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 basis of a specific interaction between disparate energy production pathways that supports pathogen fitness in shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions.IMPORTANCE Bacteria 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 a specific example of fermentative-respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.