Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Exopolysaccharide-deficient mutants of Astragali rhizobia are symbiotically effective on Astragalus sinicus, an indeterminate nodulating host

Five exopolysaccharide-deficient mutants were isolated after rhizobial strain 107 was subjected to transposon Tn5 mutagenesis. The amount of EPS produced by the mutants was dramatically decreased to between 3% and 6% of wild-type level. All mutants carried a singel copy of Tn5. Two mutants (NA3 and NA10) were complemented by the R. meliloti exoA gene and the functionally equivalent exoD gene of Rhizobium sp. strain NGR234. Two other mutants (NA7 and NA8) were complemented by the R. meliloti exoB gene and the functionally equivalent NGR234 exoC gene. The remaining mutant (NA11) was not complemented by any exo genes of R. meliloti or Rhizobium NGR234. All mutants induced normal nitrogen-fixing nodules on Astragalus sinicus, an indeterminate nodulating host.     

Host range,RFLP, and antigenic relationships betweenRhizobium fredii strains andRhizobium sp. NGR234

Rhizobium fredii is a nitrogen-fixing symbiont from China that combines broad host range for nodulation of legume species with cultivar specificity for nodulation of soybean. We have compared 10R. fredii strains withRhizobium sp. NGR234, a well known broad host range strain from Papua New Guinea. NGR234 nodulated 16 of 18 tested lugume species, and nodules on 14 of the 16 fixed nitrogen. TheR. fredii strains were not distinguishable from one another. They nodulated 13 of the legumes, and in only nine cases were nodules effective. All legumes nodulated byR. fredii were included within the host range of NGR234. Restriction fragment length polymorphisms (RFLPs) were detected with four DNA hybridization probes: the regulatory and commonnod genes,nodDABC; the soybean cultivar specificity gene,nolC; the nitrogenase structural genes, nifKDH; and RFRS1, a repetitive sequence fromR. fredii USDA257. A fifth locus, corresponding to a second set of soybean cultivar specificity genes,nolBTUVWX, was monomorphic. Using antisera against whole cells of threeR. fredii strains and NGR234, we separated the 11 strains into four serogroups. The anti-NGR234 sera reacted with a singleR. fredii strain, USDA191. Only one serogroup, which included USDA192, USDA201, USDA217, and USDA257, lacked cross reactivity with any of the others. Although genetic and phenotypic differences amongR. fredii strains were as great as those between NGR234 andR. fredii, our results confirm that NGR234 has a distinctly wider host range thanR. fredii.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Use of transposons for studying the genetic organization of Vibrio cholerae non 01]

The nonagglutinating vibrions having Tn-elements inserted into the chromosome were obtained as a result of conjugal transfer of vector plasmids carrying the different transposons (Tn9, Tn10, Tn601, Tn5-Mob) and of the consequent isolation of plasmid-free clones of Vibrio cholerae non OI. Identification of auxotrophic mutations induced by the transposons inserted into the bacterial genome made possible the construction of the primary chromosomal map of Vibrio cholerae non OI. The efficient donor strains of Vibrio cholerae non OI were constructed by introducing the transposon Tn5-Mob and the helper plasmid RP-4. The donors are capable of oriented conjugal transfer of chromosome.     

Genetic snapshots of the Rhizobium species NGR234 genome

Background  

In nitrate-poor soils, many leguminous plants form nitrogen-fixing symbioses with members of the bacterial family Rhizobiaceae. We selected Rhizobium sp. NGR234 for its exceptionally broad host range, which includes more than 112 genera of legumes. Unlike the genome of Bradyrhizobium japonicum, which is composed of a single 8.7 Mb chromosome, that of NGR234 is partitioned into three replicons: a chromosome of about 3.5 Mb, a megaplasmid of more than 2 Mb (pNGR234b) and pNGR234a, a 536,165 bp plasmid that carries most of the genes required for symbioses with legumes. Symbiotic loci represent only a small portion of all the genes coded by rhizobial genomes, however. To rapidly characterize the two largest replicons of NGR234, the genome of strain ANU265 (a derivative strain cured of pNGR234a) was analyzed by shotgun sequencing.     

Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob.

RK2::Mu plasmids and transposon Tn5-Mob were used to mobilize the Legionella pneumophila chromosome. Plate matings between L. pneumophila donors that contained RK2::Mu plasmids and auxotrophic recipients yielded recombinants at frequencies ranging from 10(-6) to 10(-7) per recipient for the markers tested. The presence of a Mu insertion in the chromosome of donors that harbored RK2::Mu plasmids increased the frequency of chromosome transfer of certain selected markers as compared with strains that contained RK2::Mu alone. Cotransfer experiments with Mu-containing donors and a thymidine and tryptophan auxotroph failed to reveal any linkage between the thy and trp loci in L. pneumophila. A strain that contained a chromosomal Tn5-Mob insertion and helper plasmid pRK24.4 transferred chromosomal markers at frequencies of 10(-7) per recipient. These findings suggest that RK2::Mu plasmids and Tn5-Mob may be useful for genetic mapping experiments with L. pneumophila.     

