Transcriptional cross talk within the mar-sox-rob regulon in Escherichia coli is limited to the rob and marRAB operons

Abstract

Bacteria possess multiple mechanisms to survive exposure to various chemical stresses and antimicrobial compounds. In the enteric bacterium Escherichia coli, three homologous transcription factors-MarA, SoxS, and Rob-play a central role in coordinating this response. Three separate systems are known to regulate the expression and activities of MarA, SoxS, and Rob. However, a number of studies have shown that the three do not function in isolation but rather are coregulated through transcriptional cross talk. In this work, we systematically investigated the extent of transcriptional cross talk in the mar-sox-rob regulon. While the three transcription factors were found to have the potential to regulate each other's expression when ectopically expressed, the only significant interactions observed under physiological conditions were between mar and rob systems. MarA, SoxS, and Rob all activate the marRAB promoter, more so when they are induced by their respective inducers: salicylate, paraquat, and decanoate. None of the three proteins affects the soxS promoter, though unexpectedly, it was mildly repressed by decanoate by an unknown mechanism. SoxS is the only one of the three proteins to repress the rob promoter. Surprisingly, salicylate somewhat activates transcription of rob, while decanoate represses it a bit. Rob, in turn, activates not only its downstream promoters in response to salicylate but also the marRAB promoter. These results demonstrate that the mar and rob systems function together in response to salicylate.

Fig 1

Regulation of mar-sox-rob gene expression by MarA, SoxS, and Rob. (A) Strains contain plasmids pBAD30, pMarA, pSoxS, and pRob and single-copy, yfp transcriptional fusions. marRAB, soxS, and rob were deleted from cells, and cells contained a constitutively active mutant of SoxR (soxR105). Cells were grown in LB–0.2% arabinose medium for 4 h prior to fluorescence and optical density measurements. Fluorescence values have been divided by the optical density and then normalized to the value for the empty-plasmid (pBAD30) negative control. (B) mar-sox-rob regulatory network inferred from the data in panel A. Dark lines, interactions found to be significant under physiological conditions; gray lines, interactions found to be significant only when regulators are overexpressed. Strains used were CR917 (marRAB promoter), CR918 (soxS promoter), CR219 (rob promoter), and CR920 (inaA promoter) harboring pBAD30, pMarA, pSoxS, and pRob, respectively.

Fig 2

Effect of autoregulation on mar-sox-rob regulon activation in the absence of genetic cross talk. Activation of the marRAB (A), soxS (B), and rob (C) promoters during induction with 5 mM salicylate, 50 μM paraquat, and 5 mM decanoate, respectively. Strains used were CR935 and CR936 (A), CR939 and CR940 (B), and CR934 and CR935 (C). A.U., absorbance units.

Fig 3

Effect of inducible cross talk on mar-sox-rob gene expression. (A) Activation of the marRAB promoter in response to paraquat (PQ) and decanoate in the presence or absence of SoxS and Rob. (B) Activation of the soxS promoter in response to salicylate and decanoate in the presence or absence of MarA and Rob. (C) Activation of the rob promoter in response to salicylate and paraquat in the presence or absence of MarA and SoxS. Salicylate, paraquat, and decanoate were used at concentrations of 5 mM, 50 μM, and 5 mM, respectively. Strains used were CR921 to CR924 (A), CR925 to CR928 (B), and CR929 to CR932 (C).

Fig 4

Effect of cross talk on activation of natively encoded systems observed through monitoring of the transcriptional responses of the marRAB (A), soxS (B), and rob (C) promoters. Each system was examined in the absence of one or both systems capable of cross regulation. Strains used were CR700 and CR933 to CR935 (A), CR914 and CR937 to CR939 (B), and CR915 and CR941 to CR943 (C).

Fig 5

Maximal activation of the downstream mar-sox-rob regulon in response to canonical inducers requires a fully intact mar-sox-rob network. Levels of transcriptional activity of two downstream promoters, the inaA (A) and micF (B) promoters, during exposure to the canonical mar-sox-rob inducers salicylate, paraquat, and decanoate. Strains used were CR916 and CR945 to CR948 (A) and CR715 and CR949 to CR952 (B).

Fig 6

Rob does not directly bind salicylate. Measurements were made using a VP-ITC (MicroCal) calorimeter with purified Rob (10 μM) and 5 mM 2,2-dipyridyl (A), 5 mM salicylate (B), and 10 mM decanoate (C). Data were collected and analyzed using the Origin-based MicroCal software suite. We also tested whether a buffer-only control with 5 mM 2,2-dipyridyl would also yield an enthalpic change due to metal chelation. While the buffer-only control yielded an enthalpic change, it was appreciably less than that with Rob present (data not shown).