Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae☆

DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiella pneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part. nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growth only in the presence of high concentrations of molybdate. This demonstrated that the gene encoding the ferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could be found in the intergenic region between nifENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusion revealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to other nitrogen-fixing organisms, R. capsulatus nifE, nifN, nifX, ORF4, ORF5, and nifQ were organized in one transcriptional unit.

Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nif gene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.

Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7.

Molecular characterization of rhizosphere and clinical isolates of Burkholderia cepacia

Nucleotide sequence and expression of the glutamine synthetase structural gene, glnA, of the archaebacterium Methanococcus voltae.

Seventeen genes specifically required for nitrogen fixation are clustered on the chromosome of Klebsiella pneumoniae and form a complex regulon that is organized into eight transcriptional units. The nif promoters are representative of a new class of promoter, the members of which lack the consensus sequences normally found in prokaryotic promoters. nif gene transcription is positively controlled and requires: (1) the ntrA gene product, which replaces the rpoD-encoded sigma subunit of RNA polymerase to allow recognition of nif promoter sequences; and (2) the product of either the nitrogen regulation gene ntrC or the specific nif regulatory gene, nifA, which are both transcriptional activators. Most nif promoters require an upstream activator sequence (UAS) for nifA-mediated activation. The UAS acts independently of orientation and can function when placed 2 kilobases upstream from the transcription start site. Current evidence suggests that activation requires an interaction between proteins bound at the UAS and at the downstream nif promoter consensus, possibly via a loop in the DNA structure. Transcription of nif is modulated by the ntrB and nifL gene products. Both proteins can `sense' environmental changes: ntrB prevents activation by ntrC in response to excess nitrogen whereas nifL prevents activation by nifA in response to fixed nitrogen and oxygen. The C-terminal end of ntrB shows clear homology at the amino acid level with a number of diverse control proteins involved in regulation or sensory transduction. Each member of this family interacts with another protein component showing homology to the N-terminal sequence of ntrC, but not to nifA. The significance of these protein homologies is discussed.

Genetics and regulation of nif and related genes in Klebsiella pneumoniae

The nitrogen-fixing bacteriumKlebsiella pneumoniae has provided a model system for analysis of the organisation and regulation of nitrogen fixation (nif) genes in diazotrophs. Expression of the 17nif genes inKlebsiella is regulated in response to the nitrogen source by the products of the nitrogen control genesntrA, ntrB andntrC. Nif transcription is also specifically regulated in response to oxygen and low levels of fixed nitrogen by the products of thenifLA operon. Promoters subject to negative regulation by thentrBC products contain a homologous sequence with dyad symmetry, indicative of a repressor binding site.Ntr-activatable promoters do not show homology with typicalEscherichia coli promoters but have a characteristic primary structure. The model we propose for regulation ofnif transcription has important consequences for genetic manipulation of existing diazotrophs and for obtaining expression of theKlebsiella nif gene cluster in other ‘prokaryotic-like’ environments such as the chloroplast.

S. MacFarlane

Papers

Genetics and regulation of nif and related genes in Klebsiella pneumoniae

Regulation of the nitrogen fixation genes in Klebsiella pneumoniae: Implications for genétic manipulation