several Escherichia coli genes involved in the phosphorylation of sugars. lsi-7 is adjacent to the lsi-6 gene, another gene involved in LOS biosynthesis. Complementation studies using Salmonella typhimurium lipopolysaccharide mutants showed lsi-6 and lsi-7 to be gonococcal homologs of S. typhimurium rfaD and rfaE, respectively. Reverse transcriptase PCR analysis demonstrated that lsi-6 and lsi-7 are part of the same transcriptional unit.