The nif Gene Operon of the Methanogenic Archaeon Methanococcus maripaludis

Abstract

Nitrogen fixation occurs in two domains, Archaea andBacteria. We have characterized a nif(nitrogen fixation) gene cluster in the methanogenic archaeonMethanococcus maripaludis. Sequence analysis revealed eight genes, six with sequence similarity to known nif genes and two with sequence similarity to glnB. The gene order,nifH, ORF105 (similar to glnB),ORF121 (similar to glnB), nifD,nifK, nifE, nifN, andnifX, was the same as that found in part in other diazotrophic methanogens and except for the presence of theglnB-like genes, also resembled the order found in many members of the Bacteria. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the nif gene cluster. Northern analysis revealed the presence of either a single 7.6-kbnif mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5′ tonifH. Distinctive features of the nif gene cluster include the presence of the six primary nif genes in a single operon, the placement of the two glnB-like genes within the cluster, the apparent physical separation of the cluster from any other nif genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens.

Sequencing and functional analysis of the nifENXorf1orf2 gene cluster of Herbaspirillum seropedicae

“…Moreover, the SalI-EcoRI fragment containing the region between nifK and nifE, was subcloned into the promoter probe vector pPW452 to give pPGK1.2, but no promoter activity was found in either E. coli (containing constitutive NifA protein) or H. seropedicae background under nitrogen-¢xing conditions (data not shown). Attempts to obtain nif transcripts, however, were unsuccessful, presumably due to the instability of the RNA [17]. Therefore the likely promoter for all these genes is upstream the nifH gene.…”

“…In B. japonicum the nifDKENX genes are organized in a single operon and a promoter sequence was found only upstream from the nifD gene [15]. The methanogenic archaeon Methanococcus maripaludis contains the nif operon nifHorf105orf121nifDKENX with only a nifH promoter sequence [17]. In R. capsulatus [4] nifX was also followed by a ferredoxin-like gene (orf4).…”

Genetics of Methanococcus: possibilities for functional genomics in Archaea

“…B. The nif operon of M. maripaludis (Kessler et al ., 1998). Inverted repeats (small arrows), the transcription start site (+) and the ribosomal binding site (RBS) are indicated.…”

“…Genes essential for N 2 fixation in M. maripaludis have been identified by a transposon mutagenesis method that should be applicable to other cloned genes (Blank et al ., 1995; Kessler et al ., 1998). The pac cassette was introduced into a derivative of a mini‐Mu plasmid, such that pac was flanked by the ends of the Mu transposon.…”

NrpRII mediates contacts between NrpRI and general transcription factors in the archaeon Methanosarcina mazei Gö1

“…Within the methanogenic archaea, studies on the regulation of nitrogen metabolism and nitrogen fixation have been pioneered in Methanococcus maripaludis. Leigh and coworkers demonstrated that the transcriptional regulation of nitrogen-regulated genes in M. maripaludis differs significantly from that known in bacteria [12][13][14][15][16]. They identified a global nitrogen regulator (NrpR), which binds as a dimer to an operator sequence and inhibits transcription in the presence of sufficient nitrogen [12,[17][18][19][20].…”