The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT

Abstract

Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT.

Fig 1

HmsT overexpression suppresses the biofilm defect of RcsA_YPSTB expression. (A) Y. pestis biofilms produced in polystyrene culture dishes and quantified by crystal violet staining (Materials and Methods). The mean and standard deviation (SD) are indicated; all of the strains except for the rcsA_YPSTB RcsAB* and RcsAB* strains differed significantly from the wild type (P < 0.05). (B) Biofilms adhering to the head of C. elegans were assayed for the ability to inhibit nematode growth by blocking feeding. Nematode eggs were deposited on Y. pestis lawns, and the fraction (mean ± SD) of animals developing to the fourth larval (L4) stage was scored (Materials and Methods).

Fig 2

Effect of Rcs on hmsT transcription in Y. pestis. hmsT mRNA levels were determined by quantitative real-time PCR (Materials and Methods) and normalized to wild type. The means and standard deviations from three independent experiments with three replicates are indicated. *, P < 0.05.

Fig 3

HmsT expression in Y. pestis. Western blots of total protein-matched lysates prepared from stationary-phase room temperature LB cultures and probed with polyclonal anti-HmsT antibody. The strain designations (Table 1) are as follows: ΔhmsT, CDY497; wild type, KIM6+; ΔrcsB, CDY326; rcsA-PSTB (rcsA_YPSTB), CDY330; and ΔrcsB rcsA-PSTB (rcsA_YPSTB), CDY421.

Fig 4

Effect of pseudogene rcsA_YPE on hmsT expression in Y. pseudotuberculosis. Shown are relative amounts of hmsT mRNA (A) and HmsT protein (B) made by the wild type (IP32953) with a functional rcsA allele (rcsA_YPSTB) and the isogenic strain (CDY564) with the pseudogene (rcsA_YPE) replacement. The transcript level was determined by qRT-PCR as described in the legend to Fig. 2, and the protein level was determined by Western blotting as described in the legend to Fig. 3. The p-hmsT strain (CDY985) was included in panel B to allow the HmsT band to be distinguished from an unidentified cross-reacting protein.

Fig 5

RcsB and RcsB-RcsA_YPSTB bind the hmsT promoter. (A) Electrophoretic mobility shift assays (EMSAs) of hmsT promoter DNA incubated with increasing concentrations of RcsB. Lanes 1 and 12, hmsT probe alone; lanes 2 to 11, hmsT probe with 100, 200, 400, 600, 800, 1,000, 1,500, 2,000, 3,000, or 5,000 ng of RcsB in the 16-μl reaction mixture. (B) Supershift of hmsT promoter by RcsA_YPSTB but not RcsA_YPE. Lane 1, hmsT probe alone; lanes 2 to 12, hmsT probe with 250 ng RcsB and with 250, 500, 1,000, 1,500, or 2,000 ng of RcsA_YPSTB (lanes 3 to 7) or RcsA_YPE (lanes 8 to 12).(C) RcsAB box mutation alters protein binding to the hmsT promoter. hmsT promoters with wild-type RcsAB box (top) or with the mutated RcsAB* box (bottom) were tested with identical protein combinations. Lanes 1 and 2, free hmsT probe in the absence or presence of bovine serum albumin (BSA); lanes 3 to 12 also contained BSA. Lanes 3 to 6 and 9 to 12 contained RcsB at 250, 500, 800, or 1,200 ng per reaction mixture; lanes 7 to 12 contained RcsA_YPSTB at 2,000 ng (lane 7) or 3,000 ng (lanes 8 to 12) per reaction mixture.

Fig 6

RcsA_YPE does not have a biofilm-related regulatory function in Y. pestis. (A) Relative amounts of adherent biofilm made by Y. pestis KIM6+ derivatives. The mean and standard deviation from at least three independent experiments with three replicates are indicated. (B) Western blots of total protein-matched lysates prepared from stationary-phase room temperature LB cultures and probed with polyclonal anti-His antibody. As predicted from genome sequence data, RcsA_YPE is 1 kDa bigger than RcsA_YPSTB because of a 10-amino-acid internal duplication. Strain designations (Table 1) are as follows: ΔrcsA, CDY327; rcsA-PE (rcsA_YPE) SY743; and rcsA-PSTB (rcsA_PSTB), SY744.