Indole pulse signalling regulates the cytoplasmic pH of E. coli in a memory-like manner

This is the final version. Available from the publisher via the DOI in this record.The data that support the findings of this study are available in Apollo (University of Cambridge Repository) at https://doi.org/10.17863/CAM.26410.Bacterial cells are critically dependent upon pH regulation. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of Escherichia coli. Indole is an aromatic molecule with diverse signalling roles. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry. We show that under conditions permitting indole production, cells maintain their cytoplasmic pH at 7.2. In contrast, under conditions where no indole is produced, the cytoplasmic pH is near 7.8. We demonstrate that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, we illustrate that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane. Additionally, we show that the efect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a “memory” to maintain the cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.Leverhulme TrustEngineering and Physical Sciences Research Council (EPSRC)Biotechnology and Biological Sciences Research Council (BBSRC)Winton Programme for the Physics of SustainabilityTrinity-Henry Barlow ScholarshipNational Physical Laboratory (UK)European Research Council (ERC

Zn(II) mediates vancomycin polymerization and potentiates its antibiotic activity against resistant bacteria

Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors.This work was supported by funding from the Royal Society, UK (516002.K5877/ROG) and the Medical Research Council, UK (G0700141). A.Z. was supported from the Said foundation, the Cambridge Trust and the Cambridge Philosophical Society

Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment

This is the final version. Available from the American Society for Microbiology via the DOI in this record. Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment.Biotechnology and Biological Sciences Research Council (BBSRC)Wellcome TrustMedical Research Council (MRC)The Royal SocietyThe Gordon and Betty Moore FoundationMarie Skłodowska‐CurieLeverhulme TrustUnited Kingdom Ministry of DefenseUniversity of Exete

Research data supporting "Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner"

This dataset was collected by Fluorescence Spectroscopy and FlowCytometryJinbo Zhu thank the support from UK Engineering and Physical Sciences Research Council (EPSRC, EP/M008258/1). Kareem Al Nahas acknowledges support from the Winton Programme for the Physics of Sustainability, Trinity-Henry Barlow Scholarship, National Physical Laboratory and ERC consolidator grant (DesignerPores 647144)