Role of the mar-sox-rob regulon in regulating outer membrane porin expression

Abstract

Multiple factors control the expression of the outer membrane porins OmpF and OmpC in Escherichia coli. In this work, we investigated the role of the mar-sox-rob regulon in regulating outer membrane porin expression in response to salicylate. We provide both genetic and physiological evidence that MarA and Rob can independently activate micF transcription in response to salicylate, leading to reduced OmpF expression. MarA was also found to repress OmpF expression through a MicF-independent pathway. In the case of OmpC, we found that its transcription was moderately increased in response to salicylate. However, this increase was independent of MarA and Rob. Finally, we found that the reduction in OmpF expression in a tolC mutant is due primarily to Rob. Collectively, this work further clarifies the coordinated role of MarA and Rob in regulating the expression of the outer membrane porins.

Fig. 1.

The levels of OmpC, OmpF, and OmpA in the outer membrane in the presence or absence of salicylate. Cells were grown overnight in medium A and subcultured 1:200 in fresh medium A in the presence or absence of 5 mM salicylate (SAL). OmpC, OmpF, and OmpA protein bands are indicated. Strains used in this experiment were MG1655 and CR720.

Fig. 2.

Full repression of ompF translation during salicylate exposure requires both MarA and Rob. (A) Levels of ompF′-′yfp translation. (B) Transcriptional activity of the P_ompF promoter. (C) Transcriptional activity of the P_micF promoter. Cells were grown overnight in medium A and subcultured 1:200 in medium A containing 5 mM salicylate for 4 h prior to fluorescence and optical density measurements. Presence or absence of genes is denoted by + or −, respectively. Strains used in this experiment were CR713 to CR715, CR737 to CR744, CR754 to CR761, and CR771 to CR778. (D) Transcriptional activity of P_micF and levels of ompF′-′yfp translation during ectopic complementation of MarA and Rob in the presence and absence of MicF. Cells were grown in medium A overnight and subcultured 1:200 in fresh medium A with and without 0.1% arabinose. Strains used in this experiment are CR715, CR774, CR776, CR781, CR714, CR757, CR759, and CR764.

Fig. 3.

Both MarA and Rob are required to fully repress OmpF expression in the outer membrane during salicylate exposure. Presence or absence of genes is indicated by + and −, respectively. Cells were grown overnight in medium A and subcultured 1:200 in fresh medium A containing 5 mM salicylate. Cultures were grown to mid-logarithmic phase prior to envelope extraction. Envelope fractions were displayed on 10% acrylamide–6 M urea-1% SDS gels and stained with Coomassie R250. Strains used were MG1655 and CR721 to CR728.

Fig. 4.

MarA functions through MicF-dependent and MicF-independent pathways to reduce the levels of OmpF during salicylate exposure. (A) Levels of ompF′-′yfp translation (strains CR757 and CR762 to CR764). (B) Transcriptional activity of the P_ompF promoter (strains CR740 and CR745 to CR747). (C) Transcriptional activity of the P_micF promoter (strains CR774 and CR779 to CR781). (D) Levels of OmpC, OmpF, and OmpA in the envelope fraction displayed on a 10% acrylamide–6 M urea-1% SDS gel (strains MG1655, CR724, and CR729 to CR731). Cells were grown in medium A overnight and subcultured 1:200 in fresh medium A with 5 mM salicylate. Cultures were grown for 4 h prior to fluorescence and optical density measurements or to mid-log phase prior to envelope extraction.

Fig. 5.

Increases in ompC transcription are independent of MarA and Rob. Cells were grown overnight in medium A and subcultured 1:200 in fresh medium A with or without 5 mM salicylate. Cultures were grown for 4 h prior to fluorescence and optical density measurements. Strains used in this experiment were MDG147 and CR788 to CR790.

Fig. 6.

Reduction in ompF translation by MicF in tolC mutants is a result of Rob-dependent activation of micF gene expression. (A) Levels of ompF′-′yfp translation. (B) Transcriptional activity of the P_ompF promoter. (C) Transcriptional activity of the P_micF promoter. Cells were grown overnight in medium A and subcultured 1:200 in medium A containing 5 mM salicylate for 4 h prior to fluorescence and optical density measurements. Strains used in this experiment were CR713 to CR715, CR748 to CR753, CR765 to CR769, and CR782 to CR787.

Fig. 7.

MicF-dependent reduction of OmpF expression in tolC mutants is a result of Rob activation of micF gene expression. Cells were grown overnight in medium A and subcultured 1:200 in fresh medium A containing 5 mM salicylate. Cultures were grown to mid-logarithmic phase prior to envelope extraction. Envelope fractions were displayed on 10% acrylamide–6 M urea-1% SDS gels and stained with Coomassie R250. Strains used were MG1655, CR702, and CR732 to CR736.