Cloning and sequencing of nifBHDKENX genes of Paenibacillus massiliensis T7 and its nif promoter analysis.
A 324 bp of nifH fragment was PCR amplified from Paenibacillus massiliensis T7 using the universal degenerate primers. The PCR-amplified nifH fragment was labeled with DIG and then used as a probe in Southern blot analysis. Southern blot result showed that there were two positive signals, indicating that there might be two copies of nifH in P. massiliensis T7. A total of 10254 bp DNA sequence containing purD and nifBHDKENX was obtained by five rounds of inverse-PCR amplification. The predicted proteins of nifBHDKENX had high homology with those from other nitrogen-fixing bacteria. Only one putative sigma54-dependent promoter sequence was detected upstream of the nifB gene and nifBHDKENX were likely to be organized in one operon. Assays of 3-galactosidase activity of P. massiliensis T7PB carrying a nifB-lacZ fusion under different concentrations of NH4+ and O2 showed that the expression of nifB-lacZ was strongly inhibited by O2.
Zhao
Hongxin
H
State Key Laboratory for Agrobiotechnology and Biological School, China Agricultural University, Beijing 100094, China.
Xie
Baoen
B
Chen
Sanfeng
S
D000643:Ammonium Chloride / Q000494:pharmacology; D001407:Bacillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D023061:Gene Order; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics*; D009876:Operon / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics; D010100:Oxygen / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C092498:NifB protein, Bacteria; D011993:Recombinant Fusion Proteins; D000643:Ammonium Chloride; D010088:Oxidoreductases; C029130:nitrogenase reductase; D001616:beta-Galactosidase; D010100:Oxygen