Abstract
Pathogenic bacteria often occupy multiple environmental niches throughout the various stages of its lifecycle that place a variety of metabolic demands on the organism, particularly in cellular redox regulation. Transitions between environmental conditions present a challenge of restoring redox homeostasis and balancing electron fluxes to meet metabolic and energetic demand. Bacteria typically use NADH dehydrogenases to oxidize NADH to NAD+. Two types of NADH dehydrogenase have been reported in bacteria: proton-pumping type I NADH dehydrogenase complex (NDH-1, complex I) and non-proton translocating type II NADH dehydrogenase (NDH-2). Both enzymes are part of the electron transport chain in bacteria and therefore play essential roles in regulating NAD+/NADH ratios, respiration and energy (ATP) generation. NDH-2 is ubiquitous in bacteria and in many cases, multiple copies of NDH-2 are present, with one copy being more important than the other for viability in particular growth conditions. These results suggest non-redundant functional differences for multiple NDH2 genes in bacteria. A unresolved question is whether organisms with multiple NDH2 genes use the corresponding enzymes to adapt to different environmental conditions.
To address this question, a study on the function of NDH-2 enzymes from the facultative intracellular saprophytic pathogen Listeria monocytogenes was undertaken. L. monocytogenes has an extracellular and intracellular (inside the cytoplasm of mammalian cells) lifestyle and is metabolically versatile, growing aerobically, anaerobically, and fermentatively. The ability of this bacterium to adapt to different environments makes it an ideal model organism to examine the distinct roles of multiple NDH-2 proteins: NDH2a (lmo2389) and NDH2b (lmo2638). This study examines the bioenergetic contribution and essentiality of each NDH-2 under a variety of conditions in L. monocytogenes. To explore this, a set of markerless deletion mutants Δndh2a, Δndh2b, Δndh2a/Δndh2b were generated. In addition, a mutant deficient in a critical extracellular electron transfer (EET) associated protein, ΔeetAB was constructed.
Using a combination of genetic, biochemical and physiological analyses it was demonstrated that although NDH2a is the primary NDH-2 involved in aerobic respiration, NDH2b can also facilitate aerobic respiration in a Δndh2a background. Both NDH-2s are individually sufficient to allow intracellular growth of L. monocytogenes. However, absence of both NDH2a and NDH2b prevents intracellular growth as the cells become bacteriostatic. Real-time measurements of ferric reduction activity showed that EET only functions under microaerophilic/anaerobic conditions and NDH2b and EETab are essential to perform EET. Electrochemical experiments suggested that both NDH2b and EETab are required for maximal EET function, but the use of riboflavin as a redox electron shuttle enables EET when either is absent. NDH-2s were predominantly expressed in the early growth phase of L. monocytogenes and eetA was expressed highly in mid-exponential phase. Loss of either NDH2b or EETab induced increased expression of superoxide dismutase A (sodA) in late exponential growth phase, indicative of reactive oxygen species (ROS) production. Metabolomic analysis of NDH-2 mutants grown with either glucose or glycerol as the sole carbon source, suggested significant impairments in glycolysis and glycerol metabolism when NDH2a is absent.
The findings from this study suggests that both NDH-2 enzymes can play a role in aerobic respiration. However, NDH2b is unique in its ability to facilitate EET-respiration using terminal electron acceptors such as Fe3+. The process of transitioning from exponential to stationary phase causes a shift from aerobic respiration to EET-mediated respiration thereby preventing the production of ROS. This underscores the separate roles each NDH-2 plays in enabling a large degree of metabolic flexibility that is integral to the dynamic adaptation when Listeria transitions from one environment to another that vastly contrasts in both nutrient makeup and oxygen availability such as the adaptation from extracellular to intracellular lifestyle.