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Pseudomonas aeruginosa has four Na+/H+ antiporters that interconvert and balance Na+ and H+ gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we constructed four strains, each of which has only one antiporter, i.e., NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na+/H+ antiporters. Although the antiporters of P. aeruginosa have been studied previously, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa. The strains expressing only NhaB or Mrp, the two electrogenic antiporters, were able to grow essentially like the wild-type strain across a range of Na+ concentrations and pH values. Strains with only NhaP or NhaP2, which are electroneutral, grew more poorly at increasing Na+ concentrations, especially at high pH values, with the strain expressing NhaP being more sensitive. The strain with no Na+/H+ antiporters was extremely sensitive to the Na+ concentration and showed essentially no Na+(Li+)/H+ antiporter activity, but it retained most K+/H+ antiporter activity of the wild-type strain at pH 7.5 and approximately one-half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. Transcriptome sequencing analysis of the quadruple deletion strain showed widespread changes, including changes in pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological roles of these proteins and their activities in P. aeruginosa. [ABSTRACT FROM AUTHOR]

The Na + -pumping NADH-ubiquinone (UQ) oxidoreductase (Na + -NQR) is an essential bacterial respiratory enzyme that generates a Na + gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na + translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na + translocation using a series of synthetic UQs with Vibrio cholerae Na + -NQR reconstituted into liposomes. UQ 0 that has no side chain and UQ CH3 and UQ C2H5 , which have methyl and ethyl side chains, respectively, were catalytically reduced by Na + -NQR, but their reduction generated no membrane potential, indicating that the overall electron transfer and Na + translocation are not coupled. While these UQs were partly reduced by electron leak from the cofactor(s) located upstream of riboflavin, this complete loss of Na + translocation cannot be explained by the electron leak. Lengthening the UQ side chain to n-propyl (C 3 H 7 ) or longer significantly restored Na + translocation. It has been considered that Na + translocation is completed when riboflavin, a terminal redox cofactor residing within the membrane, is reduced. In this view, the role of UQ is simply to accept electrons from the reduced riboflavin to regenerate the stable neutral riboflavin radical and reset the catalytic cycle. However, the present study revealed that the final UQ reduction via reduced riboflavin makes an important contribution to Na + translocation through a critical role of its side chain. Based on the results, we discuss the critical role of the UQ side chain in Na + translocation., (Copyright © 2022 Elsevier B.V. All rights reserved.)