Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis

Abstract

Background: The osmotic regulator OmpR in Escherichia coli regulates differentially the expression of major porin proteins OmpF and OmpC. In Yersinia enterocolitica and Y. pseudotuberculosis, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in Y. pestis are not yet fully understood.

Results: Y. pestis OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The ompR mutation likely did not affect the virulence of Y. pestis strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the ompR mutation, indicating the global regulatory role of OmpR in Y. pestis. Real-time RT-PCR or lacZ fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. ompC, F, X, and R were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner.

Conclusion: OmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. Y. pestis OmpR regulates ompC, F, X, and R directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and × at high osmolarity in Y. pestis, in contrast to the reciprocal regulation of them in E. coli. The main difference is that ompF expression is not repressed at high osmolarity in Y. pestis, which is likely due to the absence of a promoter-distal OmpR-binding site for ompF.

Figure 1

Phenotypes of ΔompR. a) WT or ΔompR was characterized for the ability to survive under a range of environmental stresses associated with macrophage-killing mechanisms. The '% survival' values indicate the percentage of viable bacteria after exposure to the environmental stresses. b) WT or ΔompR was used to infect macrophages so as to investigate bacterial resistance to phagocytosis in vivo and adhesion on the cell surface. The percentage of cell-associated bacteria was determined by dividing the total number of cell-associated bacteria into the total CFU in the inoculum, while the percentage of phagocytosis was calculated by dividing the number of cell-associated bacteria by the number of intracellular bacteria. Finally, student's t test was carried out to determine the statistical significance (P < 0.05).

Figure 2

Regulation of ompC, F and X by OmpR. a) Real-time RT-PCR. The mRNA levels of each indicated gene were compared between ΔompR and WT. This figure shows the increased (positive number) or decreased (minus one) mean fold for each gene in ΔompR relative to WT. b) LacZ fusion reporter. A promoter-proximal region of each indicated gene was cloned into pRW50 containing a promoterless lacZ reporter gene, and transformed into WT or ΔompR to determine the promoter activity (β-galactosidase activity in cellular extracts). The empty plasmid was also introduced into each strain as negative control, which gave extremely low promoter activity (data not shown). Positive and minus numbers indicate the increased and decreased mean folds, respectively, for the detecting promoter activity in ΔompR relative to WT. c) Primer extension. Primer extension assays were performed for each indicated gene using total RNAs isolated from the exponential-phase of WT or ΔompR. An oligonucleotide primer complementary to the RNA transcript of each gene was designed from a suitable position. The primer extension products were analyzed with 8 M urea-6% acrylamide sequencing gel. Lanes C, T, A, and G represent the Sanger sequencing reactions; on the right side, DNA sequences are shown from the bottom (5') to the top (3'), and the transcription start sites are underlined. d) DNase I footprinting. The labeled DNA probe was incubated with various amounts of purified His-OmpR (lanes 1, 2, 3, 4, and 5 contained 0, 5, 10, 15 and 20 pmol, respectively) with the addition of acetyl phosphate, and subjected to DNase I footprinting assay. Lanes G, A, T, and C represent the Sanger sequencing reactions, and theprotected regions (bold lines) are indicated on the right-hand side. The numbers indicate the nucleotide positions upstream of the transcriptional start sites.

Figure 3

Autoregulation of OmpR but not CRP. a) LacZ fusion reporter. A recombinant pRW50 that contained a promoter-proximal region of ompR was transformed into WT or ΔompR to determine the promoter activity. This figure shows the decreased mean fold for the ompR promoter activity in ΔompR relative to WT. d) DNase I footprinting. For DNase I digestion, the labeled promoter-proximal region of ompR was incubated with various amounts of purified, acetyl phosphate-treated His-OmpR (lanes 1, 2, and 3 contained 0, 10 and 20 pmol, respectively). Lanes G, A, T, and C represent the Sanger sequencing reactions, and the protected regions (bold lines) are indicated on the right-hand side. The numbers indicate the nucleotide positions upstream the transcriptional start sites.

Figure 4

Promoter activity ompC, F, X and R under different concentrations of NaCl. The lacZ fusion reporter plasmid for each of ompC, F, X, and R was transformed into WT or ΔompR to determine the β-galactosidase activity (miller unites), respectively. Bacterial cultures in the LB broth (0.5% yeast extract, 1% tryptone and 1% NaCl) at the middle exponential growth phase (an OD₆₂₀ of about 1.0) were diluted 1:50 into the fresh LB broth. Bacterial cells were grown at 26°C to an OD₆₂₀ of about 1.0, pelleted and resuspended in the fresh LB broth containing 0, 0.4, 0.6, 1, 3 and 6% NaCl, respectively, and allowed to continue growing at 26°C for 20 min for bacterial harvest.

Figure 5

OmpR consensus-like sequences within the target promoter regions. The underlined segments are OmpR binding sites determined by DNase I footprinting in Y. pestis. The boxed areas represent the sub-elements of OmpR consensus-like sequence. This figure also shows the three (e.g., C1-C2-C3) or 2 (e.g., X1-X2) tandems of OmpR consensus-like sequences, where each 20 bp tandem has been divided into two 10 bp sub-elements (boxed).