关键词: 施氏假单胞菌A1501, 电子传递链rnf, 固氮酶活, 固氮酶基因nifH

Abstract: Energy supply is one of the most important factors restricting the efficiency of biological nitrogen fixation, and electron transfer complex is a significant component in energy production process of biological nitrogen fixation. Genome analysis has identified 2 sets of rnf gene clusters coding for electron transport complex in the chromosome of nitrogen-fixing Pseudomonas stutzeri A1501, naming rnf1 and rnf2. The rnf1 gene cluster locates in the nitrogen fixation island, and rnf1 in the core genome. To clarify the function of both electron transport complexes, polar mutants of rnf1 and rnf2 gene clusters were constructed individually. Growth curves determination revealed that mutation of each cluster did not affect the growth in minimal medium, but deletion of rnf1 gene cluster decreased the nitrogenase activity by 90% of the wild type. However deletion of rnf2 gene cluster had no significant effect on nitrogenase activity, suggesting that rnf1 might be involved in the electron transfer to nitrogenase in A1501. The transcription level of nitrogenase reductase encoding gene nifH was further determined by promoter-lacZ fusion expression strategy. It was found that transcriptional activity of nifH reduced by 80% in rnf1 mutant, compared with that in the wild type. It was hypothesized that deletion of rnf1 might lead to energy lack, and an imbalance of the intracellular C/N ratio under nitrogen fixation conditions, finally affecting the expression of nif genes.

Key words: Pseudomonas stutzeri A1501, electron transfer complex rnf, nitrogenase activity, nifH gene