fixK, a gene homologous with fnr and crp from Escherichia coli, regulates nitrogen fixation genes both positively and negatively in Rhizobium meliloti.

Nitrogen fixation genes are shown to undergo a complex positive and negative regulation in Rhizobium meliloti. Activation of fixN by fixLJ is shown to require a third regulatory gene, fixK. As fixK is activated by fixLJ, we propose a cascade model for fixN regulation such that fixLJ activates fixN via fixK. In addition fixK negatively regulates expression of the nif-specific activator nifA as well as its own expression by autoregulation. Thus nifA and fixK are subject to a mixed regulation, positive (by fixLJ) and negative (by fixK). The sequence of fixK shows homology with the Escherichia coli regulators fnr and crp, which makes fixK the third characterized member of this family of prokaryotic regulators.     

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr -like gene, designated fixK , whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix⁺ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN -deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK / fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.     

苜蓿中华根瘤菌nifA基因突变影响多种细胞学过程

苜蓿中华根瘤菌nifA基因在共生固氮过程中担负着调控功能,nifA突变株Rm1354在宿主植物的根部诱导白色无效根瘤。本文报道Rm1354在自生状态下的表型变化。nifA的突变导致根瘤菌在半固体培养基上泳动变慢,胞外蛋白含量降低。有趣的是,Rm1354在延宕期间高丝氨酸内酯含量比野生型低,在指数期和静止期却比野生型高。另外,突变株Rm1354的竞争结瘤能力也大大减弱。这些结果揭示了苜蓿中华根瘤菌nifA基因对许多细胞学过程都有调控作用。     

Functional difference between Sinorhizobium meliloti NifA and Enterobacter cloacae NifA

The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the EC nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the EC nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and EC NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix-phenotype of SmY by Sm Ni     

NifA-NtrA regulatory system activates transcription of nfe,a gene locus involved in nodulation competitiveness of Rhizobium meliloti

We have previously demonstrated that the Rhizobium meliloti large plasmid pRmeGR4b carries the gene locus nodule formation efficiency (nfe) which is responsible for nodulation efficiency and competitive ability of strain GR4 on alfalfa roots. In this study we report that expression of nfe-lacZ fusions in Escherichia coli is activated in the presence of the cloned nifA gene of R. meliloti. This activation was found to be oxygen sensitive and to require the E. coli ntrA gene product. In contrast to the R. meliloti nifA, the cloned nifA gene of Klebsiella pneumoniae was able to activate expression of nfe in aerobically grown cells of both E. coli and R. meliloti. Hybridization experiments did not show homology to nfe in four R. meliloti wild-type strains tested. These strains were uncompetitive when coinoculated with a GR4 derivative carrying plasmid pRmeGR4b, but were competitive when coinoculated with a GR4 derivative carrying a single transposon mutation into the nfe region. When nfe DNA was introduced into the four wild-type strains, a significant increase in the competitive ability of two of them was observed, as deduced from their respective percentages of alfalfa root nodule occupancy in two-strains coinoculation experiments.     

Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae.

We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.