Characterization of the Pseudomonas aeruginosa NQR complex, a bacterial proton pump with roles in autopoisoning resistance

Abstract

is a Gram-negative bacterium responsible for a large number of nosocomial infections. The respiratory chain contains the ion-pumping NADH:ubiquinone oxidoreductase (NQR). This enzyme couples the transfer of electrons from NADH to ubiquinone to the pumping of sodium ions across the cell membrane, generating a gradient that drives essential cellular processes in many bacteria. In this study, we characterized NQR (Pa-NQR) to elucidate its physiologic function. Our analyses reveal that Pa-NQR, in contrast with NQR homologues from other bacterial species, is not a sodium pump, but rather a completely new form of proton pump. Homology modeling and molecular dynamics simulations suggest that cation selectivity could be determined by the exit ion channels. We also show that Pa-NQR is resistant to the inhibitor 2--heptyl-4-hydroxyquinoline -oxide (HQNO). HQNO is a quinolone secreted by during infection that acts as a quorum sensing agent and also has bactericidal properties against other bacteria. Using comparative analysis and computational modeling of the ubiquinone-binding site, we identified the specific residues that confer resistance toward this inhibitor. In summary, our findings indicate that Pa-NQR is a proton pump rather than a sodium pump and is highly resistant against the -produced compound HQNO, suggesting an important role in the adaptation against autotoxicity. These results provide a deep understanding of the metabolic role of NQR in and provide insight into the structural factors that determine the functional specialization in this family of respiratory complexes.

The aerobic respiratory chain of Pseudomonas aeruginosa cultured in artificial urine media: Role of NQR and terminal oxidases

“…The most important physiologic difference between these enzymes is that complex I is a proton pump, while NDH-2 is not involved in ion pumping [17,23]. Although in many cases it has been reported that NQR acts as a sodium pump, we recently described that P. aeruginosa NQR is an ion pump that specifically transports protons [24]. This raises questions regarding the role of each of these enzymes, especially since NQR https://doi.org/10.1371/journal.pone.0231965.g001…”

“…P. aeruginosa NQR was purified as described previously [24]. Briefly, P. aeruginosa nqrA-F NQR operon was cloned in pBAD plasmid and transformed into Vibrio cholerae O395N1 Δnqr cells [38].…”

“…Three different NADH dehydrogenases are annotated in the genome of P. aeruginosa: complex I, NDH-2 and NQR. The presence of the three types of NADH dehydrogenases is puzzling, especially since NQR and complex I are both proton pumps [24,57] and NDH-2 is not linked to the generation of an electrochemical gradient. Here we aim to elucidate the roles of these enzymes in the physiology of P. aeruginosa.…”

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

“…Bacteria are known to coordinate the composition of the electron transport chain (ETC), and in particular, levels of the terminal oxidases, according to their metabolic needs [41]. The ETC of P. aeruginosa contains three NADH dehydrogenases; the multi-subunit proton pumping NDH-1 (encoded by nuoA-N (PA2637-PA2649)), the HQNO-resistant proton pump Nqr (encoded by nqrA-F (PA2994-2999)), and a single-subunit flavoenzyme which does not participate in ion translocation (NDH-2, encoded by ndh , PA4538) [42]. All three dehydrogenases displayed increased expression during growth on acetate, particularly at the protein level (file S1).…”

Revisiting the intrageneric structure of the genus Pseudomonas with complete whole genome sequence information: Insights into Diversity and Host-related Genetic Determinants

“…aeruginosa NADH:ubiquinone oxidoreductase (NQR) is actually a completely new form of proton pump, which plays an important role in the adaptation against autotoxicity during the infection process (Raba et al, 2018).…”