Primary structure, functional organization and expression of nitrogenase structural genes of the thermophilic archaebacterium Methanococcus thermolithotrophicus

Abstract

Two regions of homology to Anabaena nifH (nitrogenase Fe protein) were detected in the total DNA of the thermophilic nitrogen-fixing archaebacterium Methanococcus thermolithotrophicus. A 2.8 kb HindIII fragment carrying one of these regions was previously cloned and shown to contain a nifH gene (Souillard et al., 1988) now referred to as ORFnifH2. A 3.4 kb PstI fragment and an overlapping 3.8 kb BglII fragment, containing the second region of homology, were cloned, and a DNA region of 4073 bp was sequenced. It contained four complete open reading frames (ORFs) (ORF nifH1, ORF105, ORF128, ORFnifD) and two truncated ORFs (ORFnifK and ORF96). Five ORFs were transcribed in the same direction in the order of ORFnifH1-ORF105-ORF128-ORFnifD-ORFnifk. ORFnifH1, ORFnifD and ORFnifK were assigned from their similarity to eubacterial nifH and nifDK (nitrogenase MoFe protein) genes. Transcription studies showed that ORFnifH1 and ORFnifD were expressed only under nitrogen-fixation conditions, whereas no ORFnifH2 mRNA was detected under the same conditions. A DNA probe containing ORFnifH1 hybridized with a 1.8 kb mRNA, as detected by a Northern blotting experiment. A transcriptional start site was localized 87 and 88 bp upstream from the ATG codon of ORFnifH1. This site is preceded, 21 bp upstream, by the sequence 5'-TTTATATA-3' already found at the same position in several archaebacterial promoters. ORFnifH1 mRNA was too small to encode ORFnifDK. This was confirmed by the fact that another transcription start site was localized 85 bp upstream from the ATG codon of ORFnifD.