Genetic dissection of the fermentative and respiratory contributions supporting Vibrio cholerae hypoxic growth

Both fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors such as nitrate (NO3-). During canonical NO3- respiration, NO3- is reduced in a stepwise manner to N2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, only requires a single periplasmic NO3- reductase (NapA) to undergo NO3- respiration, suggesting that the pathogen possesses a non-canonical NO3- respiratory chain. Here, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3- respiration in V. cholerae. We found that while the V. cholerae NO3- respiratory chain is primarily composed of homologues of established NO3- respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae. Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo. Our findings reveal the genetic bases for interactions between disparate energy production pathways that support pathogen fitness in shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions. IMPORTANCEBacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways-fermentation and respiration. Here, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3- that consists largely of components found in other NO3- respiratory systems, but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3- respiration were required for efficient pathogen growth in both laboratory conditions and in an animal infection model. These observations provide genetic evidence for fermentative-respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.