VACC, A VIRULENCE-ASSOCIATED CHROMOSOMAL LOCUS OF SHIGELLA-FLEXNERI, IS HOMOLOGOUS TO TGT, A GENE ENCODING TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE (TGT) OF ESCHERICHIA-COLI K-12

The genetic determinants required for invasion of epithelial cells by Shigella flexneri and for the subsequent bacterial spreading are encod ed by the large virulence plasmid. Expression of the virulence genes i s under the control of various genes on the large plasmid as well as o n the chromosome. We previously identified one of the virulence associ ated loci near phoBR in the NotI-C fragment of the chromosome of S. fl exneri 2a YSH6000 and designated the locus vacC. The vacC mutant showe d decreased levels of IpaB, IpaC, and IpaD proteins as well as transcr iption of ipa, an operon essential for bacterial invasion (N. Okada, C . Sasakawa, T. Tobe, M. Yamada, S. Nagai, K. A. Talukder, K. Komatsu, S. Kanegasaki, and M. Yoshikawa, Mol. Microbiol. 5:187-195, 1991). To elucidate the molecular nature of the vacC locus, we cloned the vacC r egion from YSH6000 on a 1.8-kb SalI-BamHI DNA fragment. The nucleotide sequence of the 1,822-bp vacC clone was highly (> 98%) homologous to the tgt region of Escherichia coli K-12, which is located at 9.3 min o n the linkage map. Complementation tests indicated that the vacC funct ion was encoded by an open reading frame expressing a 42.5-kDa)a prote in, which corresponded to the tgt gene off. coli K-12, coding for tRNA -guanine transglycosylase (Tgt) (K. Reuter, R. Slany, F. Ullrich, and H. Kersten, J. Bacteriol. 173:2256-2264, 1991). The cloned tgt gene fr om E. coli K-12 restored the virulence phenotype to the vacC mutant of YSH6000. Characterization of the vacC mutant indicated that levels of VirG, a protein essential for bacterial spreading, and VirF, the posi tive regulator for the expression of the virG and ipaBCD operons, decr eased significantly compared with those of the wild type. Similar phen otypic changes occurred in vacC mutants constructed by insertion of a neomycin resistance gene in shigellae and enteroinvasive E. coli strai ns, consistent with the hypothesis that the vacC (tgt) gene contribute s to the pathogenicity of Shigella flexneri.