笹川 千尋

Expression of invasion genes encoded by the large 230-kb plasmid of Shigella flexneri is controlled by the virB gene, which is itself activated by another regulator, virF. Transcription of the invasion genes is temperature regulated, since they are activated in bacteria grown at 37 but not at 30-degrees-C. Recently, we have shown that the thermoregulated expression of invasion genes is mediated by thermal activation of virB transcription (T. Tobe, S. Nagai, B. Adler, M. Yoshikawa, and C. Sasakawa, Mol. Microbiol. 5:887-893, 1991). It has also been shown that a mutation that inactivates H-NS, the product of virR (hns), derepresses transcription of virB. To elucidate the molecular mechanisms underlying virB activation, we determined the location of the transcription start site and found it to be 54 bp upstream of the 5' end of the virB coding sequence. Deletion analysis revealed that transcriptional activation by virF requires a DNA segment of 110 bp extending upstream of the transcription start site. By using a protein binding assay with crude extracts of S. flexneri harboring the malE'-'virF fusion gene, which was able to activate virB transcription, two protein species, one of 70 kDa (MalE'-'VirF fusion) and another of 16 kDa (H-NS), were shown to bind specifically to the virB promoter region. DNA footprinting analysis indicated that the VirF fusion and H-NS proteins bound to the upstream sequence spanning from -17 to -117 and to the sequence from -20 to +20, in which virB transcription starts, respectively. In an in vitro transcription assay, the VirF fusion protein was shown to activate virB transcription while the H-NS protein blocked it. virB activation was seen only when negatively supercoiled DNA was used as a template. In in vivo studies, virB transcription was significantly decreased by adding novobiocin, a gyrase inhibitor, into the culture medium while virB transcription was increased by mutating hns. These in vitro and in vivo studies indicated that transcription of virB is activated through VirF binding to the upstream sequence of the virB promoter in a DNA-topology-dependent manner and is directly repressed by H-NS binding to the virB transcription start site.

A large plasmid-encoded protein, VirG, on the bacterial surface is essential for the spreading of Shigella by eliciting polar deposition of filamentous actin in the cytoplasm of epithelial cells. VirG expression from the large plasmid is diminished greatly when it is introduced into Escherichia coli K-12 from Shigella. In an attempt to identify factors affecting VirG expression, we found that the absence of the ompT gene, encoding outer membrane protease OmpT, restored full production of VirG protein to E. coli K-12. Conversely, upon introduction of the ompT gene of E. coli K-12 into Shigella, spreading ability was completely abolished, probably because of the proteolytic degradation of VirG protein by OmpT. Analysis of the DNA sequence of the ompT region indicated that the absence of the ompT gene occurred in Shigella and enteroinvasive E. coli strains, and that the absent DNA segment corresponded to a remnant lambdoid phage structure found in E. coli K-1 2, which encompasses a 21 kb DNA segment spanning from argU through to the ompT genes. Since ompT is located near pure in E coli K-12 and a virulence locus for provoking keratoconjunctivitis in the eyes of guinea-pigs, named kcpA, is located near pure in S. flexneri, and the two loci are involved in VirG expression, the KcpA- mutants of S. flexneri 2a constructed were examined for correlation between acquisition of ompT and VirG degradation. Our data suggest that the previous recognition of a kcpA locus in S. flexneri is the result of transfer of the ompT gene from E. coli K-12, giving rise to a KcpA- phenotype. These results indicate that the lack of OmpT protease confers upon Shigella the ability to spread into adjacent epithelial cells.

Bacillus anthracis produces a gamma-linked poly-D-glutamic acid capsule that is essential for virulence. A 6.2kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low-molecular-weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region alone caused production of a high-molecular-weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51 460. By in vitro transcription-translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian gamma-glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly-D-glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low-molecular-weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.

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.

To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a NOtl restriction map of the chromosome was constructed by exploiting Notl-linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K-12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K-12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci identified previously by random Tn5 insertions were physically mapped on the chromosome. to demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K-12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5' and 3' flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison of genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K-12.

The invasion phenotype of shigellae is subject to thermoregulation that is known to be expressed through activation of some invasion (inv) genes such as ipaB, ipaC, and ipaD encoded by the large virulence plasmid of Shigella flexneri. The expression of ipa genes is regulated positively by virF through the activation of virB on the plasmid. To identify the mediator for the thermoregulation of the large plasmid, we have studied the effect of temperature on the transcription of virF and virB genes and ipa and the other two inv operons. The results showed that transcription of virB was affected by temperature more strictly than that of virF. Analysis of the mRNA level of virB at different levels of virF transcription indicated that virB transcription depended upon both temperature and virF. On the other hand, transcriptions of ipa and the other two inv operons depended on the activation of virB transcription but not on temperature. By inducing virB transcription from a tac promoter fused with the virB region, invasion ability was restored to a virF-deletion mutant at 30-degrees-C as well as at 37-degrees-C. By using conditions in which the temperature-dependent expression of the invasion phenotype was circumvented by the induction of virB transcription, intercellular spreading ability in a virF+, virB::Tn5 strain was shown to be expressed even at 30-degrees-C. These results suggest that the virB transcription stage is the main target for the thermoregulation.