Stringent response regulation of biofilm formation in Vibrio cholerae

Abstract

Biofilm formation is a key factor in Vibrio cholerae environmental survival and host colonization. Production of biofilm enables V. cholerae to survive and persist in aquatic environments and aids in the passage through the gastric acid barrier to allow access to the small intestine. The genes involved in biofilm formation are regulated by the transcriptional activators vpsR and vpsT, which are in turn transcriptionally regulated by a number of environmental signals. In this study, the role of the stringent response in biofilm formation was examined. V. cholerae mutants deficient in stringent response had a reduced ability to form biofilms, although they were not completely deficient in biofilm formation. There are three (p)ppGpp synthases in V. cholerae: RelA, SpoT, and RelV. All three synthases were necessary for vpsR transcription, with RelV showing the strongest effect. RelA was the only synthase that was necessary for vpsT expression. Stringent response regulation of vpsR and vpsT was shown to partially occur through rpoS. Biofilm formation in V. cholerae is controlled by a complex regulatory apparatus, with negative regulators of biofilm gene expression, such as quorum sensing, and positive regulators of biofilm genes, including stringent response, interacting to ensure that biofilm formation is coordinated with the environment.

Fig 1

Stringent response and V. cholerae biofilm expression. (A) Three factors regulate (p)ppGpp synthesis and hydrolysis in V. cholerae: RelA, SpoT, and RelV. All three factors are able to synthesize ppGpp and pppGpp from GDP and GTP, but only SpoT is able to hydrolyze (p)ppGpp. (B) The stringent response mediator (p)ppGpp is one of several global regulators that affect biofilm formation in V. cholerae. (p)ppGpp and c-di-GMP both induce expression of VpsR and VpsT, the transcriptional activators of biofilm formation. cAMP induces expression of VpsR but inhibits expression of VpsT. High cell density induces the quorum-sensing regulator HapR, which represses expression of VpsT and possibly VpsR (see the text for references).

Fig 2

Production of (p)ppGpp in wild-type and synthase-mutant V. cholerae strains in (A) actively growing cultures and (B) stationary biofilm cultures. N16961 and in-frame deletions of the indicated genes were used. Each culture was grown in LB medium incubated with [³²P]orthophosphate. TLC was performed on culture extracts to identify nucleotides. (C) Growth curves of the N16961 wild type (WT) and the (p)ppGpp synthase mutants.

Fig 3

Stringent response regulates biofilm formation. Biofilm assays were performed in both N16961 and C6706 backgrounds to test involvement of the relA, spoT, and relV gene products in biofilm formation. (A) N16961-derived strains. (B) C6706-derived strains. (C) Overexpression of RelA induces biofilm formation. An N16961 strain containing the relA fusion gene under the control of the P_BAD promoter was incubated with 0.025% arabinose to induce expression of relA. (A and B) Biofilm formation was normalized to cell growth by calculating the ratio of A₅₇₀/A₆₀₀ (crystal violet staining to culture density). Data were analyzed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. For panel A, the asterisk indicates a significant (P < 0.01) difference compared to the wild type. In addition, there were significant differences for relA versus relA spoT and relV versus relA relV (P < 0.05), relA relV versus relA spoT relV (P < 0.01), and relA versus relA relV, relA versus relA spoT relV, relV versus relA spoT relV, and relA relV versus relA spoT relV (P < 0.001). For panel B, the asterisk indicates a significant (P < 0.01) difference compared to every value indicated without an asterisk. For panel C, the asterisk indicates a significant (P < 0.01) difference compared to uninduced cultures.

Fig 4

Expression of vpsA and vpsL in stringent response mutants. vpsA expression (A) and vpsL expression (B) in the indicated strains were measured using qRT-PCR. Data were analyzed using a one-tailed Student's t test. Asterisks indicate a significant (P < 0.05) difference.

Fig 5

(p)ppGpp synthases have specific effects on vpsR and vpsT expression. (A and B) We constructed vpsR and vpsT promoter fusions with the lacZ gene and measured β-galactosidase activity in each N16961 strain background as indicated. (A) vpsR expression. (B) vpsT expression. (C and D) vpsR expression (C) and vpsT expression (D) were measured using qRT-PCR in the indicated strains. Serine hydroxamate was added to the culture to induce the stringent response. Solid bars indicate untreated cultures, and striped bars indicate cultures treated with serine hydroxamate. vpsR and vpsT transcript levels were normalized to gyrA levels and compared in a wild-type and relA spoT relV strain. (A and B) Data were analyzed using one-way ANOVA and Bonferroni's multiple comparison test. For panel A, asterisks indicate a significant difference compared to the wild type (at least P < 0.01). For panel B, asterisks indicate a significant difference compared to the wild type (P < 0.001). For panels C and D, data were analyzed using a one-tailed Student's t test. Asterisks indicate a significant difference between the mock-treated and serine hydroxamate-treated cultures (P < 0.05).

Fig 6

Stringent response regulation of rpoS affects vpsR and vpsT expression. Promoter fusions of rpoS, vpsR, and vpsT and β-galactosidase assays were used to measure gene expression in wild-type N16961-derived cells and the indicated mutant strains. (A) rpoS expression. (B) vpsR expression. (C) vpsT expression. Data were analyzed using one-way ANOVA and Bonferroni's multiple comparison test. For panel A, asterisks indicate a significant difference compared to the wild type (P < 0.01). In addition, there were significant differences for relA versus relV, relA versus relA spoT, relV versus relA relV, and relV versus relA spoT relV (P < 0.001). For panel B, asterisks indicate a significant difference compared to all others (P < 0.01 for all [except P < 0.05 for rpoS versus relA spoT relV rpoS]). In addition, there were significant differences for rpoS versus relA spoT relV rpoS (P < 0.05), relA spoT relV versus relA spoT relV rpoS (P < 0.01), and relA spoT relV versus rpoS (P < 0.001). (C) Asterisks indicate a significant difference compared to the wild type (P < 0.001).

Fig 7

Effects of stringent response and HapR on biofilm formation and vpsR and vpsT expression. (A) Biofilm assays were performed in the indicated C6706-derived strains. (B and C) vpsR expression (B) and vpsT expression (C) were measured using β-galactosidase assays in wild-type cultures and in hapR and (p)ppGpp-null cultures. Data were analyzed using one-way ANOVA and Bonferroni's multiple comparison test. Asterisks indicate a significant difference from all other strains (P < 0.01 for panel A and P < 0.001 for panels B and C).