The Hyb hydrogenase permits hydrogen-dependent respiratory growth of Salmonella enterica serovar Typhimurium

Abstract

Salmonella enterica serovar Typhimurium contains three distinct respiratory hydrogenases, all of which contribute to virulence. Addition of H(2) 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 H(2)-dependent growth increase was attributed to the Hyb hydrogenase, rather than to the Hya or Hyd respiratory H(2)-oxidizing enzymes. The wild type strain with H(2) had 4.0-fold greater uptake of (14)C-labeled amino acids over a period of minutes than did cells incubated without H(2). The double-uptake hydrogenase mutant containing only the Hyb hydrogenase transported amino acids H(2) dependently like the wild type. The Hyb-only-containing strain produced a membrane potential comparable to that of the wild type. The H(2)-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 H(2) 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 H(2) oxidation via the Hyb enzyme to growth, amino acid transport, and expression of periplasmic proteins that facilitate PMF-mediated transport across the outer membrane.

FIG 1

Effect of hydrogen on growth of S. enterica serovar Typhimurium WT and ALZ42 (Hyb-only-containing strain) with fumarate as an electron acceptor.

FIG 2

Comparison of membrane potentials of the WT, ALZ42, and ALZ43 strains. A small ratio indicates a larger membrane potential (n = 6; P < 0.01).

FIG 3

Immunoblot analyses of TonB-, ExbD-, and TonB/ExbD-linked complexes in WT S. enterica serovar Typhimurium. (A) TonB visualized with TonB-specific monoclonal antibodies. (B) ExbD visualized with ExbD-specific polyclonal antibodies. M, molecular mass standards.