Cloning and sequence analysis of Alcaligenes faecalis nifHDK gene cluster

Total DNA of Alcaligenes faecalis was probed with both the nifH and nifHD sequences from K. 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 pBZ1 was analyzed by physical mapping and was further subcloned for sequencing. It was found that this A. 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 1476 bp long respectively. The GC contents of these two genes were about 61.6% and 60.0%. The intergenic regions of nifH-nifD and nifD-nifK were 101 and 105 bp respectively. There were separate SD sequences upstream of all the tree genes. The deduced amino acid sequences of the nifH gene product (the Fe-protein) and the nifD gene product (the Mo-Fe-protein) were also highly homologous to other nitrogen-fixing bacteria, especially in those conserved motif. The A. faecalis sequence has the highest similarity to that of A. vinelandii.