Plasmid-determined nodulation and nitrogen-fixation abilities inRhizobium phaseoli

Summary InRhizobium phaseoli strain 8002, the 190 Md plasmid pRP2JI which determines the ability to produce nitrogen-fixing nodules onPhaseolus beans (Nod⁺ Fix⁺) and the production of melanin on L-tyrosine-containing media (Mel⁺), was shown to be transmissible by conjugation to otherRhizobium strains. When pRP2JI was transferred to Nod^- strains ofR. leguminosarum (which normally nodulates peas) the transconjugants gained the ability to nodulatePhaseolus beans and to make melanin.Out of 187 derivatives of strain 8002 carrying pRP2JI plasmids into which the transposon Tn5 had been inserted, six were Fix^- Nod⁺ Mel⁺, one was Fix^- Nod⁺ Mel^- and four were Fix⁺ Nod⁺ Mel^-. Three other derivatives of strain 8002 were Nod^- Mel^-; these had suffered deletions of c 30 Md in pRP2JI. Thus the genes for melanin production and nodulation appear to be closely linked, but melanin production is not necessary for the induction of nitrogen-fixing nodules onPhaseolus beans.     

Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids

Strains of Rhizobium leguminosarum use a cell density-dependent gene regulatory system to assess their population density. This is achieved by the accumulation of N-acyl-homoserine lactones (AHLs) in the environment during growth of the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in the late-exponential and stationary phases of growth. A genetically well-characterised strain of R. leguminosarum biovar viciae was found to have four genes, whose products synthesise different AHLs. We have analysed AHL production by four genetically distinct isolates of R. leguminosarum, three of bv. viciae and one of bv. phaseoli. Distinct differences were seen in the pattern of AHLs produced by the bv. viciae strains compared with bv. phaseoli and the increased levels and diversity of AHLs found in bv. viciae strains can be attributed to the rhiI gene, which is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rhizosphere. Additional complexity to the profile of AHLs is found to be associated with highly transmissible plasmid pRL1JI of R. leguminosarum bv. viciae, but this is not observed with some other strains, including those carrying different transmissible plasmids. In addition to AHLs produced by the products of genes on the symbiotic plasmid, there is clear evidence for the presence of other AHL production loci. Expression levels and patterns of AHLs can change markedly in different growth media. These results indicate that there is a network of quorum-sensing loci in different strains of R. leguminosarum and these loci may play a role in adapting to rhizosphere growth and plasmid transfer.     

Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum

A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRleVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRleVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix- nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRleVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.     

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

Summary Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.     

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Summary Five specific transposon-induced nodulation defective (Nod⁻) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod⁻ mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod⁻ on all their known plant hosts, (b), they could not induce root hair curling (Hac⁻) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.     

Identification of the host-range DNA which allows Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas

Summary The special ability of Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas had previously been shown to be determined by the symbiotic plasmid, pRL5JI, of this strain. A region of pRL5JI, 2.0 kb in size, was found to confer the ability to nodulate cv. Afghanistan peas when transferred to strains of R. leguminosarum which normally fail to nodulate this host. This region of pRL5JI, responsible for the extension of host-range, was closely linked to, but did not include, the genes required for root hair curling. Although extensive homology has been found between the R. leguminosarum nod genes on pRL5JI and those on the normal symbiotic plasmid pRL1JI, a fragment from the 2.0 kb region involved in nodulation of cv. Afghanistan has been identified, which was not homologous to DNA in strains which do not nodulate cv. Afghanistan.     

Genetic Characterization of a Novel Rhizobial Plasmid Conjugation System in Rhizobium leguminosarum bv. viciae Strain VF39SM

Rhizobium leguminosarum strain VF39SM contains two plasmids that have previously been shown to be self-transmissible by conjugation. One of these plasmids, pRleVF39b, is shown in this study to carry a set of plasmid transfer genes that differs significantly from conjugation systems previously studied in the rhizobia but is similar to an uncharacterized set of genes found in R. leguminosarum bv. trifolii strain WSM2304. The entire sequence of the transfer region on pRleVF39b was determined as part of a genome sequencing project, and the roles of the various genes were examined by mutagenesis. The transfer region contains a complete set of mating pair formation (Mpf) genes, a traG gene, and a relaxase gene, traA, all of which appear to be necessary for plasmid transfer. Experimental evidence suggested the presence of two putative origins of transfer within the gene cluster. A regulatory gene, trbR, was identified in the region between traA and traG and was mutated. TrbR was shown to function as a repressor of both trb gene expression and plasmid transfer.     

