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.

Further Analysis of Nitrogen Fixation (nif) Genes in Azotobacter chroococcum: Identification and Expression in Klebsiella pneumoniae of nifS, nifV, nifM and nifB Genes and Localization of nifE/N-, nifU-, nifA- and fix ABC-like Genes

“…chroococcum since a strain deleted for the nifHDK cluster was still capable of fixing N 2 in molybdenum-deficient medium (Robson, 1986). This is consistent with the presence of nzfH*, coding for a second nitrogenase Fe-protein (Robson et al, 1986a), and a second nifK-like sequence in the genome of this organism (Robson, 1986). N, fixation in the deletion strain depends upon vanadium and is catalysed by a two-component nitrogenase complex with a typical Fe-protein (component 2) but in which the conventional molybdoprotein (component 1) is replaced by a vanadoprotein.…”

“…The relationship at the genetic level between the Mo-and V-based Nz fixation systems is particularly interesting. Considering the broad similarities in the two sets of nitrogenase proteins (Robson et al, 1986a;Robson, 1986) it is probable that the two systems have arisen from one or more gene duplication events. Not all genes may be duplicated; some may be common to both systems, others unique to each system.…”

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii

“…The genetic basis of the V nitrogenase from A. vinelandii is not as well understood as it is for the V nitrogenase from A. chroococcum, but extensive similarities appear to exist within the two organisms (49, 55; our unpublished results). The vnfH genes in both organisms are organized in an operon also containing a ferredoxin-like gene downstream from vnfH (28,54). The vnJfl-ferredoxin operon is separated from an operon containing the structural genes (vnfD vnfK) for dinitrogenase 2 by approximately 2 kilobase pairs (kbp) in A. chroococcum and by 1 kbp in A. vinelandii (55 and our unpublished results).…”

“…promoter sequence (2) 1079 3115 GGG CGG GTT GGC ACT ATC AAC CCC ATC TTT ACC TGT CAA CCG G R V G T I N P I F T C Q P 3157 GCC GGT GCC CAG TTC GTC AGT ATC GGT ATC AAG GAT TGC ATC A G A Q F V S I G I K D C I 3199 GGT ATC GTG CAT GGC GGC CAA GGC TGC GTG ATG TTC GTC CGC (54), and of nifH3 from Clostridium pasteurever, the highest degree of similarity exists between the ianum (66) is shown in Fig. 4.…”

The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase

“…The target of ADP-ribosylation is not only conserved in dinitrogenase reductases of the ' conventional' molybdenum nitrogenase but also in the corresponding proteins of alternative nitrogen fixation systems (Robson et al, 1986;Joerger et al, 1989;Schuddekopf et al, 1993) and it has been shown that the alternative nitrogenase of R. rubrurn is also modified by DraT (Lehman & Roberts, 1991a). The photosynthetic non-sulphur purple bacterium Rhodobacter capsulatus also contains two nitrogenase systems : the molybdenum nitrogenase (encoded by nifHDK) and an alternative heterometal-free (' irononly ') nitrogenase (anfHDGK; Schneider et al, 199 1 b ;Gollan et al, 1993;Schuddekopf et al, 1993).…”