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Expression of a Yersinia pseudotuberculosis Type VI Secretion System Is Responsive to Envelope Stresses through the OmpR Transcriptional Activator.

Gueguen E, Durand E, Zhang XY, d'Amalric Q, Journet L, Cascales E - PLoS ONE (2013)

Bottom Line: 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.We first identified OmpR in a transposon mutagenesis screen.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM, UMR 7255), Institut de Microbiologie de la Méditerranée (IMM), Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Marseille, France.

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.

No MeSH data available.


Related in: MedlinePlus

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 [63]. (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.
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pone-0066615-g003: 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 [63]. (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.

Mentions: In silico analysis of the T6SS-4 promoter region with Virtual Footprint suggests the existence of OmpR binding motifs (Figure 3A). To test whether the control by the OmpR protein was direct, we performed electrophoretic mobility shift assays. A recombinant N-terminally 6×His-TRX-tagged variant of the Y. pseudotuberculosis OmpR protein was purified to homogeneity by metal-affinity chromatography. The native OmpRYps protein was obtained by tag proteolysis and gel filtration (see Material and Methods). OmpR being a transcriptional activator associated with a two-component system, its affinity for targets is dependent on its phosphorylation status [59]. Gel shift assays were therefore monitored in presence of acetyl-phosphate to promote OmpR auto-phosphorylation. The purified OmpRYps protein bound to the promoter region of the ompF gene (Figure 3B, upper panel, lanes 1–6), a direct target of the OmpR response regulator [60]. This protein-DNA interaction was specific as no OmpRYps binding was observed on the enteroaggregative E. coli sci-1 promoter (Figure 3B, upper panel, lane 10) whose expression is regulated by the ferric uptake regulator [61] and independent on OmpR (the sci1-lacZ fusion shares comparable β-galactosidase activities in E. coli WT and ΔompR strains; data not shown). A shift was observed when the T6SS-4 promoter was used (Figure 3B, lower panel, lanes 1–6). No shift was observed with the purified Fur protein in both ompF and T6SS-4 promoters (Figure 3B, lanes 9). The PT6SS-4-OmpRYps shift was abolished when a competitor unlabelled DNA corresponding to the ompF promoter was used (Figure 3B, lower panel, lanes 7–8). Conversely, addition of the unlabelled T6SS-4 promoter decreased OmpRYps binding on the ompF promoter (Figure 3B, upper panel, lanes 7–8). Taken together these results demonstrate that OmpR specifically binds to the T6SS-4 promoter region. However, it is worthy to note that the affinity for the purified OmpR protein is higher for the ompF promoter compared to the T6SS-4 promoter as (i) the ompF probe is retarded for lower OmpR concentrations (20 nM for ompF and 40 nM for T6SS-4) (compare upper and lower panels in Figures 3B, lanes 1–6) and (ii) the unlabelled ompF fragment has a stronger effect compared to the unlabelled T6SS-4 fragment in competition experiments (compare lanes 6–8).


Expression of a Yersinia pseudotuberculosis Type VI Secretion System Is Responsive to Envelope Stresses through the OmpR Transcriptional Activator.

Gueguen E, Durand E, Zhang XY, d'Amalric Q, Journet L, Cascales E - PLoS ONE (2013)

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 [63]. (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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3686713&req=5

pone-0066615-g003: 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 [63]. (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.
Mentions: In silico analysis of the T6SS-4 promoter region with Virtual Footprint suggests the existence of OmpR binding motifs (Figure 3A). To test whether the control by the OmpR protein was direct, we performed electrophoretic mobility shift assays. A recombinant N-terminally 6×His-TRX-tagged variant of the Y. pseudotuberculosis OmpR protein was purified to homogeneity by metal-affinity chromatography. The native OmpRYps protein was obtained by tag proteolysis and gel filtration (see Material and Methods). OmpR being a transcriptional activator associated with a two-component system, its affinity for targets is dependent on its phosphorylation status [59]. Gel shift assays were therefore monitored in presence of acetyl-phosphate to promote OmpR auto-phosphorylation. The purified OmpRYps protein bound to the promoter region of the ompF gene (Figure 3B, upper panel, lanes 1–6), a direct target of the OmpR response regulator [60]. This protein-DNA interaction was specific as no OmpRYps binding was observed on the enteroaggregative E. coli sci-1 promoter (Figure 3B, upper panel, lane 10) whose expression is regulated by the ferric uptake regulator [61] and independent on OmpR (the sci1-lacZ fusion shares comparable β-galactosidase activities in E. coli WT and ΔompR strains; data not shown). A shift was observed when the T6SS-4 promoter was used (Figure 3B, lower panel, lanes 1–6). No shift was observed with the purified Fur protein in both ompF and T6SS-4 promoters (Figure 3B, lanes 9). The PT6SS-4-OmpRYps shift was abolished when a competitor unlabelled DNA corresponding to the ompF promoter was used (Figure 3B, lower panel, lanes 7–8). Conversely, addition of the unlabelled T6SS-4 promoter decreased OmpRYps binding on the ompF promoter (Figure 3B, upper panel, lanes 7–8). Taken together these results demonstrate that OmpR specifically binds to the T6SS-4 promoter region. However, it is worthy to note that the affinity for the purified OmpR protein is higher for the ompF promoter compared to the T6SS-4 promoter as (i) the ompF probe is retarded for lower OmpR concentrations (20 nM for ompF and 40 nM for T6SS-4) (compare upper and lower panels in Figures 3B, lanes 1–6) and (ii) the unlabelled ompF fragment has a stronger effect compared to the unlabelled T6SS-4 fragment in competition experiments (compare lanes 6–8).

Bottom Line: 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.We first identified OmpR in a transposon mutagenesis screen.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM, UMR 7255), Institut de Microbiologie de la Méditerranée (IMM), Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Marseille, France.

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.

No MeSH data available.


Related in: MedlinePlus