The sym plasmid pRP2JI and at least two other loci of Rhizobium leguminosarum biovar phaseoli can confer resistance to infection by the virulent bacteriophage RL38

Abstract The virulent Rhizobium bacteriophage RL38 did not form plaques on R.leguminosarum by phaseoli but did so at high efficiency on a derivative of that strain lacking its symbiotic plasmid pRP2JI. Other strains with large deletions in pRP2JI which removed many nod and nif genes retained resistance to RL38, showing that the gene which confers phage resistance lies elsewhere on the plasmid. Although the wild-type strain of R. leguminosarum bv. phaseoli failed to plate RL38, it was possible to transduce chromosomal markers into this strain, indicating that the 'block' was not at an early stage in the infection process. Two different recombinant plasmids obtained from a clone bank of genomic DNA of R. leguminosarum bv. phaseoli , which appeared to have no DNA in common, both conferred resistance to RL38. Surprisingly, the DNA cloned in each of these plasmids did not originate from pRP2JI. Therefore, several different loci both on the Sym plasmid and elsewhere on the bacterial genome can be involved in conferring resistance to this bacteriophage.     

Localization and symbiotic function of a region on the Rhizobium leguminosarum Sym plasmid pRL1JI responsible for a secreted, flavonoid-inducible 50-kilodalton protein.

A previously described (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988) Sym plasmid-dependent, naringenin-inducible 50-kilodalton protein of Rhizobium leguminosarum biovar viciae is further characterized in this paper. The protein was overproduced by constructing a strain containing multiple copies of the R. meliloti nodD gene, which facilitated its purification. An antiserum was used to screen Tn5 insertion mutants located in the pRL1JI region found to be responsible for the production of the 50-kilodalton protein. These inserts define a new nod locus left of the nod genes identified previously. Mutations in this region affect the nodulation ability in a way which is dependent on the bacterial background as well as on the host plant. The mutants nodulate normally in a strain RBL1532 (R. leguminosarum biovar viciae strain 248, cured of its Sym plasmid) background on all three tested host plant species. In contrast, in a strain RBL5045 (R. leguminosarum biovar trifolii strain RCR5, cured of its Sym plasmid) background, nodulation on Vicia sativa is severely impaired, whereas nodulation on Vicia hirsuta and Trifolium subterraneum is apparently unaltered.     

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     

A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border

Summary The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the T_c-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the T_c-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the T_L-DNA and ends approximately 3 bp from an IS66 homolog in the T_c-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with T_c-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).     

Cloning and mutagenesis of nodulation genes from Rhizobium leguminosarum TOM, a strain with extended host range

Summary Some primitive pea lines, e.g. cultivar Afghanistan, are resistant to nodulation by most strains of Rhizobium leguminosarum. However the Turkish strain TOM can nodulate cv. Afghanistan in addition to commercial pea varieties, and this extended host range is a property of its symbiotic plasmid, pRL5JI. A gene bank was constructed using DNA from a strain of R. leguminosarum containing pRL5JI. Following transfer to a strain of R. leguminosarum that had been cured of its symbiotic plasmid, two derivatives were isolated that contained cloned nodulation determinants, and were able to nodulate both cv. Afghanistan and a commercial pea variety. In addition, these clones conferred the ability to nodulate peas to a strain of R. phaseoli that had been previously cured of its symbiotic plasmid. One of these clones was subjected to mutagenesis with transposon Tn5, and 11 mutants were identified that were affected in nodulation ability. The sites of Tn5 insertions were mapped using restriction endonucleases and all were found to be within a region of 5 kb. The mutants fell into three classes on the basis of their map positions and their phenotypes on the two different pea lines tested. One class of mutants was affected in gene functions that were common to the nodulation of both pea hosts; a second class was impaired specifically in the nodulation of the commercial pea variety; a third class of mutant failed to confer on a normal strain of R. leguminosarum the supplementary ability to nodulate cv. Afghanistan.     

psi, a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation

Summary A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region of the symbiotic plasmid close to nodulation and nitrogen fixation genes. psi is important in the symbiosis since a wild-type strain containing psi cloned on a multicopy plasmid failed to form Phaseolus nodules, and mutant strains containing psi::Tn5 mutations failed to fix nitrogen in Phaseolus nodules. It is proposed that the function of psi may be to repress in the bacteriod the expression of genes such as those for EPS synthesis which are normally expressed in free-living culture.     

Melanin Production by Rhizobium Strains

Different Rhizobium and Bradyrhizobium strains were screened for their ability to produce melanin. Pigment producers (Mel⁺) were found among strains of R. leguminosarum biovars viceae, trifolii, and phaseoli, R. meliloti, and R. fredii; none of 19 Bradyrhizobium strains examined gave a positive response. Melanin production and nod genes were plasmid borne in R. leguminosarum biovar trifolii RS24. In R. leguminosarum biovar phaseoli CFN42 and R. meliloti GR015, mel genes were located in the respective symbiotic plasmids. In R. fredii USDA 205, melanin production correlated with the presence of its smallest indigenous plasmid.     

