Second gene (nif H* ) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein

Abstract

Azotobacter chroococcum MCD1 contains a cluster of nitrogen fixation (nif) genes coding for the structural polypeptides for nitrogenase (nifH for the Fe-protein and nifD and nifK for the MoFe protein) and a second sequence in the genome homologous to nifH. DNA fragments bearing this second nifH-like sequence were cloned and the DNA sequence around the homologous region determined. Two open reading frames were identified in this region. One codes for a protein of 289 amino acid residues and is highly homologous to other Fe-proteins but is different from the gene adjacent to the nifDK genes in A. chroococcum. This putative gene we call nifH*. The following open reading frame codes for a protein of 63 amino acids, nine of which are cysteine residues. The protein is homologous to the small low-potential ferredoxins found in anaerobic bacteria, and in particular those from Chlorobium limicola. Linkage between a structural gene for nitrogenase and a small ferredoxin has not previously been observed. Sequence analysis suggests that the two genes form an operon. Transcription of the ferredoxin gene on a 1320-bp transcript was only detectable under conditions in which A. chroococcum MCD1155, which carries a chromosomal deletion of 6.3 kb removing the entire nifHDK cluster, is capable of fixing N2, i.e. in media containing no added molybdenum or high levels of NH3. The size of the observed transcript agrees well with the predicted size for a transcript encoding nifH* and the ferredoxin genes. Expression of the nifH* promoter was not significantly activated in Escherichia coli even when nifA, the positive activator of nif genes in Klebsiella pneumoniae, was supplied in multiple copies. The results are discussed in relation to an alternative pathway for N2 fixation in A. chroococcum.