Table 1.
Categories of differentially expressed genes in JB485.
Fig 1.
LeuO represses virulence in RND-deficient V. cholerae.
V. cholerae were cultured under AKI conditions and processed as described in the methods. (A) leuO expression in WT and JB485 bearing pXB266 (leuO-lacZ). (B) Deletion of leuO restores CT and TcpA production (inset) in JB485. (C) TcpP Western blot showing that leuO deletion restores TcpP production in JB485. The data presented (A & B) is the means ± SD from at least three independent experiments. *, P≤0.05.
Fig 2.
(A & B) The indicated V. cholerae strains were cultured under AKI conditions for 3.5h when total RNA was isolated and used for qRT-PCR to quantify aphA, aphB, tcpP and toxR expression. P-values are relative to a hypothetical ratio of 1.0 and were determined using the Student’s t-test. * P<0.05. (C) ToxR Western blot. The indicated strains were grown under AKI conditions for 6h when aliquots were collected, normalized by OD600, and subjected to Western blotting with anti-ToxR antibody. The extra non-specific bands serve as loading controls. (D & E) Overexpression of leuO represses aphA-lacZ, but not rxtB-lacZ expression in E. coli. E. coli containing pBAD33::leuO or pBAD33 and a (D) aphA-lacZ or (E) rtxB-lacZ reporter plasmid was cultured in LB broth to mid-log phase (5h) when β-galactosidase activity was quantified. The data are the mean ±SD of 3 independent experiments. * P≤0.05 relative to the no arabinose control.
Fig 3.
LeuO expression in JB485 is dependent upon the ToxR PPD.
(A) Expression of leuO in JB485 requires toxRS. WT, ΔtoxRS or JB485 bearing pXB266 (leuO-lacZ) were cultured under AKI conditions for 3h or 5h when β-galactosidase activity was quantified. (B) Ectopic toxRS expression complements for leuO expression. The ΔtoxRS and JB485ΔtoxRS mutants bearing pXB266 and pBAD33::toxRS were cultured under AKI conditions without or with 0.1% arabinose for 5h when β-galactosidase was quantified. (C) ToxRΔPPD production. Strains were grown under AKI conditions for 5h when aliquots were collected, normalized by OD600, and used for Western blotting with anti-ToxR antibody. A toxRS mutant carrying pBAD18::toxRΔPPDS grown under AKI conditions in the presence of 0%, 0.01% and 0.1% arabinose is included as a marker for ToxRΔPPD protein. (D & E) Differential requirements of the ToxR PPD for ompU and leuO expression. The indicated strains carrying an ompU-lacZ or leuO-lacZ reporter were cultured under AKI conditions for 5h when β-galactosidase activity was quantified. *, P≤0.05 relative to the parental strain.
Fig 4.
ToxR activates leuO transcription in response to malate.
(A) Malate quantification in WT and RND null mutant JB485 culture supernatants and cells following growth under AKI conditions for 3.5h. Malate was quantified as described in the methods. The reported results are the means ± SD of six biological replicates with each replicate consisting of two technical replicates. * P≤0.05. (B) The indicated V. cholerae strains bearing pXB266 (leuO-lacZ) were cultured under AKI conditions for 4h when malate was added to 0, 1 or 2 mM. The cultures were then incubated with shaking for an additional hour when β-galactosidase production was quantified. The data are the mean ±SD of 3 independent experiments. * P≤0.05 relative to the no malate control.
Fig 5.
Putative model for RND-mediated efflux in environmental sensing.
(A) In WT cells growing under virulence gene inducing conditions the RND transporters expel metabolites from within the periplasm and ToxR functions with TcpP to activate ToxR regulon expression. Metabolites which are produced in the cytoplasm enter the periplasmic compartment either by passive diffusion through the cytoplasmic membrane or by active transport systems localized in the cytoplasmic membrane (denoted by the blue box). Exogenous metabolites enter the periplasm by diffusion through porin channels. (B) Inhibition of RND-mediated efflux results in the accumulation of metabolites that are normally expelled by the RND transporters. The accumulated metabolites activate ToxR via interaction with its periplasmic domain. Activated ToxR then modulates target gene expression (e.g. leuO) which leads to virulence repression and altered cell physiology. The accumulated metabolites also activate select two-component regulatory systems (e.g. CpxRA, CarRS, OmpREnvZ and VieSAB) which further effect transcriptional responses and environmental adaptation. Abbreviations: CT, cholera toxin; OM, outer membrane; PPD, periplasmic domain, RR, response regulator, TCP, toxin coregulated pilus; T, metabolite transporter.
Table 2.
Strains and plasmids used in this study.