Involvement of Genes on a Megaplasmid in the Acid-Tolerant Phenotype of Rhizobium leguminosarum Biovar Trifolii

The acid-tolerant Rhizobium leguminosarum biovar trifolii strain ANU1173 exhibited several new phenotypes when cured of its symbiotic (Sym) plasmid and the second largest megaplasmid. Strain P22, which has lost these two plasmids, had reduced exopolysaccharide production and cell mobility on TY medium. The parent strain ANU1173 was able to grow easily in laboratory media at pH 4.5, whereas the derivative strain P22 was unable to grow in media at a pH of <4.7. The intracellular pH of strain ANU1173 was 6.8 when the external pH was 4.5. In contrast, strain P22 had an acidic intracellular pH of <6.4 when the external pH was <5.5. Strain P22 had a dramatically increased membrane permeability to protons and decreased proton extrusion activity. Analysis with sodium dodecyl sulfate-polyacrylamide gels showed that strain P22 lacked a slow-migrating lipopolysaccharide (LPS) banding group which was present in the parent strain. Mobilization of the second largest megaplasmid of strain ANU1173 back into strain P22 restored the altered LPS structure and physiological characteristics of strain P22. Mobilization of the Sym plasmid of strain ANU1173 into strain P22 showed that the second largest megaplasmid of strain ANU1173 was required for the establishment of nitrogen-fixing nodules on Trifolium repens and Trifolium subterraneum. Furthermore, an examination of a large number of specific exopolysaccharide- or LPS-deficient Rhizobium mutants did not show a positive correlation between exopolysaccharide or LPS synthesis and acid tolerance.     

Behavior of plasmid pJB5JI in Rhizobium huakuii under free-living and symbiotic conditions

The Rhizobium leguminosarum biovar viceae host-range plasmid pJB5JI was transferred into Rhizobium huakuii strains, both wild-type 7653R and its sym plasmid-cured mutant 7653R-1. Transconjugant 7653R-1 (pJB5JI) acquired the ability to form ineffective nodules on pea plants, whereas transconjugant 7653R (pJB5JI) could not do so, indicating that the indigenous symbiotic plasmid could restrict the functional expression of pJB5JI. On the other hand, transconjugant 7653R (pJB5JI) showed higher nitrogenase activity on A. sinicus and higher shoot dry weight than the recipient strain 7653R. The alien plasmid pJB5JI in both kinds of transconjugants remained stable during frequent transfer on culture media, but in part of the isolates from nodules formed by them the pJB5JI was not visualized on gel by the Eckhardt procedure. Southern hybridization with Tn5 and nod gene probes showed that these isolates still reserved, at least in part, DNA of pJB5JI, which was probably intergrated onto the chromosome of cells.     

A DNA element recognised by the molybdenum-responsive transcription factor ModE is conserved in Proteobacteria, green sulphur bacteria and Archaea

Background

In general, chemotaxis in Rhizobium has not been well characterized. Methyl accepting chemotaxis proteins are sensory proteins important in chemotaxis of numerous bacteria, but their involvement in Rhizobium chemotaxis is unclear and merits further investigation.

Results

A putative methyl accepting chemotaxis protein gene (mcpG) of Rhizobium leguminosarum VF39SM was isolated and characterized. The gene was found to reside on the nodulation plasmid, pRleVF39d. The predicted mcpG ORF displayed motifs common to known methyl-accepting chemotaxis proteins, such as two transmembrane domains and high homology to the conserved methylation and signaling domains of well-characterized MCPs. Phenotypic analysis of mcpG mutants using swarm plates did not identify ligands for this putative receptor. Additionally, gene knockouts of mcpG did not affect a mutant strain's ability to compete for nodulation with the wild type. Notably, mcpG was found to be plasmid-encoded in all strains of R. leguminosarum and R. etli examined, though it was found on the nodulation plasmid only in a minority of strains.

Conclusions

Based on sequence homology R. leguminosarum mcpG gene codes for a methyl accepting chemotaxis protein. The gene is plasmid localized in numerous Rhizobium spp. Although localized to the sym plasmid of VF39SM mcpG does not appear to participate in early nodulation events. A ligand for McpG remains to be found. Apparent McpG orthologs appear in a diverse range of proteobacteria. Identification and characterization of mcpG adds to the family of mcp genes already identified in this organism.     

Cloning of the nodulation (nod) genes of Rhizobium phaseoli and their homology to R. leguminosarum nod DNA

In Rhizobium phaseoli strain 8002, a large indigenous plasmid, pRP2JI, had previously been shown to carry many of the genes necessary for the induction of nitrogen-fixing nodules on Phaseolus beans. A cosmid clone library was constructed using DNA from strain 8002. From this library, two overlapping recombinant plasmids (pIJ1097 and pIJ1098) were isolated which spanned about 43 kb of pRP2JI DNA. These plasmids could restore nodulation to some, but not all nodulation-deficient strains of R. phaseoli, indicating that the nodulation genes were not clustered within one small region of pRP2JI. The cloned R. phaseoli nodulation region shared extensive DNA homology with the nodulation genes of R. leguminosarum, and on the basis of DNA hybridization, the nitrogenase genes were found to be within 10 kb of the R. phaseoli nodulation genes. Close to the nodulation genes of R. phaseoli was located a sequence that was repeated on pRP2JI but which was not present elsewhere in the genome of strain 8002.     

Symbiotic plasmid rearrangement in Rhizobium leguminosarum bv. viciae VF39SM

A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.