Characterization of a large plasmid of Rhizobium meliloti involved in enhancing nodulation

Summary The plasmid pattern of Rhizobium meliloti strain GR4 was studied and a gene bank of one of the large plasmids (pRmeGR4) of 140 Mdal, was constructed using the broad host range vector pRK290. A restriction map was established with EcoRI. Two regions of this plasmid involved in the infectivity of GR4 on Medicago sativa were identified. An EcoRI fragment hybridizing with the PstI-nif fragment of pID1 was also identified. However, no homology to the cloned Klebsiella pneumoniae nitrogenase genes (pSA30) was detected.     

Cryptic plasmid and rifampin resistance in Rhizobium meliloti influencing nodulation competitiveness.

An assessment was made of the relative contributions of a spontaneous mutation to rifampin resistance and a cryptic plasmid, pTA2, to competitive nodulation of Medicago sativa by a strain of Rhizobium meliloti. This was facilitated by use of rifampin-resistant derivatives of this strain in which pTA2 was originally present, cured, or reintroduced. Both curing of pTA2 and spontaneous mutation to rifampin resistance significantly influenced nodulating competitiveness, but the effect of rifampin resistance was greater and such that the contribution of pTA2 was evident only in cases in which paired competitors had the common rifampin resistance background. The data suggest that rifampin-resistant derivatives contain an altered RNA polymerase insensitive to the action of rifampin. All R. meliloti derivatives had symbiotic characteristics and phage susceptibility patterns similar to those of the wild type. Plasmid pTA2 transfer or other genetic interchange was not detected in nodules of M. sativa inoculated with paired competitors.     

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.     

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.     

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.     

Comparison between Rhizobium galegae and Rhizobium meliloti plasmid contents

Plasmid profiles of two strains of a newly classified rhizobial species- Rhizobium galegae -were compared with the profiles of several strains of another fast-growing Rhizobium species- Rhizobium meliloti .
The existence of a plasmid DNA band with a lower electrophoretic mobility than the R. meliloti megaplasmid band was demonstrated in the two R. galegae strains by a modified horizontal Eckhardt method. Thus R. galegae species contain giant plasmid(s) larger than the R. meliloti 1000 MD megaplasmids, previously considered to be the largest plasmids in the Rhizobiaceae family.
In one of the R. galegae strains an additional middle-size plasmid only a little smaller than 140 MD pRme41a of R. meliloti 41 was observed.     

Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency.

We examined the contribution of a cryptic plasmid, pRmeGR4b, to the nodulation of Medicago sativa by strain GR4 of Rhizobium meliloti. A 905-base-pair PstI DNA fragment in pRmeGR4b was found to hybridize DNA of the R. meliloti fixA promoter region as a probe. Sequence analysis of the PstI fragment showed a 206-base-pair region displaying high homology with the DNA upstream of the RNA start points of the P1 and P2 symbiotic promoters. Putative nif promoter consensus sequences were conserved in this DNA segment. Expression of DNA downstream of the nif promoterlike sequence, monitored by beta-galactosidase activity of different lacZ fusions, was demonstrated to depend on a functional nifA gene, both in microaerobically free-living cells and in nodules. Individual transposon Tn3-HoHo1 insertions in this DNA region caused a reduced nodulation competitiveness. This new symbiotic region, occupying approximately 5 kilobases of pRmeGR4b DNA, was called nfe (nodule formation efficiency).     

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.     

Characterization of the Rhizobium leguminosarum genes nodLMN involved in efficient host-specific nodulation

Three nodulation genes, nodL, nodM and nodN, were isolated from Rhizobium leguminosarum and their DNA sequences were determined. The three genes are in the same orientation as the previously described nodFE genes and the predicted molecular weights of their products are 20,105 (nodL), 65,795 (nodM) and 18,031 (nodN). Analysis of gene regulation using operon fusions showed that nodL, nodM and nodN are induced in response to flavanone molecules and that this induction is nodD-dependent. In addition, it was shown that the nodM and nodN genes are in one operon which is preceded by a conserved 'nod-box' sequence, whereas the nodL gene is in the same operon as the nodFE genes. DNA hybridizations using specific gene probes showed that strongly homologous genes are present in Rhizobium trifolii but not Rhizobium meliloti or Bradyrhizobium japonicum. A mutation within nodL strongly reduced nodulation of peas, Lens and Lathyrus but had little effect on nodulation of Vicia species. A slight reduction in nodulation of Vicia hirsuta was observed with strains carrying mutations in nodM or nodN.     

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.     

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.     

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.     

Transformation of Rhizobium meliloti 41 with plasmid DNA

Plasmid pGV1106, a derivative of the wide-host-range plasmid S-a of the W incompatibility group, was introduced into Rhizobium meliloti 41 by plasmid-mediated mobilization to overcome the restriction of foreign DNA. The mobilized plasmid pKK2 differed from the original pGV1106 by an extra piece of DNA of 1.3 kilobase pairs which supposedly originated from pJB3JI used for mobilization. If pKK2 was isolated from R. meliloti 41, it could be successfully reintroduced by transformation. The transformation frequency was low (10 to 54 colonies per micrograms of plasmid DNA) but reproducible, and several lines of evidence showed that it was the consequence of plasmid DNA uptake. The small size (10.3 kilobases) and elevated copy number (10 to 15 copies per cell) of pKK2 make it a potentially useful cloning vector for the study of symbiotic nitrogen fixation genes of R. meliloti 41.