Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce marked root hair curling and thick and short roots.Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered.Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26.Abbreviations Rps repression of production of small bacteriocin - Mep medium bacteriocin production - Nod nodulation - Fix fixation - Tsr thick and short roots - Flac root hair curling - Hsp host specificity - Flad root hair deformation - Tc tetracycline - Km kanamycin - Cm chloramphenicol - Sp spectinomycin - Sm streptomycin - R resistant     

Induction of the nodA promoter of Rhizobium leguminosarum Sym plasmid pRL1JI by plant flavanones and flavones.

An expression vector containing the Rhizobium leguminosarum nodA promoter cloned in front of the Escherichia coli lacZ gene was used to characterize the properties of the R. leguminosarum nodA gene-inducing compound(s) present in sterile root exudate of the host plant Vicia sativa L. subsp. nigra (L.). The major inducing compound was flavonoid in nature, most likely a flavanone. The commercially available flavonoids naringenin (5,7,4'-trihydroxyflavanone), eriodictyol (5,7,3'4'-tetrahydroxyflavanone), apigenin (5,7,4'-trihydroxyflavone), and luteolin (5,7,3',4'-tetrahydroxyflavone) induced the nodA promoter to the same level as the root exudate. On the basis of chromatographic properties, it was concluded that none of these compounds is identical to the inducer that is present in root exudate. The induction of the nodA promoter by root exudate and by the most effective inducer naringenin was very similar, as judged from the genetic requirements and the kinetics of induction.     

The replicator region of the Rhizobium leguminosarum cryptic plasmid pRL8JI

Abstract The replicator region of the cryptic plasmid pRL8JI from Rhizobium leguminosarum strain 3841 was cloned and sequenced. The recombinant plasmid (pYK3) was selected by function from a partial Eco RI library of total DNA cloned in pSUP202 and shows incompatibility with plasmid pRL8JI when conjugated into R. leguminosarum strains 3841 and its derivative 1062. The cloned insert (∼ 10.5 kb) comprises five Eco RI fragments none of which confers replicative stability when cloned individually. A single 5.0-kb Bam HI fragment, that spans all five Eco RI fragments and confers replicative stability on pSUP202 in R. leguminosarum , has been sequenced. This replicator region shows organisational and sequence similarity to the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b. It has three open reading frames ( repA, repB, repC ) and a conserved intergenic sequence.     

Recipient-induced transfer of the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae is regulated by a quorum-sensing relay

Analysis of the regulation of plasmid transfer genes on the symbiotic plasmid pRL1JI in Rhizobium leguminosarum bv. viciae has revealed a novel regulatory relay that is specifically poised to detect an N-acyl-homoserine lactone (AHL) made by different cells (potential recipients of pRL1JI). Adjacent to the traI-trbBCDEJKLFGHI plasmid transfer operon on pRL1JI are two regulatory genes, bisR and traR, which encode LuxR-type quorum-sensing regulators required for conjugation. Potential recipients of pRL1JI induce the traI-trb operon and plasmid transfer via a quorum-sensing relay involving BisR, TraR and the traI-trb operon in donor cells. BisR induces expression of traR in response to N-(3-hydroxy-7-cis-tetradecenoyl)-l-homoserine lactone (3-OH-C14:1-HSL), which is produced by CinI in potential recipient strains. In donor strains (carrying pRL1JI), BisR represses the expression of the chromosomal gene cinI; this repression results in a very low level of formation of 3-OH-C14:1-HSL and hence relatively low levels of expression of traR and the traI-trb operon in strains carrying pRL1JI. However, if 3-OH-C14:1-HSL from potential recipients is present, then traR and plasmid transfer are induced. The induction of traR occurs at very low concentrations of 3-OH-C14:1-HSL (around 1 nm). TraR then induces the traI-trb operon in a quorum-sensing dependent manner in re-sponse to the TraI-made AHLs, N-(3-oxo-octanoyl)-l-homoserine lactone and N-(octanoyl)-l-homoserine lactone. The resulting autoinduction results in high levels of expression of the traI-trb operon. Premature expression of the traI-trb operon is reduced by TraM, which probably titres out TraR preventing expression of traI when there are low levels of traR expression. Expression of traR in stationary phase cells is limited by feedback inhibition mediated by TraI-made AHLs.     

Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame.

By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified. DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56, 178 D. Based on sequence homology, this ORF was confirmed to correspond to the nifA gene. Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology. Sequence analysis also showed that the R. leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between. This novel ORF of 294 bp was found to be highly conserved also in R. meliloti. No known promoter and termination signals could be defined on the sequenced R. leguminosarum fragment.     

Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti.

Two self-transmissible Sym(biosis) plasmids, one encoding pea-specific nodulation and nitrogen-fixation functions (plasmid pJB5JI) and the other encoding clover-specific nodulation and nitrogen-fixation functions (plasmid pBR1AN) were used to determine whether the symbiotic genes encoded on these plasmids are expressed in various members of the Rhizobiaceae. The host specificity of Rhizobium trifolii and R. leguminosarum Sym plasmid-cured strains could be directly determined by the transfer to these strains of the appropriate Sym plasmid. The nodulation of white clovers was restored by either plasmid pJB5JI or pBR1AN when these plasmids were transferred to two transposon Tn5-induced hair-curling (Hac-) R. trifolii mutants. In addition, lucerne nodulation was restored to a Hac- R. meliloti mutant when either plasmid pBR1AN or pJB5JI was transferred to this strain. The phenotype of nonmucoid (Muc-) Rhizobium mutants, which had altered cell surfaces, was not influenced by the transfer to these strains of plasmid pBR1AN or plasmid pJB5JI.     

Identification of a rhizosphere protein encoded by the symbiotic plasmid of Rhizobium leguminosarum.

A protein was identified which was made by wild-type strains of Rhizobium leguminosarum but not by nodulation-deficient derivatives which had deletions of their symbiotic plasmids. The protein, which had a subunit molecular weight of ca. 24,000 ( 24K ), was found to be present in large amounts within bacteria that had been reisolated from the surface of inoculated pea roots but was not detected in bacteroids isolated from nodules. The protein could also be induced during growth of R. leguminosarum on nutrient medium and was purified from the cytoplasmic fraction of broken cells. Antiserum raised against the purified protein was used to screen transposon-induced mutants of R. leguminosarum, and four independent mutants were isolated which lacked the protein. The sites of the Tn5 insertions were found to map between the nitrogenase and nodulation genes on symbiotic plasmid pRL1JI , ca. 5 kilobases from the nitrogenase genes and 13 kilobases from the nodulation genes. Genetic determinants for the 24K protein were found to be closely linked to plasmid-borne nodulation genes for all strains of R. leguminosarum tested. However, the mutants which lacked the 24K protein still formed normal nitrogen-fixing nodules on peas, and the function of the protein is unknown.     

Selection of strains cured of the Rhizobium leguminosarum Sym-plasmid pRL1JI by using small bacteriocin

Abstract Fast growing Rhizobia are usually insensitive to a low M _r (small) bacteriocin. Introduction of the Sym-plasmid pRL1JI into these strains results in sensitivity towards small bacteriocin. Of such strains, small bacteriocin insensitive mutants (Sbs⁻) were selected.
A high percentage of these Sbs⁻ mutants appeared to be cured of pRL1JI. This selection of cured strains was feasible as well for the wild-type plasmid pRL1JI, as for a variety of transposon marked derivatives in several bacterial backgrounds.     

The function of three indigenous plasmids in Mtesorhizobium huakuii 2020 and its symbiotic interaction with Sym pJB5JI of Rhizobium leguminosarum

A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.     

Structural complexity of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli.

