Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

An insertional point mutation inactivates NolR repressor in Rhizobium meliloti 1021.

In the majority of Rhizobium meliloti isolates, nod gene expression is controlled by NolR, but this is not the case in a few strains including the widely used laboratory strain 1021. In 1021, the lack of NolR function was shown to be due to a single insertional mutation in the C-terminal coding sequence which abolished the DNA-binding ability, though the helix-turn-helix motif remained intact. This indicates that the C-terminal part of the protein is also essential for DNA binding. We conclude that in this species, control of nod gene expression involves NolR and strain 1021 represents an exception in which the NolR function was lost by a single event.     

Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation

We show that expression of common nodulation genes in Rhizobium meliloti is under positive as well as negative control. A repressor protein was found to be involved in the negative control of nod gene expression. Whereas the activator NodD protein binds to the conserved cis-regulatory element (nod-box) required for coordinated regulation of nod genes, the repressor binds to the overlapping nodD1 and nodA promoters, at the RNA polymerase binding site. A model depicting the possible interaction of the plant-derived nod gene inducer (luteolin), the NodD and the repressor with the nod promoter elements is presented. Mutants lacking the repressor exhibited delayed nodulation phenotype, indicating that fine tuning of nod gene expression is required for optimal nodulation of the plant host.     

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.     

Identification of Sinorhizobium meliloti LsrB regulon

Redox homeostasis determines cell fate for both prokaryotes and eukaryotes.In bacteria,redox state depends on aerobic respira-tion and antioxidant protection.Su...     

All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants.

Nodulation of alfalfa by exoB mutants of Rhizobium meliloti occurred without root hair curling or infection thread formation. nod exoB double mutants had the same nodulation deficiency as single nod mutants. Therefore, all the known nod genes are involved in nodule induction by exoB mutants, which apparently occurs via intercellular invasion.     

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.     

Use of a promoter-specific probe to identify two loci from the Rhizobium meliloti nodulation regulon

The nodulation regulon of Rhizobium meliloti AK631 includes several operons (nodABC, hsnABC, hsnD, efn locus) which have in common a consensus promoter sequence called the nod box. A synthetic nod box probe was used to identify two additional nod boxes, n4 and n5, which were subcloned for study. By constructing lac fusions, we show that n4 and n5 sponsor induction of downstream regions as previously shown for n1-nodABC and n2-hsnABC. Using site-directed Tn5 mutagenesis, we find that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover. Hybridization data suggest that the n5 locus is conserved among Rhizobium species. In contrast, the n4 locus seems to be unique to Rhizobium meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants. Thus, the use of a promoter probe allows us to identify nodulation genes which may be overlooked by standard methods such as random Tn5 mutagenesis.     

Expression of the nodulation gene nod C of Rhizobium meliloti in Escherichia coli: role of the nod C gene product in nodulation

The nod C gene of Rhizobium meliloti encodes a protein of mol. wt. 44 000 which is highly conserved in at least three Rhizobium species. In order to overproduce this protein, a gene fusion of lambda cI repressor sequences to a large fragment of nod C was constructed. The fusion was placed under control of the tac promoter on plasmid pEA305 to yield pJS1035. IPTG-induced Escherichia coli cells harbouring pJS1035 accumulated the cI-nod C hybrid protein up to 19% of total cellular protein. The synthesis of the hybrid protein drastically inhibits the growth rate of the bacterium. The fusion protein was purified by gel and hydroxyapatite chromatography in the presence of SDS. Antibodies raised against the purified fusion protein precipitated the mol. wt. 44 000 nod C proteins of R. meliloti and of the broad-host range Rhizobium strain NGR234, which were both expressed in E. coli mini-cells. The hybrid protein is associated with the outer membrane of E. coli cells, and the cI-nod C fusion protein appears to be an integral membrane protein. Nodulation of alfalfa by R. meliloti and of clover by R. trifolii was markedly inhibited (approximately 50%) by the addition of antibodies against the hybrid protein to plant growth medium and inoculum.     

Identification of salt- and drought-tolerant Rhizobium meliloti L. strains

The first set of experiments identified sodium chloride (NaCl) tolerance of 92 accessions of Rhizobium meliloti L. from various rhizobia collections and arid and saline areas of the Intermountain West. Accessions were incubated in salinized (0, 176, 352, 528, 616, 704 or 792 m M) yeast extract mannitol (YEM) medium. Growth was measured by turbidity at 420 nm after 3 d in culture. Rhizobial strains were classified by their growth response at an optical density (OD) of 704 m M; Groups One and Two did not exceed 0.10 and 0.33, respectively. Forty three different rhizobial strains were identified as salt-sensitive and 49 as salt-tolerant at 704 m M NaCl. None grew in a saline solution of 792 m M NaCl.The second set of experiments investigated the drought tolerance of R. meliloti accessions that exhibited differential salt tolerance. Fifteen salt-sensitive and 15 salt-tolerant strains of R. meliloti from the first experiment were exposed to simulated drought stress by adding polyethylene glycol 6,000 (PEG-6,000) to the YEM medium at concentrations of 0, –0.4, –0.8 or –1.0 MPa. Rhizobium strains were incubated for 10 days at 25°C and growth turbidity was measured at 420nm. Growth turbidity of the 30 accessions ranged from 100% at –0.4 MPa to 0% at –1.0 MPa. With one exception, strains that were more drought-tolerant (at –1.0 MPa) were also more salt-tolerant (616 m M). However, some of the more salt-tolerant strains at 616 m M were not the more drought-tolerant stains at –1.0 MPa. These salt-and drought-tolerant Rhizobium accessions are excellent models to study the mechanism(s) of such resistance, and to elucidate the role of genetics of NaCl and drought tolerance.     

Identification of the diacylglycerol kinase structural gene of Rhizobium meliloti 1021.

The cyclic beta-1,2-glucans of Rhizobium may function during legume nodulation. These molecules may become highly substituted with phosphoglycerol moieties from the head group of phosphatidylglycerol; diglyceride is a by-product of this reaction (K. J. Miller, R. S. Gore, and A. J. Benesi, J. Bacteriol. 170:4569-4575, 1988). We recently reported that R. meliloti 1021 produces a diacylglycerol kinase (EC 2.7.1.107) activity that shares several properties with the diacylglycerol kinase enzyme of Escherichia coli (W. P. Hunt, R. S. Gore, K. J. Miller, Appl. Environ. Microbiol. 57:3645-3647, 1991). A primary function of this rhizobial enzyme is to recycle diglyceride generated during cyclic beta-1,2-glucan biosynthesis. In the present study, we report the cloning and initial characterization of a single-copy gene from R. meliloti 1021 that encodes a diacylglycerol kinase homolog; this homolog can complement a diacylglycerol kinase deficient strain of E. coli. The sequence of the rhizobial diacylglycerol kinase gene was predicted to encode a protein of 137 amino acids; this protein shares 32% identity with the E. coli enzyme. Analysis of hydropathy and the potential to form specific secondary structures indicated a common overall structure for the two enzymes. Because diglyceride metabolism and cyclic beta-1,2-glucan biosynthesis are metabolically linked, future studies with diacylglycerol kinase mutants of R. meliloti 1021 should further elucidate the roles of the cyclic beta-1,2-glucans in the Rhizobium-legume symbiosis.     

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.     

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.     

Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.