Sasakawa C, Adler B, Tobe T, Okada N, Nagai S, Komatsu K, Yoshikawa M
Department of Bacteriology, University of Tokyo, Japan.
Mol Microbiol. 1989 Sep;3(9):1191-201. doi: 10.1111/j.1365-2958.1989.tb00269.x.
The 7 kb virulence Region-2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS-polyacrylamide gel electrophoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (IpaB), 41 (IpaC) and 37 (IpaD)-kiloDalton (kD) proteins. Complementation of Tn5-induced mutations in Region-2 with the above plasmid constructs indicated that Region-2 consists of two operons and that the three Ipa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the Ipa proteins revealed that Region-2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5 kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, IpaB and C proteins and the 4.5 kb RNA for the ipaD gene. In addition, the full-length RNA of 7.5 kb which covers Region-2 supplemented full expression of the Ipa proteins. The 7663 nucleotides of Region-2 were determined to confirm the five open reading frames encoding 23,655, 17,755, 62,168, 41,077 and 36,660 Dalton proteins, respectively, and their regulatory sequences.
福氏志贺菌2a大（毒力）质粒上7kb的毒力区域2编码几种侵袭肠上皮细胞所需的蛋白质。通过插入和缺失诱变、DNA亚克隆以及在小细胞中合成蛋白质的SDS-聚丙烯酰胺凝胶电泳，确定了该区域的五个基因。它们分别编码24、18、62（IpaB）、41（IpaC）和37（IpaD）千道尔顿（kD）的蛋白质。用上述质粒构建体对区域2中Tn5诱导的突变进行互补实验表明，区域2由两个操纵子组成，并且这三种Ipa蛋白对于毒力表型至关重要。通过Northern印迹、S1核酸酶保护确定的转录组织以及Tn5插入对Ipa蛋白表达的影响表明，区域2有三个启动子，转录4.0、4.5和7.5kb的RNA。4.0kb的RNA是编码24、18kD、IpaB和C蛋白的操纵子的转录本，4.5kb的RNA是ipaD基因的转录本。此外，覆盖区域2的7.5kb全长RNA补充了Ipa蛋白的完整表达。确定了区域2的7663个核苷酸，以确认分别编码23,655、17,755、62,168、41,077和36,660道尔顿蛋白质的五个开放阅读框及其调控序列。
