Characterization of the induction and cellular role of the BaeSR two-component envelope stress response of Escherichia coli

Abstract

The bacterial cell envelope is the interface between a bacterium and its environment and is constantly exposed to environmental changes. The BaeSR two-component system regulates one of six envelope stress responses in Escherichia coli and is induced by spheroplasting, overexpression of the pilin subunit PapG, and exposure to indole. The known BaeR regulon is small, consisting of eight genes, mdtABCD-baeSR, acrD, and spy, two of which encode the BaeSR two-component system itself. In this study, we investigated the molecular nature of the BaeS-inducing cue and the cellular role of the BaeSR envelope stress response. We demonstrated that at least two flavonoids and sodium tungstate are novel inducers of the BaeSR response. Interestingly, flavonoids and sodium tungstate led to much stronger induction of the BaeSR response in an mdtA efflux pump mutant, while indole did not. These findings are consistent with the hypothesis that flavonoids and sodium tungstate are natural substrates of the MdtABC efflux pump. Indole has recently been implicated in cell-cell signaling and biofilm repression through a putative interaction with the LuxR homologue SdiA. Using genetic analyses, we found that induction of the BaeSR response by indole occurs via a pathway separate from the SdiA biofilm pathway. Further, we demonstrated that the BaeSR response does not influence biofilm formation, nor is it involved in indole-mediated inhibition of biofilm formation. We hypothesize that the main function of the Bae response is to upregulate efflux pump expression in response to specific envelope-damaging agents.

Fig. 1.

The Bae pathway is specifically induced by myricetin, sodium tungstate, and zinc. β-Galactosidase assays using a spy-lacZ transcriptional reporter were done in both a wild-type (TR530) and a ΔbaeR::Kn (SL100) background. This was done for 10 μg/ml myricetin (a), 5 mM sodium tungstate (b), 1 mM zinc (c), and 0.76% ethanol (d). Induction was done for 2 h after the cultures reached mid-log phase (final OD₆₀₀ = ∼0.7). **, P < 0.001 compared to uninduced cells. The error bars indicate standard deviations.

Fig. 2.

A ΔmdtA::Kn mutant is more sensitive to sodium tungstate induction than the wild type. (a and b) β-Galactosidase assays were done with WT (TR530) and ΔmdtA::Kn (SL102) strains that were uninduced or induced by 5 mM sodium tungstate (a) or 10 μg/ml myricetin (b). (c) Complementation of ΔmdtA::Kn with pCA-mdtA (SL143) or the vector control (vc) pCA24N (SL142). Overexpression was induced with 0.1 mM IPTG either with or without 5 mM sodium tungstate. The error bars indicate standard deviations.

Fig. 3.

Normal production and transport of cellular indole appear unrelated to Bae pathway activity. β-Galactosidase assays were done to compare BaeSR pathway induction by 2 mM indole in WT (TR530) and mutant strains in the cellular indole pathway. TR530 was compared to ΔtnaA::Kn (SL113) (TnaA is tryptophanase) (a), Δmtr::Kn (SL103) (Mtr is the tryptophan/indole/H⁺ symporter) (b), ΔacrF::Kn (SL131) (AcrF is the indole efflux pump) (c), and ΔsdiA::Kn (SL114) (SdiA is a LuxR homologue predicted to interact with indole) (d). The error bars indicate standard deviations.

Fig. 4.

The effect of indole on biofilm formation is independent of the Bae pathway. Biofilms were grown using the WT strain 2K1056 and the derivatives shown for 48 h at room temperature in 96-well polystyrene plates, followed by crystal violet staining and absorbance (A₆₀₀) readings to detect the amount of biofilm present. ΔfimA::Kn is a biofilm-deficient negative control, ΔbaeR::Kn is a BaeSR pathway-null mutant, and baeS1::Tn10cam is a BaeSR pathway gain-of-function mutant. Biofilms were grown with no additions, a 0.5% ethanol solvent control, or 500 μM indole. The error bars indicate standard deviations.