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, 8 (6), e66615

Expression of a Yersinia Pseudotuberculosis Type VI Secretion System Is Responsive to Envelope Stresses Through the OmpR Transcriptional Activator

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Expression of a Yersinia Pseudotuberculosis Type VI Secretion System Is Responsive to Envelope Stresses Through the OmpR Transcriptional Activator

Erwan Gueguen et al. PLoS One.

Abstract

The Type VI secretion system (T6SS) is a macromolecular complex widespread in Gram-negative bacteria. Although several T6SS are required for virulence towards host models, most are necessary to eliminate competitor bacteria. Other functions, such as resistance to amoeba predation, biofilm formation or adaptation to environmental conditions have also been reported. This multitude of functions is reflected by the large repertoire of regulatory mechanisms shown to control T6SS expression, production or activation. Here, we demonstrate that one T6SS gene cluster encoded within the Yersinia pseudotuberculosis genome, T6SS-4, is regulated by OmpR, the response regulator of the two-component system EnvZ-OmpR. We first identified OmpR in a transposon mutagenesis screen. OmpR does not control the expression of the four other Y. pseudotuberculosis T6SS gene clusters and of an isolated vgrG gene, and responds to osmotic stresses to bind to and activate the T6SS-4 promoter. Finally, we show that T6SS-4 promotes Y. pseudotuberculosis survival in high osmolarity conditions and resistance to deoxycholate.

Conflict of interest statement

Competing Interests: Please note that Eric Cascales is a PLOS ONE Academic Editor. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and material.

Figures

Figure 1
Figure 1. Transposon mutagenesis identified OmpR and LptD as regulators of T6SS-4 expression.
(A) Location of the transposon in the three strains (Tn23, Tn31 and Tn52) displaying lower T6SS-4 expression isolated in the random screen. (B) β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of Y. pseudotuberculosis RL31748-4 (no fusion), Y.p. RL31758-41 (WT, carrying the promoter-lacZ fusion at the locus and the promoter-gfp fusion at the ara locus) and of the transposon strains carrying the pBAD24 empty vector (−) or pBAD-ompR (+). p-values obtained using paired Student’s t-test analyses are indicated (***, p≤0.0001).
Figure 2
Figure 2. OmpR does not regulate the other Y. pseudotuberculosis T6SS loci.
β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of Y. pseudotuberculosis with lacZ and gfp fusions to T6SS-1, T6SS-2, T6SS-3-rev, T6SS-3-fwd, T6SS-4, T6SS-5 and vgrG (IP31758_0696) putative promoters, and carrying the pBAD24 empty vector (−) or pBAD-ompR (+). p-values obtained using paired Student’s t-test analyses are indicated (NS, non significant [p>0.05]; ***, p≤0.0001).
Figure 3
Figure 3. OmpR binds to the promoter region of T6SS-4.
(A) Intergenic sequence upstream the first gene of the T6SS-4 operon. The TTG putative initiation codon is underlined, as the ATG initiation codon of the divergent gene upstream T6SS-4. The framed sequences in bold letters correspond to putative OmpR binding sites identified by in silico analyses using Virtual Footprint. A third OmpR binding site was experimentally identified upstream this intergenic region . (B) Electrophoretic mobility shift assays of the Y. pseudotuberculosis ompF (upper panel) or T6SS-4 (lower panel) promoters using phosphorylated purified OmpR protein (lane 1, no protein; lane 2, 10 nM; lane 3, 20 nM; lane 4, 40 nM; lane 5, 60 nM; lane 6, 80 nM). Lanes 7 and 8: competition experiments with unlabelled T6SS-4 (upper panel) or ompF (lower panel) promoter PCR fragments at a promoter:competitor 1∶4 (lane 7) or 1∶20 (lane 8) ratio, in presence of 80 nM phosphorylated purified OmpR protein. Controls include incubation with the purified ferric uptake regulator Fur (lane 9, 80 nM) or incubation of the OmpR-independent enteroaggregative E. coli sci-1 promoter PCR fragment (Psci1) with phosphorylated purified OmpR (lane 10, 80 nM). The positions of the free probes and of the shift fragments (*) are indicated.
Figure 4
Figure 4. T6SS-4 thermoregulation is OmpR-independent.
β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of the T6SS-4 promoter fusions at 28°C and 37°C in the wild-type (A) and ompR1 transposon (B) strains. Identical results were obtained for the ompR2 transposon strain (data not shown). p-values obtained using paired Student’s t-test analyses are indicated (***, p≤0.0001).
Figure 5
Figure 5. T6SS-4 gene expression is responsive to osmotic and cell envelope stresses through an OmpR-dependent mechanism.
β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of the T6SS-4 promoter in the WT or transposon ompR1 strain carrying the pBAD24 empty vector (−) or pBAD-ompR (+) after exposure – or not – to 0.6 M Sucrose or to sodium deoxycholate (DOC) for 60 min. Identical results were obtained for the ompR2 transposon strain (data not shown). p-values obtained using paired Student’s t-test analyses are indicated (NS [non significant], p>0.05; ***, p≤0.0001).
Figure 6
Figure 6. OmpR and T6SS-4 are required for survival after exposure to osmotic and cell envelope stresses.
(A) Survival to exposure to osmotic stress. Viable bacteria (relative to the initial input) of the indicated strain counted after exposure for 60 min. to 0.6 M Sucrose. (B) Survival to cell envelope stress. Highest 10-fold serial dilution for which colonies are observable on LB plates supplemented with 1% sodium deoxycholate after 24 hours of incubation at 28°C. p-values obtained using paired Student’s t-test analyses are indicated (NS [non significant], p>0.05; ***, p≤0.0001).

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Grant support

This work was supported by the Centre National de la Recherche Scientifique and a grant from the Agence Nationale de la Recherche (ANR-10-JCJC-1303-03) to E.C. E.G. and E.D. were recipients of post-doctoral fellowships from the Fondation pour la Recherche Medicale (SPF-2009-12-17571 and SPF-2010-12-21116 respectively). X.-Y.Z. was supported by the ANR-10-JCJC-1303-03 grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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