Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

The Rhizobium meliloti early nodulation genes (nodABC) are nitrogen-regulated: Isolation of a mutant strain with efficient nodulation capacity on alfalfa in the presence of ammonium

Summary The presence of combined nitrogen in the soil suppresses the formation of nitrogen-fixing root nodules by Rhizobium. We demonstrate that bacterial genes determining early nodulation functions (nodABC) as well as the regulatory gene nodD3 are under nitrogen (NH ₄ ⁺ ) control. Our results suggest that the gene product of nodD3 has a role in mediating the ammonia regulation of early nod genes. The general nitrogen regulatory (ntr) system as well as a chromosomal locus mutated in Rhizobium meliloti were also found to be involved in the regulation of nod gene expression. A R. meliloti mutant with altered sensitivity to ammonia regulation was isolated, capable of more efficient nodulation of alfalfa than the wild-type strain in the presence of 2 mM ammonium sulfate.     

Identification and cloning of nodulation genes from the wide host range Rhizobium strain MPIK3030

Summary A 70 kbp segment of the megaplasmid from a broad host range Rhizobium strain (MPIK3030) was mapped with the aid of cosmid clones made in the vector pJB8. A 7.9 kbp EcoRI fragment from this region, 55 kbp away from the nif gene cluster, was shown to hybridize to the common nod genes from R. meliloti. Using several R. meliloti nod probes it was possible to delimit an 830 bp region as being the center of greatest homology. Sequence data from two sections of this region gave a nucleotide homology of 73.7% to the nodC gene of R. meliloti. Using Tn5 mutagenesis a clone was isolated carrying Tn5 in the highly homologous region. When tested on Macroptilium atropurpureum, this MPIK3030 derivative was shown to have a Nod^– phenotype. When the wild-type allele was reintroduced into the Tn5 mutant, nodulation was restored. Interspecies complementation also showed that both R. meliloti and Rhizobium sp. MPIK3030 nod regions were able to restore nodulation to Tn5-induced nodC mutants from either strain.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Chemotaxis of Rhizobium meliloti towards Nodulation Gene-Inducing Compounds from Alfalfa Roots

Luteolin, a flavone present in seed exudates of alfalfa, induces nodulation genes (nod) in Rhizobium meliloti and also serves as a biochemically specific chemoattractant for the bacterium. The present work shows that R. meliloti RCR2011 is capable of very similar chemotactic responses towards 4′,7-dihydroxyflavone, 4′,7-Dihydroxyflavanone, and 4,4′-dihydroxy-2-methoxychalcone, the three principal nod gene inducers secreted by alfalfa roots. Chemotactic responses to the root-secreted nod inducers in capillary assays were usually two- to four-fold above background and, for the flavone and flavonone, occurred at concentrations lower than those required for half-maximal induction of the nodABC genes. Complementation experiments indicated that the lack of chemotactic responsiveness to luteolin seen in nodD1 and nodA mutants of R. meliloti was not due to mutations in the nod genes, as previously thought. Thus, while nod gene induction and flavonoid chemotaxis have the same biochemical specificity, these two functions appear to have independent receptors or transduction pathways. The wild-type strain was found to suffer selective, spontaneous loss of chemotaxis towards flavonoids during laboratory subculture.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic β-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.     

Host-specificity mutants of Rhizobium meliloti have additive effects in situ on initiation of alfalfa nodules

Pairs of Rhizobium meliloti nod mutants were co-inoculated onto alfalfa (Medicago saliva L.) roots to determine whether one nod mutant could correct, in situ, for defects in nodule initiation of another nod mutant. None of the Tn5 or nod deletion mutants were able to help each other form nodules when co-inoculated together in the absence of the wild-type. However, as previously observed, individual nod mutants significantly increased nodule initiation by low dosages of co-inoculated wild-type cells. Thus, nod mutants do produce certain signal substances or other factors which overcome limits to nodule initiation by the wild-type. When pairs of nod mutants were co-inoculated together with the wild-type, the stimulation of nodulation provided by individual nodABC mutants was not additive. However, clearly additive or synergistic stimulation was observed between pairs of mutants with a defective host-specificity gene (nodE, nodF, or nodH). Each pair of host-specificity mutants stimulated first nodule formation to nearly the maximum levels obtainable with high dosages of the wild-type. Mutant bacteria were recovered from only about 10% of these nodules, whereas the co-inoculated wild-type was present in all these nodules and substantially outnumbered mutant bacteria in nodules occupied by both. Thus, these mutant co-inoculants appeared to help their parent in situ even though they could not help each other. Sterile culture filtrates from wild-type cells stimulated nodule initiation by low dosages of the wild-type, but only when a host-specificity mutant was also present. The results from our studies seem consistent with the possibility that pairs of host-specificity mutants are able to help the wild-type initiate nodule formation by sustained production of complementary signals required for induction of symbiotic host responses.     

Identification of host range determinants in the Rhizobium species MPIK3030

Summary R. meliloti primarily nodulates Medicago sativa but cannot nodulate Macroptilium atropurpureum. By introducing an 11.4 kb region into R. meliloti from the Symplasmid of Rhizobium strain MPIK3030, the host range of the R. meliloti transconjugants were shown to be extended to M. atropurpureum, one of the hosts of MPIK3030 but not normally nodulated by R. meliloti. The region responsible for host range extension was isolated by mass conjugating a clone bank from MPIK3030 into the R. meliloti wild type, and subsequent screening for nodulation on M. atropurpureum. Using deleted derivatives of a plasmid reisolated from endosymbiotic bacteria, the host range region was further narrowed down to three EcoRI fragments. Tn5 mutagenesis allowed the isolation of three discrete regions on an 11.4 kb section, which are involved in the extension of host range to M. atropurpureum. Finally, complementation experiments performed with R. meliloti common nod and hsn mutants indicated that none of the genes involved in the early steps of nodulation, including host-range functions, can be complemented by genes carried on the 11.4 kb fragment derived from MPIK3030.     

Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in Sinorhizobium meliloti

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

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.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti 1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [¹⁴C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [¹⁴C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxAB fusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.     

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.     

Root exuded nod-gene inducing signals limit the nodulation capacity of different alfalfa varieties with Rhizobium meliloti

Summary Different nodulation capacities were found among nine different varieties of alfalfa, cultivated in the Central region of Mexico, by Rhizobium meliloti 2011. A correlation between nodulation capacity and foliar dry weight was observed, which points to a genotype dependance on these parameters. A correlation between the nodulation capacity and the R. meliloti nod-gene inducing activity of the root exudates from the different varieties, as measured by -galactosidase induction in a test system consisting of a R. meliloti nodC-lacZ strain incubated with each root exudate, was established. When the root exudate from the best nodulating variety was added to the four poorest nodulating varieties, an increase in nodule formation was observed. We conclude that root exuded nod-gene inducing signals are a symbiotically-limiting component in natural populations of the poorest nodulating varieties of alfalfa.     

Expression of an Exogenous 1-Aminocyclopropane-1-Carboxylate Deaminase Gene in Sinorhizobium meliloti Increases Its Ability To Nodulate Alfalfa

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.