固氮类芽孢杆菌(Paenibacillus sabinae T27) nifH基因的克隆，鉴定及启动子分析

Paenibacillus sabinae T27 (CCBAU 10202=DSM 17841) is a gram-positive, spore-forming diazotroph with potential for use as a bacterial fertilizer in agriculture. In this study, three nifH clusters were cloned from P. sabinae T27. The first nifH cluster is composed of nifB1nifH1nifDnifK, the second cluster contains nifNnifB2nifH2, and the third contains nifH3 and t1 t2 encoding ABC transporters. The DNA sequence identities between nifH1 and nifH2, between nifH1 and nifH3, and between nifH2 and nifH3 are 96.57％, 78％ and 78.81％, respectively. The coding regions of nifH1, nifH2 and nifH3 from P. sabinae under the control of the nifH promoter of Klebsiella pneumonia could partially restore nitrogenase activity of K. pneumonia nifH- mutant strain 1795 which has no nitrogenase activity. This suggests that the three nifH genes from P. sabinae T27 have function in nitrogen fixation. RT-PCR showed that all three nifH genes were transcribed under the excess of fixed nitrogen and oxygen. Further, we demonstrated that nifB1 with nifH1, nifB2 with nifH2, and nifH3 with t2 were co-transcribed using RT-PCR. DNA sequence analysis showed that among the three nifH clusters, there are five putative promoter regions for nifH genes: one in the nifH1 cluster, two in the nifH2 cluster and the other two in the nifH3 cluster. Analysis of β-galactosidase activity showed that the five different promoter-lacZ transcriptional fusions were expressed in Escherichia coli JM109, E. coli σ54 mutant strain TH1 and Bacillus cereus B905, suggesting that transcription of nifH genes of P. sabinae T27 are independent of both nif-specific activator NifA and transcriptional factor σ54 which are required for expression of nifH in most diazotrophs.