The complete physical map of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli strain CFN42 was established. The data support the concept that Rhizobium symbiotic genes are part of a complex genomic structure which contains a large amount of reiterated DNA sequences. This plasmid is a circular structure of 390 kb with approximately 10 families of internally reiterated DNA sequences of two to three elements each. One family includes two directly oriented nitrogenase operons situated 120 kb apart. We also found several stretches of pSym that are reiterated in other replicons of the cell. Localization of symbiotic gene sequences by heterologous hybridization revealed that nodABC sequences are separated in two regions, each of which contains a nod boxlike element, and it also suggested the presence of two copies of the nifA and nodD gene sequences. We propose that the complex structure of the symbiotic plasmid allows interactions between repeated DNA sequences which, in turn, might result in frequent rearrangements.     

Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid

Rhizobium trifolii T37 contains at least three plasmids with sizes of greater than 250 megadaltons. Southern blots of agarose gels of these plasmids probed with Rhizobium meliloti nif DNA indicated that the smallest plasmid, pRtT37a, contains the nif genes. Transfer of the Rhizobium leguminosarum plasmid pJB5JI, which codes for pea nodulation and the nif genes and is genetically marked with Tn5, into R. trifolii T37 generated transconjugants containing a variety of plasmid profiles. The plasmid profiles and symbiotic properties of all of the transconjugants were stably maintained even after reisolation from nodules. The transconjugant strains were placed into three groups based on their plasmid profiles and symbiotic properties. The first group harbored a plasmid similar in size to pJB5JI (130 megadaltons) and lacked a plasmid corresponding to pRtT37a. These strains formed effective nodules on peas but were unable to nodulate clover and lacked the R. trifolii nif genes. This suggests that genes essential for clover nodulation as well as the R. trifolii nif genes are located on pRtT37a and have been deleted. The second group harbored hybrid plasmids formed from pRtT37a and pJB5JI which ranged in size from 140 to ca. 250 megadaltons. These transconjugants had lost the R. leguminosarum nif genes but retained the R. trifolii nif genes. Strains in this group nodulated both peas and clover but formed effective nodules only on clover. The third group of transconjugants contained a hybrid plasmid similar in size to pRtT37b. These strains contained the R. trifolii and R. leguminosarum nif genes and formed N2-fixing nodules on both peas and clover.     

The influence of the symbiotic plasmid pRL1JI on the distribution of GM rhizobia in soil and crop rhizospheres,and implications for gene flow

The distribution of two genetically modified Rhizobium leguminosarum strains was investigated in the field. One, RSM2004, released in 1987, carries a Tn5 marker on its conjugative symbiotic plasmid (pSym). The second, CT0370, released at the same site in 1994, has a gusA gene integrated into its chromosome but no pSym. Plate counts indicated that the CT0370 population became established at a higher level than RSM2004. However, when peas, alfalfa and barley were grown, RSM2004 was found to outnumber CT0370 on all roots and by 100-fold on pea. Although the transfer of pSym from RSM2004 to CT0370 could be detected on plates and in microcosm studies with high inoculum densities, no transfer was detected in the field. Subsequent transfer of pSym from RSM2004 to CT0370 demonstrated that it conferred an advantage in the rhizosphere. In addition to increasing host fitness, plasmids may transfer, or mobilise other genetic elements, to other bacteria. This is more likely in sites such as the rhizosphere, where cells are active and numbers are high. The distribution of pSym and other genetic elements associated with rhizobia, in bacterial sub-populations from the soil and roots of the different plants, was investigated using PCR. The genetic elements studied were: ISRm3, an insertion element from Sinorhizobium meliloti; pSB102, a broad host range mer plasmid; the Rhizobium nodC gene (carried on pSym) and plasmid replication origins repCI and repCII. As expected, ISRm3 was detected in rhizoflora cultured from alfalfa but not the other plants. The mer gene was ubiquitous but the transfer region of pSB102 was not detected. The nodC and both repC primers amplified products from all the plants, giving further evidence for the occurrence of plasmids originating from Rhizobium in the rhizoflora of non-host plants. Despite the abundance of elements associated with transferable plasmids in rhizobia, none was detected in either inoculant strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transposon mediation allows a symbiotic plasmid of Rhizobium leguminosarum bv. trifolii to become a symbiosis island in Agrobacterium and Rhizobium

