Cloning and sequencing of nifBHDKENX genes of Paenibacillus massiliensis T7 and its nif promoter analysis

Abstract

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.