Reduction of Cellular Stress by TolC-Dependent Efflux Pumps in Escherichia coli Indicated by BaeSR and CpxARP Activation of spy in Efflux Mutants

Abstract

Escherichia coli has nine inner membrane efflux pumps which complex with the outer membrane protein TolC and cognate membrane fusion proteins to form tripartite transperiplasmic pumps with diverse functions, including the expulsion of antibiotics. We recently observed that tolC mutants have elevated activities for three stress response regulators, MarA, SoxS, and Rob, and we suggested that TolC-dependent efflux is required to prevent the accumulation of stressful cellular metabolites. Here, we used spy::lacZ fusions to show that two systems for sensing/repairing extracytoplasmic stress, BaeRS and CpxARP, are activated in the absence of TolC-dependent efflux. In either tolC mutants or bacteria with mutations in the genes for four TolC-dependent efflux pumps, spy expression was increased 6-to 8-fold. spy encodes a periplasmic chaperone regulated by the BaeRS and CpxARP stress response systems. The overexpression of spy in tolC or multiple efflux pump mutants also depended on these systems. spy overexpression was not due to acetate, ethanol, or indole accumulation, since external acetate had only a minor effect on wild-type cells, ethanol had a large effect that was not CpxA dependent, and a tolC tnaA mutant which cannot accumulate internal indole overexpressed spy. We propose that, unless TolC-dependent pumps excrete certain metabolites, the metabolites accumulate and activate at least five different stress response systems.

Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

“…Such regulation is not unprecedented. For example, a high level of spy gene expression requires that both the Cpx and the Bae stress responses be activated (44). Thus, E. coli is able to fine-tune transcription levels and cellular physiology based on environmental stimuli, the degree of stress-induced damage, and the specific stress responses that become activated.…”

The Vibrio cholerae Cpx Envelope Stress Response Senses and Mediates Adaptation to Low Iron

“…These data support the hypothesis put forth here and by others that Cpx induction in these mutants is due to the accumulation of a specific metabolite that can be handled only by these efflux pumps. Toxic, Cpx-inducing metabolites likely also induce the Cpx response in other organisms, since it has been demonstrated in E. coli that the Cpx pathway is activated in a tolC mutant (84), and in Sinorhizobium meliloti, a symbiotic partner of the leguminous plant Medicago sativa, the absence of a functional TolC protein leads to an increased susceptibility to antimicrobial agents and to activation of the Cpx response (85). Surprisingly, we found that Cpx induction by diverse cues could be reversed in all cases by the addition of exogenous iron (Fig.…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…Tannins, secondary metabolites of plants, can also induce the expression of MdtABC via BaeSR (761). Of the BaeSR regulon, there is a spy gene encoding a periplasmic chaperone (Spy), which shows increased levels in TolC or pump mutants and requires BaeSR and CpxAR systems for full activation (762). The expression of MdtEF is positively regulated by two AraC family activators, YdeO and GadX (763)(764)(765), and is also stimulated by N-acetyl-D-glucosamine through catabolite activation (763) and overexpression of a small (85-nucleotide) noncoding DsrA RNA (766).…”