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.     

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.     

Regulation of Syrm and Nodd3 in Rhizobium Meliloti

The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont. NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers. The third, NodD3, is an inducer-independent activator of nod operons. We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression. Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM. The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule. We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma. This regulatory circuit may be important for regulation of nod genes within the developing nodule.     

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.     

Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti.

In Rhizobium meliloti, expression of the nodulation genes (nod and nol genes) is under both positive and negative controls. These genes are activated by the products of the three related nodD genes, in conjunction with signal molecules from the host plants. We showed that negative regulation is mediated by a repressor protein, binding to the overlapping nodD1 and nodA as well as to the nodD2 promoters. The encoding gene, termed nolR, was identified and cloned from strain 41. By subcloning, deletion and Tn5 mutagenesis, a region of 594 base-pairs was found to be necessary and sufficient for repressor production in strains of R. meliloti lacking the repressor or in Escherichia coli. Sequence analysis revealed that nolR encodes a 13,349 Da protein, which is in agreement with the molecular weight of the NolR protein, determined after purification by affinity chromatography, utilizing long synthetic DNA multimers of the 21 base-pair conserved repressor-binding sequence. Our data suggest that the native NolR binds to the operator site in dimeric form. The NolR contains a helix-turn-helix motif, which shows homology to the DNA-binding sequences of numerous prokaryotic regulatory proteins such as the repressor XylR or the activator NodD and other members of the LysR family. Comparison of the putative DNA-binding helix-turn-helix motifs of a large number of regulatory proteins pointed to a number of novel regularities in this sequence. Hybridizations with an internal nolR fragment showed that sequences homologous to the nolR gene are present in all R. meliloti isolates tested, even in those that do not produce the repressor. In another species, such as Rhizobium leguminosarum, where NodD is autoregulated, however, such sequences were not detected.     

Structural and functional analysis of two different nodD genes in Bradyrhizobium japonicum USDA110.

Bradyrhizobium japonicum has two closely linked homologs of the nodulation regulatory gene, nodD; these homologs are located upstream of and in divergent orientation to the nodYABCSUIJ gene cluster. We report here the nucleotide sequence and mutational analyses of both nodD copies. The predicted NodD1 and NodD2 proteins shared 62% identical amino acid residues at corresponding positions and exhibited different degrees of homology with NodD proteins of other Bradyrhizobium, Azorhizobium, and Rhizobium strains. Induction of the nodYABCSUIJ operon, as measured by expression of a translational nodC'-'lacZ fusion, required the nodD1 gene, but not nodD2. A B. japonicum mutant deleted for both nodD copies (strain delta 1267) still showed residual nodulation activity; however, nodulation of soybean was significantly delayed, and nodulation of mung bean and siratro resulted in strongly reduced nodule numbers. Fully efficient nodulation of mung bean and siratro by strain delta 1267 was restored by genetic complementation with the nodD1 gene, but not with nodD2. We conclude from these data that nodD1 is the critical gene that contributes to maximal nodulation efficiency, whereas the nodD2 gene does not play any obvious role in nodulation of the host plants tested.     

Role of the nodD and syrM genes in the activation of the regulatory gene nodD3, and of the common and host-specific nod genes of Rhizobium meliloti

To analyse the regulation of the nodulation (nod) genes of Rhizobium meliloti RCR2011 we have isolated lacZ gene fusions to a number of common, host-range and regulatory nod genes, using the mini-Mu-lac bacteriophage transposon MudII1734. Common (nodA, nodC, nod region IIa) and host-range (nodE, nodG, nodH) genes were found to be regulated similarly. They were activated (i) by the regulatory nodD1 gene in the presence of flavones such as chrysoeriol, luteolin and 7,3',4'-trihydroxyflavone, (ii) by nodD2 in the presence of alfalfa root exudate but not with the NodD1-activating flavones, and (iii) by the regulatory genes syrM-nodD3 even in the absence of plant inducers. Thus common and host-range nod genes belong to the same regulon. In contrast to the nodD1 gene, the regulatory nodD3 gene was not expressed constitutively and exhibited a complex regulation. It required syrM for expression, was activated by nodD1 in the presence of luteolin and was positively autoregulated.     

Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD 1 is preceded by nolE, a gene whose product is secreted from the cytoplasm

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia. Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences. Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.     

Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816.

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.     

nodD alleles of Sinorhizobium fredii USDA191 differentially influence soybean nodulation, nodC expression, and production of exopolysaccharides

All Rhizobium strains examined to date have one or multiple alleles of nodD. At least one copy of nodD and the presence of flavonoid exudates are required for nod gene induction and nodulation. Sinorhizobium fredii USDA191 has two copies of nodD. In this study, we demonstrate that inactivation of either copy of nodD caused a reduction in basal levels of expression of nodC. Extra copies of nodD1 had no effect on the expression of nodC when compared with the wild type, but extra copies of nodD2 abolished the inducer requirement, thereby rendering nodC constitutive. A nodD1 mutant was unable to nodulate soybean cultivars 'Peking' and 'McCall'. Inactivation of nodD2 or addition of extra copies of nodD1 or nodD2 caused delayed nodulation on Peking, and reduced the number of nodules on McCall. Both nodD alleles of S. fredii USDA191 appear to be involved in regulation of exopolysaccharide production; however, nodD2 appears to be more important in this respect than nodD1.     

Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules

The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds. We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123. Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes. Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene. Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression. In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains. The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD. An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro. Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R. l. bv. trifolii to the nonlegume Parasponia. DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined. Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.