Differential regulation of the hmsCDE operon in Yersinia pestis and Yersinia pseudotuberculosis by the Rcs phosphorelay system

Abstract

Yersinia pestis, the agent of plague, forms a biofilm in its flea vector to enhance transmission. Y. pestis biofilm development is positively regulated by hmsT and hmsD, encoding diguanylate cyclases (DGCs) involved in synthesis of the bacterial second messenger c-di-GMP. rcsA, encoding an auxiliary protein in Rcs phosphorelay, is nonfunctional in Y. pestis, while in Yersinia pseudotuberculosis, rcsA is functional and represses biofilms. Previously we showed that Rcs phosphorelay negatively regulates transcription of hmsT in Y. pestis and its ancestor Yersinia pseudotuberculosis. In this study, we show that Rcs positively regulates hmsCDE operon (encoding HmsD) in Y. pestis; while in the presence of functional rcsA, Rcs represses hmsCDE operon in Y. pseudotuberculosis. Loss of rcsA's function in Y. pestis not only causes derepression of hmsT but also causes activation of hmsD, which may account for the increased biofilm formation in Y. pestis. In addition, differential regulation of the two DGCs, HmsT and HmsD by Rcs may help Y. pestis to adapt to different environment.

Figure 1. Regulation of hmsCDE by Rcs.

(a) β-galactosidase activities of hmsC::lacZ reporter in Y. pestis. Y. pestis KIM6+ (WT) transformed with empty vector (vector) and functional RcsA (p-rcsA), RcsB deletion mutant transformed with empty vector (vector), functional RcsA (p-rcsA), wild-type RcsB (p-rcsB), inactive RcsB (p-rcsB (D56Q)) and active RcsB (p-rcsB (D56E)). (b) mRNA levels of hmsD regulated by Rcs in Y. pestis. hmsD mRNA levels were determined by qRT- PCR (Methods), and normalized to wild type. The mean and standard deviation of three independent experiments with three replicates are indicated. *P < 0.05. (c) Expression of HmsD regulated by Rcs in Y. pestis. Western blots of total protein-matched lysates prepared from stationary phase LB cultures and probed with anti-Myc antibody. Levels of HmsD were quantitated by densitometry using ImageJ from at least two independent experiments: numbers below blots indicate the ratio of HmsD from the indicated strain compared to that from wild-type hmsD-Myc₂ strain (WT). ns, none specific band. Strain designations (Supplementary Table S1) are: Control, KIM6+ without Myc tag; WT, hmsD-Myc₂; rcsA+, functional rcsA hmsD-Myc₂; ΔrcsB, ΔrcsB hmsD-Myc₂; ΔrcsB Vector, ΔrcsB hmsD-Myc₂/pUC19; ΔrcsB p-rcsB, ΔrcsB hmsD-Myc₂/pYC332; ΔrcsD Vector, ΔrcsD-N-terminal hmsD- Myc₂/pET-32a; ΔrcsD p-rcsD, ΔrcsD-N-terminal hmsD- Myc₂/pYC225. Full-length blots are presented in Supplementary Fig. S5 online.

Figure 2. hmsCDE positively regulates biofilm formation in Y. pestis.

Y. pestis biofilms produced in polystyrene culture dishes and quantified by crystal violet staining (Methods). The mean and standard deviation of three independent experiments with three replicates are indicated. *P < 0.05, **P < 0.01.

Figure 3. Organization of hmsCDE promoter-proximal region and the predicted or verified cis-acting elements.

Top, the consensus RcsAB box TaAGaatatTCcta. The capital letters indicate conserved nucleotides. The right half is reported as the RcsB binding site, named B for short while the left part might be the RcsA binding site, named A for short. Middle, the organization of hmsCDE promoter-proximal region. Transcription start site G (+1) (under bent arrow) was mapped 64-bp upstream of the translation initiator codon ATG by 5′ RACE. By alignment with the consensus RcsAB box, the putative Rcs box of hmsC is located at 59-bp upstream of the transcription start site. A repeat of the left half of the conserved putative RcsA binding site is located immediately upstream of RcsAB box, thus it is designated as the RcsAAB box. Bottom, Rcs box mutations were introduced into the putative binding sites located at positions indicated. X indicates the binding site mutated.

Figure 4. Role of the RcsAAB box on transcriptional regulation of hmsCDE by Rcs.

β-galactosidase activities of hmsC::lacZ reporters with the RcsAAB box mutated to (a) RcsXXX, (b) RcsAXX, (c) RcsABX or (d) RcsXAB were analyzed. The mean and standard deviation of three independent experiments with three replicates are indicated. The mean and standard deviation of three independent experiments with three replicates are indicated. *P < 0.05, **P < 0.01.

Figure 5. Role of the RcsAAB box on regulation of expression of HmsD by Rcs in Y. pestis.

Western blots of total protein-matched lysates prepared from cells with mutation of (a) RcsXXX, (b) RcsAXX, (c) RcsABX or (d) RcsXAB were analyzed by anti-Myc antibody. Levels of HmsD were quantitated by using ImageJ from at least two independent experiments: numbers below blots indicate the ratio of HmsD from the indicated strain compared to that from wild-type hmsD-Myc₂ strain (WT). Strain designations (Supplementary Table S1) are: 1, KIM6+; 2, hmsD-Myc₂; 3, Rcs box mutation, hmsD-Myc₂; 4, functional rcsA, Rcs box mutation, hmsD-Myc₂; 5, ΔrcsB, Rcs box mutation, hmsD-Myc₂; 6, ΔrcsB, Rcs box mutation, hmsD-Myc₂/pUC19; 7, ΔrcsB, Rcs box mutation, hmsD-Myc₂/pYC332. pYC332: p-rcsB. Full-length blots are presented in Supplementary Fig. S6–S9 online.

Figure 6. RcsB binds to the hmsC promoter.

Electrophoretic mobility shift assays (EMSA) of hmsC promoter DNA incubated with increasing concentrations of RcsB. hmsD promoters with wild-type RcsAAB box (a) or with the mutated RcsAAB box RcsXXX (b), RcsAXX (c), RcsABX (d), or RcsXAB (e) were tested with identical protein combinations. Lane 1, hmsD probe alone; lanes 2–10, 100 ng hmsCDE probe with 400, 800, 1200, 1600, 2000, 2400, 4000, 6000 or 8000 ng of RcsB in the 16 μL reaction.

Figure 7. Regulation of hmsCDE transcription by Rcs in Y. pseudotuberculosis and Y. pestis.

In Y. pseudotuberculosis (top), RcsB together with RcsA indirectly repress the transcription of hmsCDE independent of the RcsAB binding site, while RcsB activates transcription of hmsCDE dependent on the RcsB binding site. As a consequence, Rcs negatively regulates the hmsCDE operon in Y. pseudotuberculosis. In Y. pestis (bottom), as rcsA is mutated to nonfunctional pseudogene, RcsB alone directly positively but indirectly negatively regulates hmsCDE operon. As a consequence, Rcs positively regulates the hmsCDE operon in Y. pestis.