Three regulatory nodD alleles of diverged flavonoid-specificity are involved in host-dependent nodulation by Rhizobium meliloti

Summary Rhizobium meliloti infective on Medicago, Melilotus and Trigonella plants has three copies of the nodulation regulatory gene nodD. Strains containing mutations in nodD1 exhibited a delayed and/or decreased nodulation on Melilotus albus (Ma), Medicago sativa (Ms), Medicago quasifalcata (Mqu) and Trigonella coerulea (Tc), while on Medicago truncatula (Mt) they nodulated similarly to the wild-type R. meliloti. Delayed nodulation was observed also when nodD2 mutants were inoculated onto Ms, Mt and Tc, but not on Ma and Mqu. A nodD3 mutant exhibited delayed nodulation on Ms and Ma. Using a nodC-lacZ fusion and cloned nodD genes on plasmids, high induction levels were detected in R. meliloti when nodD1 was present with seed exudates from Ms, Ma and Mqu, nodD2 with those from Ms and Mt, and nodD3 with those from Ms, Ma and Mqu. NOne of the nodD copies exhibited high levels of nodC-lacZ induction when present with seed exudate from Tc. Only nodD1 induced nodC-lacZ expression in conjunction with the flavone, luteolin. The plant hosts used in this study exude different flavonoids and correlation between nodulation and nodC-lacZ induction abilities of the host exudates was observed. We concluded that all the three nodD copies of R. meliloti have common nod-promoter activating but diverged flavonoid-recognizing abilities. Thus, the three nodD alleles contribute to the activation of nodulation genes in a host-dependent manner.     

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.     

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.     

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.     

Nucleotide sequence of Rhizobium meliloti nodulation genes

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.     

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.     

Nucleotide sequence of Rhizobium meliloti RCR2011 genes involved in host specificity of nodulation.

A 6 kb DNA segment of the R. meliloti 2011 pSym megaplasmid, which contains genes controlling host specificity of root hair infection and of nodulation, was cloned and sequenced. The DNA sequence analysis, in conjunction with previous genetic data, allowed identification of four nod genes designated as E, F, G and H. nodH is divergently transcribed with respect to nodFE and nodG. A conserved nucleotide sequence was found around 200 bp upstream of the translation start of nodF, nodH and nodA. This sequence is also present upstream of common nodA and species specific nodF genes of other Rhizobium species. The predicted protein products of nodF and nodG show homology with acyl carrier protein and ribitol dehydrogenase, respectively. The nodH product contains a rare sequence of four contiguous proline residues. Comparison with the nod gene products of R. leguminosarum shows that species specific nodFE products are as well conserved as those of common nodABC and nodD genes.     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

NoIR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis

The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated. A negative control of plasmid-borne nod gene expression is provided by the NoIR repressor encoded by the chromosomal noIR gene. NoIR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod-box (Kondorosi et al., 1989). We demonstrate here that NoIR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host-specific nod genes located downstream of the n2, n3 and n5 nod-boxes. Thus, the nod genes are differentially regulated by NoIR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation. Furthermore, NoIR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine-tuning mechanism for optimal nodulation, specific to host-plant genotypes. In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C₁₆ fatty acids. Expression of noIR was high both in the free-living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin.     

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.     

Involvement of the syrM and nodD3 genes of Rhizobium meliloti in nod gene activation and in optimal nodulation of the plant host

We identified and sequenced the regulatory syrM and nodD3 genes of Rhizobium meliloti 41. Both genes were shown to contribute to optimal nodulation of alfalfa. In R. meliloti strains carrying syrM and nodD3 on plasmid, the nod genes are expressed constitutively, resulting in host-range extension to siratro. This is due to the presence of multiple syrM copies, suggesting that SyrM participates directly in nod gene activation. NodD3 activates nod genes in conjunction with flavonoids and enhances syrM expression, which is controlled also by its own product, NodD2, and two putative trans-acting factors. nodD3 is regulated by SyrM, NodD1, nodD3, the repressor NoIR, and two putative factors.     

The rkpGHI and -J genes are involved in capsular polysaccharide production by Rhizobium meliloti.

The first complementation unit of the fix-23 region of Rhizobium meliloti, which comprises six genes (rkpAB-CDEF) exhibiting similarity to fatty acid synthase genes, is required for the production of a novel type of capsular polysaccharide that is involved in root nodule development and structurally analogous to group II K antigens found in Escherichia coli (G. Petrovics, P. Putnoky, R. Reuhs, J. Kim, T. A. Thorp, K. D. Noel, R. W. Carlson, and A. Kondorosi, Mol. Microbiol. 8:1083-1094, 1993; B. L. Reuhs, R. W. Carlson, and J. S. Kim, J. Bacteriol. 175:3570-3580, 1993). Here we present the nucleotide sequence for the other three complementation units of the fix-23 locus, revealing the presence of four additional open reading frames assigned to genes rkpGHI and -J. The putative RkpG protein shares similarity with acyltransferases, RkpH is homologous to short-chain alcohol dehydrogenases, and RkpJ shows significant sequence identity with bacterial polysaccharide transport proteins, such as KpsS of E. coli. No significant homology was found for RkpI. Biochemical and immunological analysis of Tn5 derivatives for each gene demonstrated partial or complete loss of capsular polysaccharides from the cell surface; on this basis, we suggest that all genes in the fix-23 region are required for K-antigen synthesis or transport.     

Localization of nodulation gene on the chromosome of Rhizobium meliloti

Using N-methyl-N'-nitro-N-nitrosoguanidine mutant RM54 of Rhizobium meliloti L5-30 defective in the nodulation process (Nod-) and in the biosynthesis of adenine was obtained. Nod- phenotype of this mutant was not caused by the auxotrophic mutation. The nod gene is located on the chromosome. The wild type strain of R. meliloti and Nod- mutant RM54 harbour two indigenous plasmids having a molecular weight of 90 Mdal and about 300 Mdal.