Genetic Dissection of the Fermentative and Respiratory Contributions Supporting Vibrio cholerae Hypoxic Growth

Abstract

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, requires only a single periplasmic NO₃^- reductase (NapA) to undergo NO₃^- respiration, suggesting that the pathogen possesses a noncanonical NO₃^- respiratory chain. In this study, 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 under 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. In this study, 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 under 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.

Keywords: Vibrio cholerae; anaerobic respiration; fermentation; hypoxia; nitrate.

FIG 1

Identification of V. cholerae genetic determinants for hypoxic metabolism and NO₃⁻ reduction. (A) (Left) Experimental workflow for TIS screen comparing V. cholerae mutant libraries grown under normoxic or hypoxic conditions. (Right) Volcano plot of Con-ARTIST analysis output depicting read fold change (FC) and inverse Mann-Whitney U test P value for each gene queried in the TIS screen. Dashed lines indicate arbitrary thresholds of FC of >10 or <0.1 and an inverse P value of >20. Red circles indicate genes known to be involved in NO₃⁻ respiration. (B) (Left) Experimental workflow for arrayed transposon mutant screen to identify genes required for NO₃⁻reduction to NO₂⁻ in V. cholerae. (Right) List of mutant strains queried in the arrayed screen (see Table S3 in the supplemental material for an extended version). (C) Proposed organization of the NO₃⁻ reduction machinery in V. cholerae based on results from the transposon screens.

FIG 2

Genes involved in NO₃⁻ reduction in V. cholerae are required for divergent pH-dependent responses to NO₃⁻ under hypoxia. (A) Optical density (OD at 600 nm [OD₆₀₀]) growth curves and endpoint CFU plating for C6706 WT V. cholerae and napC, tatA, ccmF, narP, and moaA transposon insertion strains. Cultures were grown hypoxically in M9 minimal medium (initial pH 7.0) plus 1% glucose (fermentative conditions) in the absence (red, -N) or presence (black, +N) of 3 mM NO₃⁻. Endpoint pH and NO₂⁻ levels were measured at the time of plating (20 h). (B) OD₆₀₀ growth curves for strains in panel A grown in LB medium at pH 8 (NO₃⁻ respiratory conditions) with or without 3 mM NO₃⁻. Endpoint NO₂⁻ accumulation is shown in blue on the right y axis. Data are the means ± SEs from 3 biological replicates.

FIG 3

The NADH dehydrogenase Na⁺-Nqr participates in NO₃⁻ respiration in V. cholerae. (A) OD₆₀₀ growth curve of V. cholerae WT, ΔnapA, ΔnqrF, and ΔnqrF ΔnapA strains grown hypoxically in LB medium buffered at pH 8 in the presence (black and blue) or absence (red) of 20 mM NO₃⁻. Induction of the nitrate respiratory pathway is labeled as “NO₃^—RI” in each growth curve. (B) Methyl viologen-dependent in vitro nitrate reductase (NR) activity (nanomoles of NO₂⁻ produced per minute per milligram of protein). Aliquots from cultures in panel A were collected after 2 h of incubation and assayed for NR activity. (C) Time course in vivo NR activity. (D) Representative membrane potential flow cytometry plots from DiOC₂-stained cells. (E) Quantification of green/red fluorescence ratios in cells indicated by dashed boxes in panel D. Data are the means ± SEs from 3 biological replicates. Statistical significance was determined by the Student t test.

FIG 4

VC1890, VC0786, VC1581, and VC1511 do not contribute to NO₃⁻ respiration in V. cholerae. (A) Proposed schematic of NO₃⁻-terminated electron transport chain in V. cholerae under hypoxic NO₃-replete conditions. Redox equivalents are channeled into the ETC by one or more membrane-bound dehydrogenases that feed the ubiquinone (UQ) pool. Electrons from the UQ pool are ultimately transferred to NO₃⁻ by specific terminal reductases. (B) OD₆₀₀ growth curves of V. cholerae WT and dehydrogenase-deficient strains. Each dehydrogenase was also deleted in combination with Na⁺-Nqr (right graphs). Strains were grown hypoxically in LB medium buffered at pH 8 in the presence (black) or absence (red) of 20 mM NO₃⁻. Data are the means ± SEs from 3 biological replicates.

FIG 5

V. cholerae fermentation is driven by AdhE. (A) Schematic of fermentative branches in V. cholerae. (B) OD₆₀₀ growth curve of V. cholerae WT and fermentative mutant strains: Δack1(left), ΔldhA (right), and ΔadhE (bottom). (C) OD₆₀₀ growth curve of E. coli K-12 WT and Δadh strains. Cultures were grown hypoxically in LB medium buffered at pH 8 in the presence (black) or absence (red and blue) of 20 mM NO₃⁻. Data are the means ± SEs from 3 biological replicates.

FIG 6

NO₃⁻ respiration in V. cholerae supports lack of fermentation in vitro and in vivo fitness. (A) In vitro competitive indices (CI) of WT V. cholerae versus ΔnapA, ΔadhE, or ΔadhE ΔnapA strains during hypoxic growth in LB medium buffered at pH 8. CIs were determined at 8 h postinoculation. (B) In vivo competition of WT V. cholerae versus ΔnapA, ΔadhE, and ΔadhE ΔnapA strains in the streptomycin-treated adult mouse colonization model. The WT control (WT versus WT lacZ-) and ΔnapA competition data (left data sets) are reproduced from reference . CIs were determined at 24 h postinoculation from harvested colon (co) and cecum (ce) samples. Statistical significance was determined by the Student t test (A) or Mann-Whitney U test (B). A P value of less than 0.05 was considered statistically significant. Bars represent geometric means ± SE.