Conserved plasmid/chromosome sequences in fast- and slow-growing rhizobia that nodulate the same plant

Apart from the ability to nodulate legumes, fast-and slow-growing rhizobia have few bacteriological traits in common. Given that there is only one pathway to nodulation, DNA sequences conserved in fast- and slow-growing organisms that nodulate the same host should be strongly enriched in infectivity genes. We tested this hypothesis with seven fast-growing and five slow-growing strains that produced responses varying from fully effective nodulation through various ineffective associations to non-nodulation on four different hosts (Lotus pedunculatus, Lupinus nanus, Macroptilium atropurpureum, and Vigna unguiculata). When restriction enzyme digested total DNA from 10 of the strains was separately hybridized with nick-translated plasmid DNA isolated from 4 fast-growing strains, variable but significant homologies were found with all 10 strains. Part of this homology was shown to be associated with the nifKDH genes for nitrogenase and part with putative nodulation genes carried on pC2, a cosmid clone containing a 37 kbp region of the large sym plasmid present in the fast-growing broad-host range Rhizobium sp. strain NGR234. Analysis of the extent of homology between the plasmids of 3 fastgrowing strains (NGR234, TAL 996 and UMKL 19) able to effectively nodulate Vigna unguiculata showed them to have homologous DNA fragments totalling 47 kbp. This core homology represents less than 12% of the total coding capacity of the sym plasmid present in each of these strains.Abbreviations Sym symbiotic sequences/plasmids - nod genes required for nodulation - nod putative nod genes - nif genes required for the synthesis of the enzyme nitrogenase     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

The role of Rhizobium conserved and host specific nodulation genes in the infection of the non-legume Parasponia andersonii

Summary Rhizobium and Bradyrhizobium bacteria gain intercellular entry into roots of the non-legume Parasponia andersonii by stimulating localized sites of cell division which disrupt the epidermis. Infection threads are then initiated from intercellular colonies within the cortex. Infection via the information of infection threads within curled root hairs, which commonly occurs in legumes, was not observed in Parasponia. The conserved nodulation genes nodABC, necded for the curling of legume root hairs, were not essential for the initiation of infection, however, these genes were required for Parasponia prenodule development. In contrast, the nodD gene of Rhizobium strain NGR234 was essential for the initiation of infection. In addition, successful infection required not only nodD but a region of the NGR234 symbiotic plasmid which is not needed for the nodulation of legumes. Agrobacterium tumefaciens carrying this Parasponia specific region, as well as legume nod genes, was able to form nodules on Parasponia which reached an advanced stage of development.     

A continuous culture study of the phosphorus nutrition of Rhizobium trifollii WU95, Rhizobium NGR234 and Bradyrhizobium CB756

With continuous cultures in a fully defined minimal salts medium steady states were achieved at both limiting and non-limiting concentrations of phosphate in the inflowing medium for Rhizobium trifolii WU95, cowpea Rhizobium NGR234, and Bradyrhizobium CB756.Millimolar growth yields obtained from P-limited cultures varied over 2-fold from 3.2 g dry weight·(mmol P)^-1 for WU95 to 5.3 g dry weight·(mmol P)^-1 for CB756 and 7.2 g dry weight·(mmol P)^-1 for NGR234.For both WU95 and NGR234 growth under P-excess conditions resulted in elevated levels of total biomass P and the storage compound polyphosphate, compared with P-limited cultures. However, P-limited cultures of these two strains still contained significant quantities of polyphosphate. The P-status for CB756 cultures did not affect either total biomass P or polyphosphate levels. Alkaline phosphatase was maximally derepressed in P-limited cultures of WU95 and NGR234. However, in CB756 alkaline phosphatase was not detected at significant levels regardless of its P supply.These data suggest that growth of rhizobia is controlled predominantly by the attainment of a critical internal P level.Abbreviation HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid     

Molecular cloning of thentrA gene of the broad host-rangeRhizobium sp. NGR234, and phenotypes of a site-directed mutant

Summary The clonedntrA (rpoN) gene andntrA mutants ofRhizobium meliloti were used to isolate the homologous gene from the broad-host rangeRhizobium sp. NGR234 by hybridization and interspecies complementation. The NGR234 locus was analyzed by deletion and insertional mutagenesis. A site-directedntrA mutant, NGR234rn1, was made with an interposon, GmI, and its phenotype was examined ex planta and in symbiosis. NGR234rn1 formed Fix⁻ nodules on six genera tested from among its legume hosts, including both indeterminate and determinate nodule-type plants. Formation of nodules onMacroptilium was delayed, and expression of anR. meliloti nodABC-lacZ fusion was reduced by the mutant allele.     

