A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti.

BACKGROUND: Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. RESULTS: We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis. CONCLUSIONS: Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

Inhibition of the FixL sensor kinase by the FixT protein in Sinorhizobium meliloti.

Nitrogen fixation in symbiotic rhizobia is subject to multiple levels of gene regulation. In Sinorhizobium meliloti, the alfalfa symbiont, the FixLJ two-component regulatory system plays a major role in inducing nitrogen fixation and respiration gene expression in response to the low ambient O(2) concentration of the nodule. Here we report on the mode of action of the FixT protein, a recently identified repressor of nitrogen fixation gene expression in S. meliloti. First, we provide evidence that FixT prevents transcription of the intermediate key regulatory genes nifA and fixK by counteracting the activity of the FixLJ two-component system under otherwise inducing microoxic conditions. Second, we demonstrate that FixT acts as an inhibitor of the sensor hemoprotein kinase FixL, preventing the production or the accumulation of its phosphorylated form. FixT is thus a new example of a regulatory protein that blocks signal transduction in two-component systems at the level of the sensor kinase.

Negative autoregulation of the Rhizobium meliloti fixK gene is indirect and requires a newly identified regulator, FixT.

fixK genes are crp/fnr homologues that have been discovered in diverse Rhizobium spp., in which they are usually essential for symbiotic nitrogen fixation. One recurrent function of fixK genes in rhizobia is to activate the transcription of operons required for respiration in the microoxic environment of the nodule. In a similar manner to its Escherichia coli crp and fnr homologues, R. meliloti fixK regulates its own expression negatively. However, we demonstrate here that fixK negative autoregulation is not direct and, instead, involves a newly identified gene, fixT, the expression of which depends on fixK. Inactivation of fixT resulted in derepression of fixK expression under free-living microoxic conditions. Furthermore, constitutively expressed fixT strongly repressed fixK-lacZ expression in the absence of a functional fixK gene. Several lines of evidence indicate that fixT is active via its protein product FixT. FixT does not resemble any protein present in databases so far. Nodules induced by a fixT mutant were Fix+, thus demonstrating that fixT is not essential for symbiotic nitrogen fixation.

Phosphorylation of the Rhizobium meliloti FixJ protein induces its binding to a compound regulatory region at the fixK promoter.

The FixJ protein is a member of the regulator class of two-component systems involved in the transcriptional activation of nitrogen fixation genes in Rhizobium meliloti. Phosphorylation of FixJ was previously demonstrated to dramatically enhance its transcriptional activity at the nifA and fixK promoters. Here we show that the isolated carboxyl-terminal domain of FixJ, FixJC, binds the fixK promoter, whereas binding of the full-length FixJ protein requires its phosphorylation. By analyzing the DNase I and Exonuclease III protection patterns of the wild-type and a mutant fixK promoter, we have identified two overlapping binding regions for both phosphorylated FixJ and FixJC. A higher affinity region is located between positions -69 and -44 relative to the transcription start site, and a lower affinity region, between positions -57 and -31, overlaps the -35 region of the promoter.

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 meliloti regulatory gene fixJ activates transcription of R. meliloti nifA and fixK genes in Escherichia coli.

When present in Escherichia coli on the multicopy expression vector pUC19, a Rhizobium meliloti regulatory gene, fixJ, belonging to a two-component regulatory system, activated the expression of two R. meliloti symbiotic genes, nifA and fixK. Primer extension by reverse transcription showed that FixJ stimulates nifA expression in E. coli by activating pnifA.

A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus.

A 290-kilobase (kb) region of the Rhizobium meliloti 2011 pSym megaplasmid, which contains nodulation genes (nod) as well as genes involved in nitrogen fixation (nif and fix), was shown to carry at least six sequences repeated elsewhere in the genome. One of these reiterated sequences, about 5 kb in size, had previously been identified as part of a cluster of fix genes located 220 kb downstream of the nifHDK promoter. Deletion of the reiterated part of this fix cluster does not alter the symbiotic phenotype. Deletion of the second copy of this reiterated sequence, which maps on pSym 40 kb upstream of the nifHDK promoter, also has no effect. Deletion of both of these copies however leads to a Fix- phenotype, indicating that both sequences carry functionally reiterated fix gene(s). The fix copy 40 kb upstream of nifHDK is part of a symbiotic cluster which also carries a nod locus, the deletion of which produces a marked delay in nodulation.

Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation.

In Rhizobium meliloti 2011 nodulation genes (nod) required to nodulate specifically alfalfa are located on a pSym megaplasmid. Nod- derivatives carrying large pSym deletions were isolated. By complementation of these strains with in vivo- and in vitro-constructed episomes containing pSym of sequences and introduction of these episomes into Agrobacterium tumefaciens, we show (i) that from a region of pSym of about 360 kilobases, genes required for specific alfalfa nodulation are clustered in a DNA fragment of less than 30 kilobases and (ii) that a nod region located between nifHDK and the common nod genes is absolutely required for alfalfa nodulation and controls the specificity of root hair curling and nodule organogenesis initiation.