Department of Biology, Kenyon College, Gambier, Ohio, United States of America.
PLoS One. 2010 Apr 12;5(4):e10132. doi: 10.1371/journal.pone.0010132.
Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H(2) production involves consumption of 2H(+), hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2-2.5) that are three pH units lower than the pH limit of growth (pH 5-6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms.
We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H(2) to 2H(+). Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H(2) production and consumption was tested using a H(2)-specific Clark-type electrode. Hyd-3-dependent H(2) production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H(2) consumption was maximal at alkaline pH. H(2) production, was unaffected by a shift in external or internal pH. H(2) production was associated with hycE expression levels as a function of external pH.
Anaerobic growing cultures of E. coli generate H(2) via Hyd-3 at low external pH, and consume H(2) via Hyd-2 at high external pH. Hyd-3 proton conversion to H(2) is required for acid resistance in anaerobic cultures of E. coli.
通过发酵细菌（如大肠杆菌）生产氢气提供了一种潜在的氢生物燃料来源。由于 H(2) 的产生涉及到 2H(+)的消耗，因此氢气酶的表达可能涉及到 pH 响应和调节。在大肠杆菌中，氢气酶消耗质子与耐酸性有关，即在比生长 pH 限制（pH 5-6）低三个 pH 单位的 pH 2-2.5 水平下，能够存活并暴露于酸中。在酸性环境中的生存能力增强，使更大的感染性接种物能够通过胃并定植于肠道。大多数酸抗性机制已在好氧培养物中定义，但使用厌氧培养物将揭示新的酸抗性机制。
我们分析了大肠杆菌 K-12 W3110 活培养物中细菌氢气酶的 pH 调节。在 pH 5 到 6.5 的厌氧生长过程中，大肠杆菌表达三种可逆地将 H(2)氧化为 2H(+)的氢化酶同工酶。在缺氧条件下，我们确定了哪些氢化酶复合物有助于耐酸，通过在没有通气的情况下在 pH 5.5 下生长的培养物的存活率来衡量，并且在 pH 2 或 pH 2.5 下暴露 2 小时。在低氧条件下，所有菌株在极端酸性条件下的存活率明显低于需氧培养物。在 pH 2.5 时，hyd-3（Hyd-3）的缺失使缺氧酸存活率降低了 3 倍，在 pH 2 时降低了 20 倍，但对需氧培养物的酸存活率没有影响。hydb（Hyd-2）的缺失对酸存活率没有显著影响。使用 H(2)-特异性克拉克型电极测试了 H(2)产生和消耗的 pH 依赖性。与 pH 6.5 相比，pH 5.5 时 Hyd-3 依赖性 H(2)的产生增加了 70 倍，而 Hyd-2 依赖性 H(2)的消耗在碱性 pH 时达到最大值。H(2)的产生不受外部或内部 pH 变化的影响。H(2)的产生与外部 pH 作为函数的 hycE 表达水平有关。
在低外部 pH 下，大肠杆菌的厌氧生长培养物通过 Hyd-3 产生 H(2)，在高外部 pH 下通过 Hyd-2 消耗 H(2)。在大肠杆菌的厌氧培养物中，Hyd-3 质子转化为 H(2)是耐酸所必需的。
