1887

Abstract

Hydrogenases play many roles in bacterial physiology, and use of H by the uptake-type enzymes of animal pathogens is of particular interest. Hydrogenases have never been studied in the pathogen , so targeted mutant strains were individually generated in the two H-uptake enzymes (Hya and Hyb) and in the H-evolving enzyme (Hyc) to address their roles. Under anaerobic fermentative conditions, a Hya mutant strain () was unable to oxidize H, while a Hyb mutant strain oxidized H like the wild-type. A 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 . The 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 restored survivability, and this ability was attributed in part to the GDAR system (removes intracellular protons) by mutant strain (e.g. 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 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 . Indeed, the very high rates of H oxidation by Hya in acid can supply each cell with 2.4×10 protons min. Electrons generated from Hya-mediated H 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.

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2012-08-01
2019-10-20
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