Structural analysis of the acfA and acfD genes of Vibrio cholerae: effects of DNA topology and transcriptional activators on expression

Abstract

The Vrbrio cholerae acfA, B, C, and D genes are involved in the synthesis of a colonization factor; their expression is under the control of ToxR, the cholera toxin transcriptional activator. By a combination of Southern blot analysis, cloning, and nucleotide sequence analysis, we determined that the acf genes are clustered on a 5-kb region, the aefA and acpD genes are transcribed divergently, and the translation start sites of the two genes are separated by only 173 bp. Expression from the aefA and acJD promoters in V. cholerae was studied by using acfA:phoA translational and a.flD-lacZ transcriptional fusions; when carried by the chromosome, the acfA-acJD intergenic region flanked by the two reporter genes was found to contain the cis-acting element(s) necessary for the environmental regulation of the two promoters. However, this regulation was almost completely abolished when the same construction was carried by a low-copy-number plasmid. These results suggested that differences in DNA topology between the plasmid versus the chromosomal constructs might influence the expression of the acfA and acJD promoters. Support for this conclusion was obtained by showing that ToxR-dependent but not basal expression of both promoters was strongly inhibited by nalidixic acid and novobiocin, two DNA gyrase inhibitors, suggesting that the activation of these promoters is affected by changes in DNA supercoiling. Expression of the acfA and aciD promoters was also investigated in the heterologous host Escherichia coli harboring plasmids expressing either ToxR or ToxT, two transcriptional activators of the V. cholerae virulence genes. ToxR activated the aajD promoter 2.5-fold but inhibited the aefA promoter 2-fold. In contrast, the expression of the acfA promoter was activated 10-fold and that of the acJD promoter was activated 3-fold by ToxT, supporting the previously proposed cascade model for organization of the ToxR regulon.Vibrio cholerae is the causative agent of the diarrheal disease Asiatic cholera. This diarrhea is largely induced by the action of cholera toxin, a heat-labile enterotoxin composed of two different subunits encoded by the ctxA and ctxB genes (4,14). In addition, V. cholerae adheres to and colonizes the small intestine of the host, a virulence property that has been associated with the synthesis of the toxincoregulated pilus (TCP) (8,23,28).Expression of the ctx4B operon is under the control of the transcriptional activator encoded by the toxR locus (17). This locus is composed of two genes, toxR and toxS, that are clustered in an operon (16). toxR encodes a transmembrane DNA-binding protein that recognizes a heptanucleotide sequence tandemly repeated in the clx regulatory region (19).The product of toxS, a membrane protein, was shown to act in conjunction with ToxR to activate c&xAB expression (16).The ToxR regulon, defined as the set of genes whose expression is under the control of ToxR, comprises, in addition to ctxAB, the tcp genes required for the production of the TCP, the tag genes (ToxR-activated...

Oscillations in supercoiling drive circadian gene expression in cyanobacteria

“…Changes in the level of supercoiling have been observed during a variety of stress responses-temperature, peroxide, and osmotic-as well as growth conditions (23)(24)(25)28), and changes in supercoiling have been shown to elicit a global transcriptional response (29). A role for supercoiling in mediating changes in expression is not limited to E. coli and has been observed in a variety of bacteria including Salmonella typhimurium, Vibrio cholera, Bacillus subtilis, and Synechocystis PCC 6803 (30)(31)(32)(33).…”

A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains

“…6). Based on these data and data from previously published work, the ToxT-dependent branch includes ctxAB, tcp, at least one of the acf genes, acfA (Parsot and Mekalanos, 1992), and aldA, the gene encoding aldehyde dehydrogenase (DiRita et al, 1991;Parsot and Mekalanos, 1991). The ToxT-independent branch consists of ompU, as its product is detected at wild-type levels in the toxT mutant background (this work) and the toxT mutant strain VJ740 supports a wild-type level of expression of an ompU-lacZ transcriptional fusion (J.…”

“…Also, as described above, OmpU appears to require ToxR, but not ToxT, for its activation, as determined by the observation that ToxT does not restore OmpU production when expressed in a toxR mutant background (DiRita et al, 1991). Finally, the acfD promoter is only weakly activated by ToxT and DNA supercoiling may play a role in its expression (Parsot and Mekalanos, 1992).…”

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression

“…Examples have come from studies in Bordetella pertussis (Graeff-Wohlleben et al 1995), Campylobacter jejuni (Dedieu et al 2002), Chlamydia (Niehus et al 2008), Dickeya dadantii (Hérault et al 2014), E. coli (Beltrametti et al 1999;Sánchez-Céspedes et al 2015), Helicobacter pylori (Ye et al 2007), Salmonella spp. (Bang et al 2002;Galán and Curtiss 1990;Leclerc et al 1998;Ó Cróinín et al 2006;Webber et al 2013), Shigella flexneri (Dorman et al 1990), Vibrio cholerae (Parsot and Mekalanos 1992), Staphylococcus aureus (Fournier and Klier 2004;Schröder et al 2014;Sheehan et al 1992) and Yersinia enterocolitica (Rohde et al 1994).…”