Fluorescent Protein Sensors Illuminate Cellular pH Changes in Enterobacter cloacae: Unravelling the Mechanisms of TolC-Dependent Acid Survival

TolC is the outer membrane component of the tripartite RND efflux pump AcrAB-TolC. The underlying mechanism of TolC-dependent acid survival at physiological pH remains unclear. The present study aimed at understanding mechanism of TolC mediated acid survival in logarithmic phase culture of Enterobacter cloacae subsp. cloacae ATCC 13047 not preconditioned to acidic pH. Of the three distinct loci encoding tolC and tolC like proteins, only ECL_04363 (hereafter referred to as EctolC2) exhibited 86% identity with TolC protein from E. coli K12; deletion of which compromised survival at pH4.0. Tracking the periplasmic and cytoplasmic pH changes with fluorescent protein sensors indicated deletion of EctolC2 resulted in sustained cytoplasmic acidification and loss of pH homeostasis causing cell death. Exogenous supplementation of lysine significantly rescued acid induced lethality in EcΔtolC2 mutants. ΔmarRAB mutants exhibited slower death rate compared to Δrob mutants; underpinning role of the latter in upregulating EctolC2 at early time point of growth at pH4.0. Results of the study construe that EctolC2 provides reinforcement to the outer membrane preventing extreme acidification of the periplasm and thereby restoring cytoplasmic pH homeostasis. This study provides significant insight into mechanism of TolC dependent acid survival in E. cloacae.