8 GENES IN REGION-5 THAT FORM AN OPERON ARE ESSENTIAL FOR INVASION OF EPITHELIAL-CELLS BY SHIGELLA-FLEXNERI 2A
8 GENES IN REGION-5 THAT FORM AN OPERON ARE ESSENTIAL FOR INVASION OF EPITHELIAL-CELLS BY SHIGELLA-FLEXNERI 2A
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DOI：
10.1128/jb.175.8.2334-2346.1993
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发表时间：
1993-04-01
影响因子：
3.2
通讯作者：
YOSHIKAWA, M
中科院分区：
文献类型：
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作者：
SASAKAWA, C;KOMATSU, K;YOSHIKAWA, M
福氏志贺菌2a YSH 6000中230-kb大质粒pMYSH 6000上的7-kb区域5是上皮细胞侵袭所需的毒力相关DNA片段之一（C. Sasakawa，K. Kamata，T.酒井，S. Makino，M. Yamada，N. Okada和M. Yoshikawa，J. Bacteriol. 170：2480-2484，1988）。为了阐明第5区的功能结构和确定第5区编码的毒力相关基因，我们对第5区进行了插入和缺失突变、DNA亚克隆和全核苷酸测序，发现第5区包含11个开放阅读框架（ORF），命名为ORF-1至ORF-11，它们可以翻译成分子量为15.1、47.5、47.5、47.5和47.5的蛋白质。13.2、33.0、33.4、24.2、9.4、28.5、39.9、9. 1和10.4kDa。用上述质粒构建体对14个Tn 5诱导的5区非侵入性突变体进行的互补试验表明，5区由操纵子组成，ORF-2至ORF-9，而不是ORF-1、ORF-10和ORF-11，是侵入所必需的，和8个ORF中的7个（ORF-2和ORF-4至ORF-9）和推测剩余的ORF（ORF-3）是分泌Ipa蛋白所需的。通过启动子-证明载体、S1核酸酶保护和引物延伸RNA测序分析确定的转录组织显示，区域5是从位于ORF-2 5'端上游47 bp的启动子转录的，并且鉴定的启动子活性由230-kb质粒上的转录激活因子virB基因调节。
The 7-kb region 5 on the large 230-kb plasmid pMYSH6000 in Shigella flexneri 2a YSH6000 is one of the virulence-associated DNA segments required for the invasion of epithelial cells (C. Sasakawa, K. Kamata, T. Sakai, S. Makino, M. Yamada, N. Okada, and M. Yoshikawa, J. Bacteriol. 170:2480-2484, 1988). To elucidate the functional organization of region 5 and to determine the virulence-associated genes encoded by region 5, we performed insertion and deletion mutagenesis, DNA subcloning, and complete nucleotide sequencing of region 5 and found that region 5 contained 11 open reading frames (ORFs) named ORF-1 through ORF-11 which could be translated into proteins with molecular masses of 15.1, 47.5, 13.2, 33.0, 33.4, 24.2, 9.4, 28.5, 39.9, 9. 1, and 10.4 kDa, respectively. Complementation tests of the 14 Tn5-induced noninvasive mutants of region 5 with the above plasmid constructs have indicated that region 5 consists of an operon and that ORF-2 through ORF-9, but not ORF-1, ORF-10, and ORF-11, are essential for invasion, and 7 of 8 ORFs (ORF-2 and ORF-4 through ORF-9) and presumably the remaining ORF (ORF-3) are required for secretion of the Ipa proteins. The transcriptional organization, as determined by a promoter-proving vector, S1 nuclease protection, and primer extension RNA sequencing analysis revealed that region 5 is transcribed from a promoter located 47 bp upstream of the 5' end of ORF-2 for the 47.5-kDa protein and that the promoter activity identified was regulated by the virB gene, the transcriptional activator on the 230-kb plasmid.