OmpR, a response regulator of the two-component signal transduction pathway, influences inv gene expression in Yersinia enterocolitica O9

Abstract

The environmental control of invasin (inv) expression in Yersinia enterocolitica is mediated by a regulatory network composed of negative and positive regulators of inv gene transcription. Previously, we demonstrated that OmpR, a response regulator of the two-component signal transduction pathway EnvZ/OmpR, negatively regulates inv gene expression in Y. enterocolitica O9 by direct interaction with the inv promoter region. This study was undertaken to clarify the role of OmpR in the inv regulatory circuit in which RovA protein has been shown to positively regulate inv transcription. Using ompR, rovA, and ompR rovA Y. enterocolitica mutant backgrounds we showed that the inhibitory effect of OmpR on inv transcription may be observed only when RovA is present/active in Y. enterocolitica cells. To extend our research on inv regulation we examined the effect of OmpR on rovA gene expression. Analysis of rovA-lacZ transcriptional fusion in Y. enterocolitica wild-type and ompR background indicated that OmpR does not influence rovA expression. Thus, our results indicate that OmpR influences inv expression directly via binding to the inv promoter, but not through modulation of rovA expression.

Keywords: OmpR regulator; RovA regulator; Yersinia enterocolitica; invasin; signal transduction pathway.

Figure 1

Effect of pH and temperature on inv transcription in the wild-type strain Ye9. Cells were grown to stationary phase at 25°C or 37°C in LB medium buffered to pH 7.0 or 5.5. Total RNA was extracted and used in sqRT-PCR to assess inv mRNA levels. PCRs for inv and 16S rRNA were carried out for 23 cycles and 10 cycles, respectively. (A) Lanes: MM—DNA molecular mass marker (100 bp ladder); lane 1–25°C, pH 7.0; lane 2—37°C, pH 7.0; lane 3—25°C, pH 5.5; lane 4—37°C, pH 5.5. (B) The densities of inv bands relative to those of the 16S rRNA bands on the gel in part A. Values are means ± SD, n = 2–3; a, b, c, d—results of Tukey post-hoc multiple mean comparison test. Means without a common letter differ significantly (p < 0.05).

Figure 2

Effect of pH and temperature on ompR transcription in the wild-type strain Ye9. Cells were grown to early stationary phase at 25°C or 37°C in LB medium buffered to pH 7.0 or 5.5. Total RNA was extracted and used in sqRT-PCR to assess ompR mRNA levels. PCRs for ompR and 16S rRNA were carried out for 25 cycles and 10 cycles, respectively. Lanes: 1—DNA molecular mass marker (100 bp ladder); lane 2—25°C, pH 7.0; lane 3—37°C, pH 7.0; lane 4—25°C, pH 5.5; lane 5—37°C, pH 5.5.

Figure 3

OmpR protein levels present in Ye9 cells grown under different conditions. Cytoplasmic extracts of cells grown to early stationary phase in buffered LB medium were Western blotted and probed with anti-OmpR antibody. Lanes: 1—PageRuler Prestained Protein Ladder Plus (Fermentas); lane 2—25°C, pH 7.0; lane 3—37°C, pH 7.0; lane 4—25°C, pH 5.5; lane 5—37°C, pH 5.5; lane 6—OmpR-His₆ protein (1.5 μg).

Figure 4

Effect of OmpR and different temperature and pH conditions on rovA expression determined using a rovA::lacZYA operon fusion. β-galactosidase activities were measured in strain YeR2 (OmpR⁺) and in the isogenic mutant strain ARR8 (OmpR⁻) grown to early stationary phase in LB medium at different pH at 25°C (A) or 37°C (B). The data presented are the means of three-independent experiments ± SD. Statistical significance was calculated using Student's t-test. ^**p < 0.01; ^***p < 0.001.

