Molecular linkage of the nif/fix and nod gene regions in Rhizobium leguminosarum biovar trifolii.

Nucleotide sequence analysis of a 2.5kb region downstream of the nifA gene from Rhizobium leguminosarum biovar trifolii has resulted in linkage, at the DNA sequence level, of the nifEN, nifHDK, fixABCX, nifA gene cluster with the nodEF, nodD, nodABCIJ genes. Four genes have been identified within this intervening region. Immediately 3' to the nifA gene is the nifB gene and the nifB-linked ferredoxin-encoding fdxN gene. Downstream of fdxN in R. leguminosarum bv. trifolii and in Rhizobium meliloti, we have identified an open reading frame which has not been described previously and which we propose to designate fixU. Downstream of fixU in R. leguminosarum bv. trifolii is a nod gene, nodT, which is contiguous with nodJ (B. Surin et al., manuscript in preparation). As a result of this study, the linkage relationships of 22 symbiotic genes spanning a 24 kb region of the symbiotic plasmid from R. leguminosarum bv. trifolii are now known.

Iismaa SE ，Ealing PM ，Scott KF ，Watson JM ... -  《MOLECULAR MICROBIOLOGY》

The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N-terminal domain found in other NifA proteins.

The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N-terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Bradyrhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N-terminal domain is not essential for NifA function in R.I. bv. trifolii.

Iismaa SE ，Watson JM 《MOLECULAR MICROBIOLOGY》

The Rhizobium meliloti fdxN gene encoding a ferredoxin-like protein is necessary for nitrogen fixation and is cotranscribed with nifA and nifB.

Sequencing of the Rhizobium meliloti DNA region downstream of nifA revealed the existence of nifB, fdxN and ORF3. The molecular weight of the fdxN protein (Mr 6830) and the distribution of cysteine residues in its deduced amino acid sequence is typical for low molecular weight bacterial ferredoxins. Interposon insertion and plasmid integration mutagenesis demonstrated that FdxN is essential for nitrogen fixation in R. meliloti, whereas the predicted translation product of ORF3 (Mr 8708) is not necessary for this process. In contrast, ferredoxin-like proteins, which are encoded by nifB-associated genes, are not required for nitrogen fixation in all other organisms analysed so far. Plasmid integration mutagenesis additionally revealed that nifA, nifB and fdxN form one transcriptional unit. This result was confirmed by complementation analysis of polar interposon insertion mutants of nifA, nifB and fdxN and by complementation of a non-polar nifA deletion mutant. A DNA sequence resembling a typical nif consensus promoter, which is preceded by two putative NifA-binding sites, is located in front of nifB. This nifB promoter can be activated in Escherichia coli by the nifA gene product of Klebsiella pneumoniae to the same level as that of the R. meliloti nifH promoter. In contrast, R. meliloti NifA stimulates the nifH promoter more efficiently than the nifB promoter. This low-level activation of the nifB promoter may be the reason why transcription of nifB and fdxN is initiated primarily at a promoter in front of nifA.

Klipp W ，Reiländer H ，Schlüter A ，Krey R ，Pühler A ... -  《molecular and general genetics》

Host-specific regulation of symbiotic nitrogen fixation in Rhizobium leguminosarum biovar trifolii.

Strains of Rhizobium leguminosarum bv. trifolii (Rlt) able to form effective nodules on Trifolium ambiguum (Caucasian clover, CC) form ineffective nodules on Trifolium repens (white clover, WC), whereas strains that form effective nodules on WC usually do not nodulate CC. Here, we investigate the genetic basis of the host-specific nitrogen-fixation phenotype of CC rhizobia. A cosmid library of the symbiotic plasmid from the WC rhizobium strain Rlt NZP514 was introduced into the CC rhizobium strain Rlt ICC105. An 18 kb Asp718 fragment containing the nifABHDKEN and fixABCX genes of NZP514 that imparted the Fix(+) phenotype was identified. Tn5 mutagenesis of this region revealed that the nifHDKEN, fixABC and nifB genes were required for the Fix(+) phenotype, but that the nifA gene was not. Introduction of several plasmids containing NZP514 nif/fix genes into an ICC105 nifA mutant strain demonstrated that the NifA protein of ICC105 was able to activate expression of the NZP514 nif/fix genes but not the ICC105 nif/fix genes in WC nodules. Reporter gene fusion studies showed that the host-specific regulation of the nif/fix genes depended on the DNA region between the promoters of the divergently transcribed nifH and fixA genes. We hypothesize that a protein acting either in response to a host-specific signal or in the absence of such a signal is able to bind upstream of the NifA-binding sites and interact with NifA to prevent it activating nif/fix gene expression.

Miller SH ，Elliot RM ，Sullivan JT ，Ronson CW ... -  《MICROBIOLOGY-SGM》

Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid.

Innes RW ，Hirose MA ，Kuempel PL 《-》