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Primary structure, functional organization and expression of nitrogenase structural genes of the thermophilic archaebacterium Methanococcus thermolithotrophicus
Author(s) -
Souillard N.,
Sibold L.
Publication year - 1989
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1989.tb00200.x
Subject(s) - orfs , open reading frame , biology , genetics , gene , homology (biology) , start codon , methanococcus , promoter , microbiology and biotechnology , stop codon , peptide sequence , messenger rna , gene expression , archaea
Summary 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 Hindlll fragment carrying one of these regions was previously cloned and shown to contain a nifH gene (Souillard et al. , 1988) now referred to as 0RF nifH2. A 3.4kb Pstl fragment and an overlapping 3.B kb BglU fragment, containing the second region of homology, were cloned, and a DNA region of 4073bp was sequenced. It contained four complete open reading frames (ORFs) (ORFnifH1, ORF105, ORF128, ORFnifD) and two truncated ORFs (ORFnifK and ORF96). Five ORFs were transcribed in the same direction in the order of ORF nifH1 ‐ORF105‐ORF128‐ORF nifD ‐ORF nifK. ORF nifH1 , ORF nifD and ORF nifK were assigned from their similarity to eubacterial nifH and nifDK (nitrogenase MoFe protein) genes. Transcription studies showed that ORFnifH1 and ORF nifD were expressed only under nitrogen‐fixation conditions, whereas no ORF nifH2 mRNA was detected under the same conditions. A DNA probe containing ORF nifH1 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 ORF nifH1 , This site is preceded, 21 bp upstream, by the sequence 5′‐TTTATATA‐3′ already found at the same position in several archaebacterial promoters. ORF nifH1 mRNA was too small to encode ORF nifDK. This was confirmed by the fact that another transcription start site was localized 85bp upstream from the ATG codon of ORFnifD.