Key Laboratory of Synthetic Biology, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, China.
Biotechnol Bioeng. 2010 Jul 1;106(4):564-72. doi: 10.1002/bit.22722.
Purple non-sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in wastewater. However, NH(4)(+) in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria. In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245-1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH(4)(+) concentrations (15-40 mM), whereas neither was detected in the wild-type strain under the same conditions. Further analysis indicated that high NH(4)(+) concentrations greatly inhibited the expression of nifA and nitrogenase gene in the wild-type strain but not in the glnA1(-) mutant. These observations suggest that GS is essential to NH(4)(+) repression of nitrogenase and that deletion of glnA1 results in the complete derepression of nitrogenase by preventing NH(4)(+) assimilation in vivo, thus relieving the inhibition of nifA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphaeroides and possibly in other hydrogen-producing PNS bacteria.
紫色非硫（PNS）细菌通过在废水中发酵有机酸来产生氢气。然而，废水中的 NH(4)(+) 可能通过抑制固氮酶的表达和活性来抑制氢气的合成，固氮酶是 PNS 细菌中催化氢气生成的酶。在这项研究中，通过同源重组敲除了 Rhodobacter sphaeroides 6016 的 glnA 基因，该基因编码谷氨酰胺合成酶（GS），并检测了其对产氢和固氮酶活性的影响。在有相对较高 NH(4)(+) 浓度（15-40mM）的情况下，使用 3mM 谷氨酰胺作为氮源，突变体中可以检测到氢气生成（1,245-1,588mL 氢气/L 培养物）和固氮酶活性，而在相同条件下野生型菌株中均未检测到。进一步分析表明，高浓度 NH(4)(+) 极大地抑制了野生型菌株中 nifA 和固氮酶基因的表达，但在 glnA1(-) 突变体中则没有。这些观察结果表明，GS 对于 NH(4)(+) 对固氮酶的抑制作用是必需的，并且 glnA1 的缺失通过防止体内 NH(4)(+) 同化作用而导致固氮酶的完全去抑制作用，从而缓解了 nifA 和固氮酶基因表达的抑制。因此，敲除 glnA1 为去除 R. sphaeroides 中以及可能在其他产氢 PNS 细菌中铵离子的抑制作用提供了一种有效的方法。