The Rhizobium sp. strain NGR234 systemically suppresses arbuscular mycorrhizal root colonization in a split‐root system of barley (Hordeum vulgare)

Nitrogen‐fixing bacteria (rhizobia) form a nodule symbiosis with legumes, but also induce certain effects on non‐host plants. Here, we used a split‐root system of barley to examine whether inoculation with Rhizobium sp. strain NGR234 on one side of a split‐root system systemically affects arbuscular mycorrhizal (AM) root colonization on the other side. Mutant strains of NGR234 deficient in Nod factor production (strain NGRΔnodABC), perception of flavonoids (strain NGRΔnodD1) and secretion of type 3 effector proteins (strain NGRΩrhcN) were included in this study. Inoculation resulted in a systemic reduction of AM root colonization with all tested strains. However, the suppressive effect of strain NGRΩrhcN was less pronounced. Moreover, levels of salicylic acid, an endogenous molecule related to plant defense, were increased in roots challenged with rhizobia. These data indicate that barley roots perceived NGR234 and that a systemic regulatory mechanism of AM root colonization was activated. The suppressive effect appears to be Nod factor independent, but enhanced by type 3 effector proteins of NGR234.     

Three Replicons of Rhizobium sp. Strain NGR234 Harbor Symbiotic Gene Sequences

Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b.     

Symbiotic mutants of USDA191, a fast-growing Rhizobium that nodulates soybeans

Summary Symbiotic and auxotrophic mutants of Rhizobium japonicum strain USDA191 were isolated using Tn5 mutagenesis and techniques that cause plasmid deletions and plasmid curing. Characterization of several mutants that are unable to nodulate (Nod^-) or unable to fix nitrogen (Fix^_) showed that nod and nif genes are located within one regions of a 200 MD plasmid (pSym191). Blot hybridization analysis of plasmids in other fast-growing R. japonicum strains showed that nod as well as nif sequences are located on plasmids in eight strains but are apparently carried in the chromosome in two strains.     

The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfalfa

Summary We have shown by physical and genetic means that there are two megaplasmids in all strains of Rhizobium meliloti that we have studied. Megaplasmids from several strains of R. meliloti were mobilized to Agrobacterium tumefaciens and to other Rhizobium strains using the Tn5-Mob system. We were also able to resolve these two megaplasmids in agarose gels for most strains, and to show that only one of them hybridized to nif and nod genes. Transfer of this plasmid, the pSym, to Agrobacterium, R. leguminosarum, and R. trifolii strains conferred on these recipients the ability to nodulate alfalfa ineffectively. The second megaplasmid did not appear to have a direct role in nodule initiation. However, we were able to complement extracellular polysaccharide (EPS^-) mutants of R. meliloti by transferring this second megaplasmid into them. Furthermore, Tn5-induced EPS^- mutants of R. meliloti 2011, which produced ineffective (Fix^-) nodules of abnormal morphology, were shown by hybridization and complementation to carry mutations in this second megaplasmid. This demonstrates that both megaplasmids of R. meliloti are necessary for the effective nodulation of alfalfa.     

Analysis of R-primes demonstrates that genes for broad host range nodulation of Rhizobium strain NGR234 are dispersed on the Sym plasmid

Summary R-prime plasmids carrying regions of the symbiotic (Sym) plasmid of the broad host range Rhizobium strain NGR234 were isolated in intergeneric matings with Escherichia coli K12. Three R-primes carrying approximately 180 kb (pMN23), 220 kb (pMN31) and 330 kb (pMN49) of Sym DNA were characterized in more detail. Restriction enzyme analysis and hybridization studies showed that these R-primes carried large overlapping regions of the Sym plasmid, and had the symbiotic genes (two copies of nifH, D and K; nodA, B, C and D; region II; host specific nodulation (hsn) genes) located over half of the 470 kb Sym plasmid. Only the largest of these R-primes (pMN49) contained the complete nodulation host range of the original parent strain NGR234. This broad host range was shown to be present on plasmid pMN49 by being expressed in Agrobacterium tumefaciens strain A136. Furthermore the R-prime plasmids were shown to contain different regions of distinctive host specific nodulation (hsn) for tropical legume infection and for the nodulation of the non-legume Parasponia. Nodulation of soybeans, however, required an additional region that was not essential for the nodulation of other tropical legumes. Strain NGR234 was also found to nodulate the stem and roots of the tropical legume Sesbania rostrata at a very low efficiency. However, the R-prime mini Sym plasmid constructions enabled a greater efficiency of nodulation of Sesbania rostrata to occur.