Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: activation by virF and repression by H-NS

Abstract

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.

Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli

“…It was proposed that the physical basis for the thermoregulated expression of virF resides in a temperature-dependent structural modification of the virF promoter, resulting from a temperature-sensitive curved DNA structure (Falconi et al, 1998). H-NS also mediates repression of the virB gene downstream in the Shigella regulatory cascade (Tobe et al, 1993). The regulatory cascade in EPEC, whereby the expression of Ler is thermoregulated by H-NS, is apparently analogous to the Shigella system.…”

H‐NS: a modulator of environmentally regulated gene expression

“…1. Role of H-NS in the VirF/ VirB regulatory cascade (Colonna et al, 1995;Tobe et al, 1993;. Genes are indicated as boxes and regulatory proteins as balls; P, promoter; black straight arrow, activation of transcription; stippled straight lines with blunt ends, repression of transcription.…”

“…Those for the virB promoter are based on Fig. 7 in Tobe et al (1993). Positions are given relative to the transcription start site.…”

Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri

“…Expression of genes of the entry region is regulated by the growth temperature, these genes being expressed at 37 u C but not at 30 u C (Maurelli et al, 1984), and by a cascade involving two VP gene products, VirF and VirB. VirF, a member of the AraC family of transcriptional activators, is required for transcription of virB (Sakai et al, 1986;Tobe et al, 1993) and VirB, a member of the ParB family of partitioning proteins, is required for transcription of genes of the entry region (Adler et al, 1989; Tobe et al, 1991). Control by temperature is dependent upon the chromosomally encoded protein H-NS, as inactivation of hns leads to expression of genes of the entry region at both 30 and 37 u C (Maurelli & Sansonetti, 1988;Prosseda et al, 1998).…”