Expression of invasion genes encoded by the large 230-kb plasmid of ShigeUlajflxneri 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°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 ofvirR (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 (MaIE'-'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 V`irF 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.

By using genetic complementation tests with various in vitro-constructed mutants with mutations in the cap region (which is essential for encapsulation in Bacillus anthracis), we identified three cistrons, capB, capC, and capA, in this order of arrangement. Minicell analysis revealed that these cistrons produce proteins of 44, 16, and 46 kilodaltons, respectively. The complete nucleotide sequence of 3,244 base pairs covering the whole cap region was determined and revealed the existence of the three open reading frames of capB (397 amino acid residues; molecular weight, 44,872), capC (149 amino acid residues; molecular weight, 16,522), and capA (411 amino acid residues; molecular weight, 46,420) arranged in the order predicted by complementation tests. These three cistrons were all transcribed in the same direction from promoters unique to each cistron. Judging from the predicted amino acid sequence of the three proteins and from their localization and their sensitivity to various physicochemical treatments, they appeared to be membrane-associated enzymes mediating the polymerization of D-glutamic acid via the membrane. Capsular peptides immunologically identical to that of B. anthracis were found in B. subtilis, B. megaterium, and B. licheniformis, but no sequence homologous to the cap region was found in any of these bacilli other than B. anthracis. Using strains of B. anthracis with or without insertional inactivation of the cap region, we found that the capsule of B. anthracis conferred strong resistance to phagocytosis upon the bacterial host.

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On the 230-kilobase-pair (kb) virulence plasmid of Shigella flexneri 2a strain YSH6000, at least seven separate genetic determinants have been identified. One of them, an approximately 4-kb region, virG, that is required for the Sereny reaction, was extensively studied to examine the role of the virG region. The phenotype of a VirG-mutant (M94) of YSH6000 in the cytoplasm of cultured MK cels was characterized by a kinetic study of the invading shigellae. The observed phenotype of M94 in the cytoplasm indicated that the virG locus is not required for multiplication of the invading shigellae, but is essential for their spread to adjacent cells. The DNA region necessary for the VirG function was localized to a 3.6-kb DNA sequence on the 230-kb plasmid. A 130-kilodalton polypeptide was confirmed to be the virG product. External labeling of bacteria with 1251 indicated that the 130-kilodalton virG protein is exposed on the bacterial surface. The nucleotide sequence of 4,472 bp, which contains the functional virG gene and its own regulatory sequence, was determined, and a large open reading frame encoding 1,102 amino acid residues was identified.