YqjA is a Putative Transporter Essential for Alkaline pH Tolerance in Escherichia Coli
Authors: Kumar Sujeet , Doerrler William T. 
Published: November 2023

Alkaline pH tolerance is a vital factor in the epidemiology of pathogenic bacteria, for existence in oceanic environments and for industrial applications and bioremediation processes. Bacteria must retain a steady cytoplasmic pH of about 7.6 in wide range of external pH including alkaline milieu. Although several mechanisms are available for bacteria to survive in alkaline environments, cytoplasmic membrane cation/H+ antiporters play a key role by bringing protons into the cytoplasm in exchange of cations to help maintain a cytoplasm that is acidic relative to the outside environment. To date, a number of cation/H+ antiporters including NhaA, NhaB, ChaA, MdfA and MdtM have been reported to function in alkaline pH homeostasis in Escherichia coli. The poorly understood DedA/Tvp38 membrane protein family (DedA for short) is present in most sequenced genomes including E. coli. Most are predicted to possess 4–6 membrane spanning domains, although structural studies have not been carried out for any DedA family member. We have previously shown that the partially redundant E. coli DedA family members YghB and YqjA (displaying 62% amino acid identity) are required for proper cell division, drug/biocide efflux and growth at elevated pH. Growth at alkaline pH (ranging from pH 8.5 to 9.5) requires only the YqjA protein with YghB playing only a minor role. Function of both YqjA and YghB requires acidic amino acids present in the first predicted transmembrane domain (E39 and D51 of both proteins). Overexpression of yqjA (or yghB) from a plasmid restores growth at alkaline pH, but only when millimolar concentrations of sodium or potassium are present in the growth medium. Moreover, overexpression of mdfA, encoding a Na+/K+‐H+ antiporter belonging to the unrelated major facilitator superfamily, partially complements the alkaline pH sensitivity of the ΔyqjA mutant. The objective of this study was to clarify the mechanism and regulation of YqjA in alkaline pH tolerance. We employed site‐directed mutagenesis and gene complementation to analyze conserved, charged amino acids of YqjA. We found that at least two membrane embedded arginines (R130 and R136) are needed for the function of YqjA. Importantly, while lysine can functionally substitute for R130 indicating only the necessity of a positive charge at this position, it could not at position R136 indicating a specific role here for arginine in the transport mechanism. All mutant proteins were well expressed and membrane associated. Additionally, using a reporter gene placed under the control of the yqjA promoter, we found that yqjA expression was higher at alkaline pH than at acidic and neutral pH. Moreover, increased expression of yqjA at elevated pH also required sodium or potassium salts. We observed that the transcriptional regulator CpxR is required for the expression of yqjA at alkaline pH in the presence of Na+/K+. Such regulation was not observed for the closely related YghB. Based on these results, we suggest that YqjA, a member of DedA/Tvp38 family, is a proton‐dependent membrane transporter required for survival of E. coli at alkaline pH.Support or Funding InformationFinancial support provided by the Nation Science Foundation.