An Excretory Function for the Escherichia coli Outer Membrane Pore TolC: Upregulation of marA and soxS Transcription and Rob Activity Due to Metabolites Accumulated in tolC Mutants

Bacteria use efflux pumps to export a variety of xenobiotics (37). Some of these pumps have major clinical significance because they export multiple antibiotics (41). Recently, it has been noted that these pumps also export substances, such as bile salts and steroids, which occur in the environment of enteric bacteria (11; for a review, see reference 42). An essential component of several efflux systems is TolC. TolC forms a multifunctional outer membrane channel (for a review, see reference 23) with roles in colicin uptake and secretion, bacteriophage adsorption, efflux of multiple antibiotics, detergents, dyes, and organic solvents, and export of hemolysin, heat-stable enterotoxin II (61), microcin J25 (9), and enterobactin (6). Export through the TolC channel requires interaction with two other proteins, an inner membrane transporter (e.g., AcrB) and a periplasmic membrane fusion protein (e.g., AcrA) that links the transporter to TolC. By means of this tripartite structure, xenobiotics or cellular products are pumped directly out of the cell from the cytosol or inner membrane. Basal levels of the AcrAB-TolC pump are important in providing the intrinsic resistance of Escherichia coli to many xenobiotics. Upregulation of the AcrAB-TolC pump engenders a multiple-antibiotic-resistance phenotype which is clinically significant. However, at least seven other sets of proteins in E. coli, such as AcrEF, EmrAB, and MacAB, form similar tripartite pumps with TolC, but they have different substrate specificities. The structures of TolC, AcrB, and AcrA have been solved, and a docking mechanism for AcrAB-TolC has been proposed (12, 32). tolC and acrAB are members of the marA/soxS/rob regulon, which includes over 40 genes that promote resistance to multiple antibiotics, to numerous other xenobiotics, and to superoxides (3, 13, 24, 27, 38; for comprehensive reviews, see articles cited in reference 54). These genes are transcriptionally activated by three paralogous proteins, MarA, SoxS, and Rob, that bind a sequence upstream of the regulon promoter called the marbox. Each of these transcriptional activators is regulated in a distinct manner. MarA and SoxS are transcriptionally regulated. The marRAB operon is repressed by MarR and autoactivated by MarA (the role of MarB is unknown). The operon can be derepressed by treating cells with salicylate and related phenolics which decrease the affinity of MarR for its binding sites (1, 28). However, a “mar-independent effect” of salicylate that increases the transcription of marRAB and of inaA, another member of the marA/soxS/rob regulon, has also been described (7, 49). The effect on marRAB transcription was found even in strains with combined deletions or null mutations of marRAB, soxRS, rob, and emrAB (29), indicating the existence of an additional mechanism for activating the regulon. soxS transcription is activated by SoxR after SoxR is activated by exposure to superoxides or nitric oxide (43). Rob is a very abundant and stable protein in E. coli (∼10,000 molecules per cell) but has very little activity in vivo (2, 19, 46). Its activity is increased posttranslationally by treatment with 2,2′-dipyridyl, 4,4′-dipyridyl, bile salts, or decanoate (45, 46). Thus, each activator is activated in response to different environmental signals. Upregulation of these transcriptional activators engenders a low but significant level of multiple antibiotic, superoxide, and organic solvent resistance. The antibiotic resistance and solvent resistance are due primarily to the AcrAB-TolC pump (4, 13, 55). tolC has four known promoters, two of which (p3 and p4) are activated by MarA, SoxS, and Rob via a single, uniquely configured marbox (10, 25, 62). The acrAB promoter is also activated by MarA, SoxS, and Rob (24). Here, we examined the effects of tolC on the regulation of MarA, SoxS, and Rob during growth in standard laboratory media. We found elevated levels of transcription of marA and soxS and elevated activity of the Rob protein in tolC efflux mutants. From these findings, we infer that the following homeostatic loop occurs in wild-type bacteria: (i) normal metabolism results in the generation of certain intracellular metabolites that trigger the upregulation of the transcriptional activators MarA, SoxS, and Rob; (ii) these activators, in turn, upregulate tolC, increasing the capacity for excretion of the metabolites via TolC; and (iii) the resulting reduction in the concentrations of the trigger metabolites (TMs) restores the basal levels of the activators. In tolC mutants, the metabolites are not as effectively excreted, and the activator levels remain elevated.