Fig. 1.

The BumSR TCS is required for butyrate-modulated expression of peb3. (A) Expression of the peb3::astA transcriptional reporter in WT C. jejuni ΔastA and isogenic bumS and bumR mutants. The bumS and bumR mutants examined include a representative bumS::Tn and bumR::Tn mutant isolated in our genetic screen to identify Tn mutants defective for butyrate-dependent repression of peb3::astA expression and previously constructed C. jejuni ΔbumS and ΔbumR mutants with in-frame deletion of the coding sequence of each gene. C. jejuni strains were grown for 16 h in CDM alone (solid blue bars) or CDM supplemented with 12.5 mM butyrate (hatched blue bars) as indicated. The level of peb3::astA expression in each mutant is relative to the level of expression in WT C. jejuni grown without butyrate, which was set to 100 units. Results from a representative assay with each sample analyzed in triplicate are shown. Error bars indicate SDs of the average level of expression from three samples. Statistical significance was calculated in GraphPad Prism by ANOVA with Tukey’s test: * indicates the mutant grown in CDM alone had significantly increased or decreased expression relative to the WT strain grown in CDM alone; ** indicates the mutant grown with butyrate had a significantly increased or decreased expression relative to the WT strain grown with butyrate; *** indicates a strain showed a significantly different level of expression when grown in the presence of butyrate compared to growth in CDM alone (P < 0.05). (B) Production of BumR in WT and isogenic bumS and bumR mutants. Immunoblot analysis of the level of BumR in whole-cell lysates of WT and mutant strains after growth in CDM alone or CDM supplemented with 12.5 mM butyrate. RpoA served as a control for protein loading. Immunoblots were performed in triplicate, and values underneath the immunoblots represent quantified BumR mean signals relative to RpoA and normalized to WT, which was set to 100.