Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the archaeon Methanosarcina barkeri 227

• DOI: 10.1128/jb.178.1.143-148.1996
• Publication Date: 2016-11-14T10:49:13Z
• Authors: Y T Chien; S H Zinder
• Keywords: Azotobacter vinelandii; Structural gene; Gene cluster; Primer extension

Determination of the nucleotide sequence nitrogenase structural genes (nifHDK2) from Methanosarcina barkeri 227 was completed in this study by cloning and sequencing a 2.7-kb BamHI fragment containing 3' end nifK2 1,390 bp nifE2-homologous genes. Open reading frame is 1,371 long including stop codon TAA encodes polypeptide 456 amino acids. Phylogenetic analysis deduced acid sequences nifE2 gene products M. showed that both cluster most closely with corresponding nif-1 Clostridium pasteurianum, consistent our previous analyses nifH2 nifD2. The nifE product known to be homologous nifD, shows branching pattern for proteins resembles nifD (with exception vnfE Azotobacter vinelandii), suggesting duplication occurred before divergence nitrogenases. Primer extension had single transcription start site located 34 nucleotides upstream ATG translation nifH2, resembling archaeal consensus promoter [TTTA(A/T)ATA] found 32 site. A tract four T's, previously identified as termination archaea, immediately downstream gene, potential gene. Hybridization nifDK2 probes RNA revealed 4.6-kb transcript N2-grown cells, large enough harbor nifHDK their internal open frames, while no detected NH4(+)-grown cells. These results support model which are cotranscribed NH4(+)-repressed operon.