| Biological nitrogen fixation is a high energy consumption precess. Reduction of N2 to NH3 requires consuming 16 ATP, which greatly limits efficiency of fixation nitrogen. Pseudomonas stutzeri A1501, isolated from rice paddy soils in south China, fixes nitrogen under microaerobic conditions in the free-living state. In this study, the function and expression regulation of P. stutzeri A1501 rnf gene clusters encoding electronic transport complex involved in nitrogen fixation were studied, based on the complete genome sequencing and gene functional annotation.Bioinformatics analysis of the central metabolic pathways, energy production and environmental adaptation in P. stutzeri A1501 showed that its metabolic pathway were diversed, which contains all components of complete Entner-Doudorofff, pentose phosphate and tricarboxylic acid cycle pathways, but Embden-Meyerhof-Parnas pathway for hexose catabolism. All of the metabolic pathways, especially TCA pathway, provide the energy during the process of the growth and nitrogen fixation.The production of energy involved in the process of nitrogen fixation is closely related to electron transport.The sequence analysis showed that its genome carries one set of nqr and two sets of rnf cluster encoding electron transport complex. rnf1 gene cluster consists of 7 genes (rnfABCDEGH), rnf2 gene cluster consists of 6 genes (rnfABCDEG), and nqr gene cluster consists of 6 genes (rnfABCDEF). To understand the function of rnf gene clusters involved in nitrogen fixation process, we constructed three polar mutants (nqr,rnf1 and rnf2) by suicide plasmid pK18mob, respectively. Physiological and biochemical analysis indicated that all the mutants had the same growth rates as the wild type, but the inactivation of rnf1 gene cluster resulted in a significant decrease in nitrogenase activity (about 10% of wild type nitrogenase activity), while the rnf2 cluster and nqr cluster mutant had no significant effect on the nitrogenase activity. Since rnf1 gene cluster is located in a nitrogen fixation island, rnf1 products may play an essential role in nitrogen fixation. While nqr and rnf2 are not loacted within the island, their products may relate with oxygen stress to adapt to environment. In addition, nifF gene in the nitrogen fixation island, may participate in the multiple pathways mediate electron flow to nitrogenase, inactivation of nifF gene led to 46.9% decrease in nitrogenase activity, suggesting that unknown complement pathway of electron transport possiblly existed in a nitrogen fixation island.To further understand the function of rnf1 gene cluster, we constructed 7 non-polar mutants of rnf1 genes (rnfABCDEGH), respectively. Analysis of nitrogenase activities showed that the disruption of any gene in rnf1 gene cluster could cause a significant decrease in nitrogenase activity 3.3-14.9%,suggesting that the rnf1 gene products, RnfA, RnfB, RnfC, RnfD, RnfG and RnfE proteins might act together in electron transport as an intact complex, and any inactivied gene in this cluster could loss the function of electron transport and affected the fixation nitrogenase activity in P. stutzeri A1501. Sequence analysis of the rnfH, the terminal gene in rnf1 cluster, shows it has the same direction of transcription with nifY2 and PST1323, adjacent open reading frame (ORF) to rnf1 operon. The RT-PCR results showed that rnfH, nifY2 and PST1323 is a co-transcribed unit.NifA, as a positive regulator of nif gene expression, may participate in expression and regulation of rnf1 gene cluster. A bacterial one-hybrid selection system to identify protein-DNA interaction indicated that NifA protein could interact with rnf1 promoter region. Electrophoresis mobility shift assasy also showed that NifA protein could bind to the rnf1 promotor region in vitro. Our results suggested that Rnf1 electron transport complex play an essential role in nitrogen fixation, and the expression of rnf1 gene cluster was activated by NifA.
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