Figure 5

Influence of OmpR and RovA proteins on inv transcription in wild-type Y. enterocolitica, rovA, ompR and rovAompR mutants, and complemented strains. Cells were grown to early stationary phase at 25°C in LB medium (pH 7.0). Total RNA was extracted and used in sqRT-PCR to assess inv mRNA levels. PCRs for inv and 16S rRNA were carried out for 28 cycles and 16 cycles, respectively. The PCR reactions were mixed before loading onto the gel. (A) Lanes: MM—DNA molecular mass marker (100 bp ladder); 1—Ye9 (WT); 2—AS3 (rovA mutant); 3—AS3/pETRlac; 4—Ye9/pETRlac; 5—AR4 (ompR mutant); 6—AC1 (ompRrovA mutant). (B) The densities of inv bands relative to those of the 16S rRNA bands on the gel in part A. RT-PCR signals were averaged from 3 replicates (lanes 1, 2, 5, 6). Values are means ± SD; a, b, c, d, e—results of Tukey post-hoc multiple mean comparison test. Means without a common letter differ significantly (p < 0.05).

Figure 6

OmpR, RovA and H-NS binding sites in the promoter region of inv in Y. enterocolitica. Two RovA and H-NS binding sites (I and II), (Ellison and Miller, 2006b) and the putative OmpR binding site (Brzostek et al., 2007) are underlined. The transcriptional start site of the inv promoter (+1), ATG start codon and Shine-Dalgarno (SD) sequences are indicated.

Figure 7

Interaction of purified RovA from Ye9 strain with the inv promoter region examined in EMSA. EMSA showing the binding of increasing quantities of RovA-His₆ to the inv promoter region, using a 553-bp inv DNA fragment (−328 to +225) encompassing the RovA binding sites. The amount of RovA added was 0, 0.125, 0.25, 0.5, and 1.0 μg (lanes 1–5). A 300-bp fragment of the ngoA302V gene from Neisserria gonorrhoeae FA1090 was used as a negative control. DNA-protein complexes were separated by electrophoresis in 6% native polyacrylamide gels and silver stained.

Figure 8

Interaction of purified OmpR with the inv promoter region examined in EMSA. EMSA showing the binding of increasing quantities of non-phosphorylated (OmpR; 0.1, 0.2, 0.3, 0.4 μg—lanes 2–5) or phosphorylated (OmpR-P; 0.1, 0.2, 0.3, 0.4 μg—lanes 6–9) OmpR-His₆ protein to the inv promoter region. A 553-bp inv DNA fragment (−328 to +225) encompassing the OmpR and RovA binding sites was used. A 307-bp fragment of 16S rDNA of Y. enterocolitica was used as a negative control. Lane 1- inv promoter fragment and control DNA incubated without proteins. DNA-protein complexes were separated by electrophoresis in 6% native polyacrylamide gels and silver stained.

Figure 9

Competition for binding the inv promoter fragment between OmpR and RovA proteins. EMSAs examining competition for binding the inv promoter fragment between OmpR-P, which was added first (0.3 μg—lanes 2–5), and RovA (0.05, 0.125, 0.25 μg, lanes 3–5 respectively); and between RovA, which was added first (0.25 μg, lanes 6–9), and OmpR-P (0.05, 0.15, 0.3 μg—lanes 7–9). A 553-bp inv DNA fragment (−328 to + 225) encompassing the OmpR and RovA binding sites was used. Lane 1—inv promoter fragment incubated without proteins. DNA-protein complexes were separated by electrophoresis in 6% native polyacrylamide gels and silver stained. The arrowhead indicates the band excised for MS/MS analysis.

Figure 10

Amino acid sequencing and bioinformatic analysis of proteins identified by the LC-MS/MS. The DNA-protein complexes (indicated by the arrowhead at Figure 9) were subject to the MS/MS analysis. MS/MS data were used to search protein database. (A) The amino acid sequences of OmpR Y. enterocolitica strain Ye9 (GI 28912448) and RovA Y. enterocolitica 8081(GI 123442405) derived from the NCBI database (B). Peptides detected by MS/MS analysis are indicated in bold. Sixty-nine percentage of OmpR and forty-five percentage of RovA protein sequence are covered by matching peptides.