Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a.
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.
Sasakawa
C
C
Department of Bacteriology, University of Tokyo, Japan.
Komatsu
K
K
Tobe
T
T
Suzuki
T
T
Yoshikawa
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D001483:Base Sequence; D004251:DNA Transposable Elements; D004848:Epithelium / Q000382:microbiology; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D011401:Promoter Regions, Genetic; D012763:Shigella flexneri / Q000235:genetics* / Q000472:pathogenicity; D014774:Virulence
D001426:Bacterial Proteins; D004251:DNA Transposable Elements