Cloning and sequence analysis ofAlcaligenes faecalis nifHDK gene cluster

Total DNA ofAlcaligenes faeculis was probed with both the nifH and nifHD sequences fromK. pneumoniae.One positive band of about 4. 6 kb was discovered. This nifH homologous fragment was cloned into the vector pBluescript SK(+) to construct the recombinant plasmid pBZ1. The inserted fragment in pBZl was analyzed by physical mapping and was further subcloned for sequencing. It was found that thisA. faecalis nifHDK homology possessed a typical Sigma(54)-dependent promoter region with upstream activator sequence (UAS) and A-T rich region. The nifH and nifD ORFs were 888 and 1 476 bp long respectively. The GC contents of these two genes were about 61.6% and 60.0%. The intergenic regions of nifH-nifD and nifn-nifK were 101 and 105 bp respectively. There were separate SD sequences upstream of all the three genes. The deduced amino acid sequences of the nifH gene product (the Feprotein) and the nifD gene product (the Mo-Fe-protein) were also highly homologous to other nitrogen-fixing bacteria, especially in those conserved motif. TheA. faeculis sequence has the highest similarity to that ofA. uinelandii.