The Hyb Hydrogenase Permits Hydrogen-Dependent Respiratory Growth of <named-content content-type="genus-species">Salmonella enterica</named-content> Serovar Typhimurium

Abstract

ABSTRACT Salmonella enterica serovar Typhimurium contains three distinct respiratory hydrogenases, all of which contribute to virulence. Addition of H2 significantly enhanced the growth rate and yield of S. Typhimurium in an amino acid-containing medium; this occurred with three different terminal respiratory electron acceptors. Based on studies with site-specific double-hydrogenase mutant strains, most of this H2-dependent growth increase was attributed to the Hyb hydrogenase, rather than to the Hya or Hyd respiratory H2-oxidizing enzymes. The wild type strain with H2 had 4.0-fold greater uptake of 14C-labeled amino acids over a period of minutes than did cells incubated without H2. The double-uptake hydrogenase mutant containing only the Hyb hydrogenase transported amino acids H2 dependently like the wild type. The Hyb-only-containing strain produced a membrane potential comparable to that of the wild type. The H2-stimulated amino acid uptake of the wild type and the Hyb-only strain was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but was less affected by the ATP synthase inhibitor sodium orthovanadate. In the wild type, proteins TonB and ExbD, which are known to couple proton motive force (PMF) to transport processes, were induced by H2 exposure, as were the genes corresponding to these periplasmic PMF-coupling factors. However, studies on tonB and exbD single mutant strains could not confirm a major role for these proteins in amino acid transport. The results link H2 oxidation via the Hyb enzyme to growth, amino acid transport, and expression of periplasmic proteins that facilitate PMF-mediated transport across the outer membrane. IMPORTANCE Complex carbohydrates consumed by animals are fermented by intestinal microflora, and this leads to molecular hydrogen production. Salmonella enterica serovar Typhimurium can utilize this gas via three distinct respiratory hydrogenases, all of which contribute to virulence. Since H2 oxidation can be used to conserve energy, we predicted that its use may augment bacterial growth in nutrient-poor media or in competitive environments within H2-containing host tissues. We thus investigated the effect of added H2 on the growth of Salmonella Typhimurium in carbon-poor media with various terminal respiratory electron acceptors. The positive effects of H2 on growth led to the realization that Salmonella has mechanisms to increase carbon acquisition when oxidizing H2. We found that H2 oxidation via one of the respiration-linked enzymes, the Hyb hydrogenase, led to increased growth, amino acid transport, and expression of periplasmic proteins that facilitate proton motive force-mediated transport across the outer membrane.