QstR-dependent regulation of natural competence and type VI secretion in Vibrio cholerae

Abstract

During growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is a transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall, these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.

Ecological implications of gene regulation by TfoX and TfoY among diverse Vibrio species

“…1F). Notably, comEA expression in V. cholerae is regulated by an intermediate transcription factor, QstR, which is dependent on the production of both TfoX and HapR (Lo Scrudato and Blokesch, 2013;Jaskólska et al, 2018). HapR is the master regulator of the QS system, meaning that comEA expression is dependent on a functional QS system and on high cell density.…”

“…We, therefore, speculate that an increase in LitR levels can compensate for the lack of the intermediate regulator QstR in V. fischeri. Indeed, the QstR protein of V. cholerae is required for competence-mediated DNA uptake in two ways: (i) through direct and indirect induction of certain competence genes, such as comEA, as described above; and (ii) due to its ability to downregulate the dns gene (Lo Scrudato and Blokesch, 2013;Jaskólska et al, 2018) encoding for the extracellular nuclease Dns, which degrades transforming material outside the cell and within the periplasmic space (Blokesch and Schoolnik, 2008;Seitz and Blokesch, 2014). Notably, the HapR protein of V. cholerae also silences dns expression, but only partially in the absence of QstR (Lo Scrudato and Blokesch, 2013).…”

“…first diluted the cells to low densities. The rationale behind our experimental setup was the knowledge that the TfoXmediated pathway requires co-induction by HapR in V. cholerae (homologues are LuxR in V. alginolyticus and OpaR in V. parahaemolyticus) (Lo Scrudato and Blokesch, 2012;Jaskólska et al, 2018), which we assumed to be a conserved feature in other Vibrio species (see data below). In addition to T6SS activation, we also tested the level of competence genes transcripts and bacterial motility upon TfoX and TfoY induction.…”

Conserved type VI secretion regulation in diverseVibriospecies by the regulatory proteins TfoX and TfoY

“…1F). Notably, comEA expression in V. cholerae is regulated by an intermediate 192 transcription factor, QstR, which is dependent on the production of both TfoX and HapR (Lo 193 Scrudato and Blokesch, 2013; Jaskólska et al, 2018). HapR is the master regulator of the QS 194 system, meaning that comEA expression is dependent on a functional QS system and on high cell 195 density.…”

Analysis of Vibrio cholerae genomes using a novel bioinformatic tool identifies new, active Type VI Secretion System gene clusters

“…Clinical strains, such as C6706 and A1552, show little T6SS activity in rich growth medium (24)(25)(26). Expression of genes encoded on the large T6SS cluster is upregulated by the QstR protein, which integrates signals from three other regulators: CytR (responding to nucleoside starvation), HapR (responding to quorum sensing signals) and TfoX (responding to chitin oligomers) (27)(28)(29)(30). By contrast, the majority of V. cholerae that have no history of human pathogenicity (referred to as environmental strains) express the T6SS constitutively in rich growth medium and can kill other bacterial cells in a contact-dependent manner (25).…”