ALBERT

Notes: The membrane proteins ToxR and ToxS regulate a variety of genes associated with the virulence of Vibrio cholerae, the agent of human cholera. One of the ToxRS-regulated genes is the ompU gene, which encodes a porin that may also act as an adhesin. To begin to understand the mechanism of ompU transcription activation by ToxRS, we performed genetic and biochemical studies on the ompU promoter. Deletions with a 5′ end-point at or downstream of −128, relative to the start site for transcription, did not direct expression of a lacZ reporter gene in wild-type V. cholerae, although the −128 promoter fragment did direct ToxRS-dependent reporter gene activity under conditions of ToxR overexpression in E. coli. Consistent with the activation data is that membranes containing ToxR and ToxS caused a gel electrophoretic mobility shift when mixed at low concentrations with deletion fragments whose end-point is at −211, but not with −128 or −68 fragments. ToxRS membranes did shift the −128 fragment when added at higher concentrations. DNase I footprinting analysis of ompU promoter DNA complexed with ToxRS membranes demonstrated protection of three sites: an upstream site ranging from −238 to −139, and two downstream sites ranging from −116 to −58 and −53 to −24. Within the DNA protected from DNase I digestion by ToxRS membranes, there are no elements bearing similarity to those identified previously within the promoters of two other ToxR-dependent genes, ctxA and toxT. We suggest a model for transcription activation that involves sequential ToxR-binding events to distinct regions in the ompU promoter.

Notes: In pathogenic Vibrio cholerae, at least 17 genes are co-ordinately regulated by ToxR. Most of these genes, including those that encode cholera toxin (CT), toxin co-regulated pilus (TCP), accessory colonization factor (ACF) and OmpU, are positively regulated. OmpT is the only identified protein under negative regulation of ToxR. To understand the molecular mechanism by which ToxR represses OmpT expression, we cloned ompT and characterized the ompT promoter and its interaction with ToxR. Sequence analysis revealed that ompT encodes a predicted 35.8 kDa outer membrane porin of V. cholerae. Primer extension analysis identified a transcriptional start site 104 bp upstream of the translational start codon. Both primer extension analysis and promoter fusion studies showed that ToxR represses OmpT expression at the transcriptional level. Promoter fusion studies also suggest that cyclic AMP receptor protein (CRP) is involved in ompT activation. Gel mobility shift assays combined with DNase I footprinting analysis demonstrated that ToxR mediates repression of ompT transcription by directly binding to an A/T-rich region between −95 and −30 of the ompT promoter. To further understand how the interaction of ToxR with different promoters results in its function as an activator or repressor, we have also mapped the regions on the ctxAB and toxT promoters to which ToxR binds. The regions protected by ToxR on each of these promoters are all A/T rich and large in size, although they are positioned differently relative to each transcriptional start site.

Notes: Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping −10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than −247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the −300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the −300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3::lacZ fusions extended to what would be −246 bp of the LEE2 operon. A lacZ fusion from the −300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.

Notes: ToxR is a bitopic membrane protein that controls virulence gene expression in Vibrio cholerae. Its cytoplasmic domain is homologous to the winged helix–turn–helix (‘winged helix’) DNA-binding/transcription activation domain found in a variety of prokaryotic and eukaryotic regulators, whereas its periplasmic domain is of ill-defined function. Several genes in V. cholerae are regulated by ToxR, but by apparently different mechanisms. Whereas ToxR directly controls the transcription of genes encoding two outer membrane proteins, OmpU and OmpT, it co-operates with a second membrane-localized transcription factor called TcpP to activate transcription of the gene encoding ToxT, which regulates transcription of cholera toxin (ctxAB) and the toxin-co-regulated pilus (tcp). To determine the requirements for gene activation by ToxR, different domains of the protein were analysed for their ability to control expression of toxT, ompU and ompT. Soluble forms of the cytoplasmic winged-helix domain regulated ompU and ompT gene expression properly but did not activate toxT transcription. Membrane localization of the winged helix was sufficient for both omp gene regulation and TcpP-dependent toxT transcription, irrespective of the type of periplasmic domain or even the presence of a periplasmic domain. These results suggest that (i) the major function for membrane localization of ToxR is for its winged-helix domain to co-operate with TcpP to activate transcription; (ii) the periplasmic domain of ToxR is not required for TcpP-dependent activation of toxT transcription; and (iii) membrane localization is not a strict requirement for DNA binding and transcription activation by ToxR.

Notes: Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to translocate into host cells several effector molecules that are required for virulence. One of these, the translocated intimin receptor, Tir, inserts into the host cell cytoplasmic membrane, where it functions as the receptor for intimin, an outer membrane adhesin expressed by EPEC. A chaperone for Tir, called CesT, is required for stability of Tir in the EPEC cytoplasm. In this study, the cyaA gene reporter system was used to identify domains in Tir that mediate secretion into the culture supernatant and translocation into host cells. A Tir–CyaA fusion containing the first 15 N-terminal residues of Tir was secreted and translocated into HeLa cells by a ΔtirΔcesT mutant; however, maximal secretion and translocation was observed with the first 26 N-terminal residues of Tir. Fusions containing progressively larger N-terminal sequences of Tir were also efficiently secreted and translocated into HeLa cells by the ΔtirΔcesT strain. However, in a Δtir mutant that expresses CesT, Tir26–CyaA and an additional fusion containing the first 69 N-terminal residues of Tir were not secreted or translocated, but fusions containing larger N-terminal Tir sequences were secreted and translocated by the Δtir mutant. Wild-type EPEC secreted and translocated the Tir15–CyaA fusion, whereas longer fusions, such as Tir26–CyaA and Tir69–CyaA, were translocated to higher levels, similar to what was observed with the ΔtirΔcesT mutant. A Tir–CyaA fusion containing the CesT binding domain was translocated into HeLa cells more rapidly in the presence of CesT compared with translocation in the absence of CesT. Collectively, these results suggest that an N-terminal domain of 26 amino acids functions as a CesT-independent signal that is capable of delivering Tir into both the culture supernatant and the cytosol of host cells. Furthermore, in addition to its role in the stability of Tir, CesT may function in translocation by mediating rapid delivery of Tir into host cells.

Notes: Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.