Roles of H2 uptake hydrogenases in Shigella flexneri acid tolerance

Hydrogenases play many roles in bacterial physiology, and use of H-2 by the uptake-type enzymes of animal pathogens is of particular interest. Hydrogenases have never been studied in the pathogen Shigella, so targeted mutant strains were individually generated in the two Shigella flexneri H-2-uptake enzymes (Hya and Hyb) and in the H-2-evolving enzyme (Hyc) to address their roles. Under anaerobic fermentative conditions, a Hya mutant strain (hya) was unable to oxidize H-2, while a Hyb mutant strain oxidized H-2 like the wild-type. A hyc strain oxidized more exogenously added hydrogen than the parent. Fluorescence ratio imaging with dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) showed that the parent strain generated a membrane potential 15 times greater than hya. The hya mutant was also by far the most acid-sensitive strain, being even more acid-sensitive than a mutant strain in the known acid-combating glutamate-dependent acid-resistance pathway (GDAR pathway). In severe acid-challenge experiments, the addition of glutamate to hya restored survivability, and this ability was attributed in part to the GDAR system (removes intracellular protons) by mutant strain (e.g. hya/gadBC double mutant) analyses. However, mutant strain phenotypes indicated that a larger portion of the glutamate-rescued acid tolerance was independent of GadBC. The acid tolerance of the hya strains was aided by adding chloride ions to the growth medium. The whole-cell Hya enzyme became more active upon acid exposure (20 min), based on assays of hyc. Indeed, the very high rates of Shigella H-2 oxidation by Hya in acid can supply each cell with 2.4x10(8) protons min(-1). Electrons generated from Hya-mediated H-2 oxidation at the inner membrane likely counteract cytoplasmic positive charge stress, while abundant proton pools deposited periplasmically likely repel proton influx during severe acid stress.