Department of Microbiology, University of Georgia, Athens, GA, USA.
Microbiology (Reading). 2012 Aug;158(Pt 8):2204-2212. doi: 10.1099/mic.0.058248-0. Epub 2012 May 24.
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 2.4×10(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.
氢化酶在细菌生理学中发挥着多种作用，动物病原体摄取型氢化酶利用氢气的作用尤其引人关注。在志贺氏菌中从未研究过氢化酶，因此分别针对两种志贺氏菌 H2 摄取酶（Hya 和 Hyb）和 H2 产生酶（Hyc）生成了靶向突变株，以确定它们的作用。在厌氧发酵条件下，Hya 突变株（hya）不能氧化 H2，而 Hyb 突变株像野生型一样氧化 H2。与亲本相比，hyc 菌株氧化的外加氢气更多。用 JC-1 染料（5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物）进行荧光比率成像显示，亲本菌株产生的膜电位比 hya 高 15 倍。hya 突变株也是迄今为止最敏感的酸菌株，比已知的抗酸谷氨酸依赖酸抗性途径（GDAR 途径）中的突变株更为敏感。在严重的酸挑战实验中，向 hya 中添加谷氨酸恢复了存活率，并且通过突变株（例如 hya/gadBC 双突变株）分析，该能力部分归因于 GDAR 系统（去除细胞内质子）。然而，突变株表型表明，谷氨酸挽救的酸耐受性的更大部分独立于 GadBC。向生长培养基中添加氯离子有助于 hya 菌株的酸耐受性。根据 hyc 的测定，全细胞 Hya 酶在酸暴露（20 分钟）后变得更加活跃。事实上，在酸性条件下，Shigella H2 通过 Hya 的氧化速率非常高，每个细胞每 min(-1)可提供 2.4×10(8)个质子。内膜中由 Hya 介导的 H2 氧化产生的电子可能抵消细胞质正电荷应激，而大量质子池在周质中沉积可能在严重的酸应激期间排斥质子流入。
