Bacteroides fragilis Maintains Concurrent Capability for Anaerobic and Nanaerobic Respiration.

Bacteroides species can use fumarate and oxygen as terminal electron acceptors during cellular respiration. In the human gut, oxygen diffuses from intestinal epithelial cells supplying "nanaerobic" oxygen levels. Many components of the anaerobic respiratory pathway have been determined, but such analyses have not been performed for nanaerobic respiration. Here, we present genetic, biochemical, enzymatic, and mass spectrometry analyses to elucidate the nanaerobic respiratory pathway in Bacteroides fragilis. Under anaerobic conditions, the transfer of electrons from NADH to the quinone pool has been shown to be contributed by two enzymes, NQR and NDH2. We find that the activity contributed by each under nanaerobic conditions is 77 and 23%, respectively, similar to the activity levels under anaerobic conditions. Using mass spectrometry, we show that the quinone pool also does not differ under these two conditions and consists of a mixture of menaquinone-8 to menaquinone-11, with menaquinone-10 predominant under both conditions. Analysis of fumarate reductase showed that it is synthesized and active under anaerobic and nanaerobic conditions. Previous RNA sequencing data and new transcription reporter assays show that expression of the cytochrome bd oxidase gene does not change under these conditions. Under nanaerobic conditions, we find both increased CydA protein and increased cytochrome bd activity. Reduced-minus-oxidized spectra of membranes showed the presence of heme d when the bacteria were grown in the presence of protoporphyrin IX and iron under both anaerobic and nanaerobic conditions, suggesting that the active oxidase can be assembled with or without oxygen. IMPORTANCE By performing a comprehensive analysis of nanaerobic respiration in Bacteroides fragilis, we show that this organism maintains capabilities for anaerobic respiration on fumarate and nanaerobic respiration on oxygen simultaneously. The contribution of the two NADH:quinone oxidoreductases and the composition of the quinone pool are the same under both conditions. Fumarate reductase and cytochrome bd are both present, and which of these terminal enzymes is active in electron transfer depends on the availability of the final electron acceptor: fumarate or oxygen. The synthesis of cytochrome bd and fumarate reductase under both conditions serves as an adaptation to an environment with low oxygen concentrations so that the bacteria can maximize energy conservation during fluctuating environmental conditions or occupation of different spatial niches.

Genetic and Biochemical Characterization of the Na+/H+ Antiporters of Pseudomonas aeruginosa.

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. IMPORTANCE Pseudomonas aeruginosa has four Na+/H+ antiporters that connect and interconvert its Na+ and H+ gradients. We have constructed four deletion mutants, each of which has only one of the four Na+/H+ antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH values and Na+ concentrations, whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH values. We also constructed a quadruple mutant lacking all four antiporters, in which the H+ and Na+ gradients are disconnected. This will make it possible to study the role of the two gradients independently.

The side chain of ubiquinone plays a critical role in Na+ translocation by the NADH-ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae.

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. UQ0 that has no side chain and UQCH3 and UQC2H5, 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 (C3H7) 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.

The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.