The symbiotic plasmid (pSym) of Rhizobium leguminosarum bv. trifolii 4S5, which carries Tn5-mob, was successfully transferred into Agrobacterium tumefaciens A136 by using a conjugation method. The resulting transconjugants induced the development of ineffective nitrogen-fixing nodules on the roots of white clover seedlings. Depending on the manner in which the pSym was retained, the transconjugants were divided into two groups of strains, Afp and Afcs. pSym was retained as a plasmid in the Afp strains but was integrated into the int gene encoding a phage-related integrase on the linear chromosome of A. tumefaciens A136 in strain Afcs1 (one of the Afcs strains) to form a symbiosis island. Conjugation was performed between strain Afcs1 and R. leguminosarum bv. trifolii H1 (a pSym-cured derivative of wild-type strain 4S), and the Rhizobium H1tr strains were screened as transconjugants. Eighteen of the H1tr strains induced effective nitrogen-fixing nodules on the roots of the host plants. pSym was transferred into all of the transconjugants, except for strain H1tr1, at the same size as pSym of strain 4S5. In strain H1tr1, pSym was integrated into the chromosome as a symbiosis island. These data suggest that pSym can exist among Rhizobium and Agrobacterium strains both as a plasmid and as a symbiosis island with transposon mediation.     

Derivatives of Rhizobium meliloti strains carrying a plasmid of Rhizobium leguminosarum specifying hydrogen uptake and pea-specific symbiotic functions

pIJ1008, a Rhizobium leguminosarum plasmid which determines hydrogen uptake ability and symbiotic functions in pea was transferable to three of seven natural isolates of R. meliloti tested. In these three strains, pIJ1008 was maintained stably with the respective sym megaplasmid indigenous to each R. meliloti strain. These strains carrying both plasmids nodulated alfalfa but not pea. By reisolation and examination of the strains from alfalfa nodule tissue, it was shown that pIJ1008 continued to be maintained but that pea-nodulation ability was suppressed.In one strain of R. meliloti which carries a 200 kb cryptic plasmid (in addition to a megaplasmid), the transfer and selection for pIJ1008 resulted in the loss of the cryptic plasmid.In three separate plant growth experiments, alfalfa nodules induced by each of the R. meliloti strain carrying both sym plasmids were assayed for hydrogen uptake activity. The average activity was 40-, 3.5-and 2-fold higher than with the respective pIJ1008-free strains. However, this higher activity was not accompanied by an increase in plant biomass or nitrogen content of shoots.C.B.R.I. Contribution Number: 1478     

Amplification and deletion of a nod-nif region in the symbiotic plasmid of Rhizobium phaseoli.

One remarkable characteristic of the genomes of some Rhizobium species is the frequent occurrence of rearrangements. In some instances these rearrangements alter the symbiotic properties of the strains. However, no detailed molecular mechanisms have been proposed for the generation of these rearrangements. To understand the mechanisms involved in the formation of rearrangements in the genome of Rhizobium phaseoli, we have designed a system which allows the positive selection for amplification and deletion events. We have applied this system to investigate the stability of the symbiotic plasmid of R. phaseoli. High-frequency amplification events were detected which increase the copy number of a 120-kb region carrying nodulation and nitrogen fixation genes two to eight times. Deletion events that affect the same region were also found, albeit at a lower frequency. Both kinds of rearrangements are generated by recombination between reiterated nitrogenase (nifHDK) operons flanking the 120-kb region.     

Transfer of the Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a to a strain of Rhizobium sp. that nodulates on Hedysarum coronarium

The Rhizobium leguminosarum biovar trifolii symbiotic plasmid pRtr5a was transferred to the Rhizobium sp. (Hedysarum coronarium) strain RB16. Transconjugants carrying pRtr5a ineffectively nodulated Trifolium repens, T. pratense and T. alexandrinum and were unable to nodulate H. coronarium plants. Agarose gel electrophoresis of transconjugants showed that all had lost an indigenous plasmid (230 Md). These results suggest that this plasmid harbours the symbiotic determinants for nodulation on H. coronarium.