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》

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 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》

Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein nifA.

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a -24/-12 type promoter (hupSp). A regulatory region located in the -173 to -88 region was essential for promoter activity in R. leguminosarum. Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential -173 to -88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule.

Brito B ，Martínez M ，Fernández D ，Rey L ，Cabrera E ，Palacios JM ，Imperial J ，Ruiz-Argüeso T ... -  《-》

Transcription of a Rhizobium leguminosarum biovar phaseoli gene needed for melanin synthesis is activated by nifA of Rhizobium and Klebsiella pneumoniae.

The Rhizobium leguminosarum biovar phaseoli symbiotic plasmid pRP2JI carries a gene, melA, specifying the enzyme tyrosinase, which is responsible for the production of the pigment melanin in these bacteria. Transcription of melA is activated by the nifA gene of Rhizobium and, when the cloned melA gene is transferred to Escherichia coli, melA is expressed if the recipients contain nifA gene of Klebsiella pneumoniae. This nifA-dependent activation was temperature sensitive and required the ntrA gene. The cloned nifA gene of K. pneumoniae, when transferred to a nifA mutant of Rhizobium phaseoli biovar phaseoli, corrected the Mel- but not the Fix- phenotype. nifA of R. leguminosarum biovar phaseoli activated melA at higher levels in cells grown in low concentrations of oxygen. Also, nifA of R. leguminosarum biovar phaseoli activated nifH of K. pneumoniae in Escherichia coli cells grown in low-oxygen concentrations.

Hawkins FK ，Johnston AW 《MOLECULAR MICROBIOLOGY》