Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

Summary R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod^-) or nitrogen fixation (Fix^-) have been readily obtained. In some Nod^- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. ³²P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod^- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod^- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod^- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Contribution of calystegine catabolic plasmid to competitive colonization of the rhizosphere of calystegine-producing plants by Sinorhizobium meliloti Rm41

Calystegines are plant secondary metabolites produced by the roots of a few plant species, and the ability to catabolize calystegines is infrequent in rhizosphere bacteria. In Sinorhizobium meliloti Rm41, the endosymbiont of the legume Medicago sativa, this ability results from the presence of the genes cac (for calystegine catabolism) located on the nonsymbiotic plasmid pRme41a. The effect of the cac catabolic plasmid pRme41a on the ability of Rm41 to colonize the rhizosphere of calystegine-positive plants was studied using derivatives of Rm41 with or without cac catabolic plasmid. When strains were inoculated alone, the presence of a cac catabolic plasmid had no effect on their colonization of the rhizosphere, regardless of whether plants produced calystegines or not. However, a spontaneous rifampicin-resistant mutant of Rm41 containing a cac catabolic plasmid reached population levels in the rhizosphere of calystegine-positive plants that were several orders of magnitude higher than those of the same strain without the plasmid, when each was co-inoculated with a derivative of Rm41 cured of pRme41a. In contrast, the cac catabolic plasmid provided little or no selective advantage in the rhizosphere of calystegine-negative plants. In conclusion, the cac catabolic plasmid pRme41a can contribute to the ability of S. meliloti Rm41 to colonize the rhizosphere of alternative, nonlegume plant hosts producing calystegines.     

A chromosomal linkage map of Agrobacterium tumefaciens and a comparison with the maps of Rhizobium spp

Summary Plasmid R68.45 was found to mobilize the Agrobacterium tumefaciens chromosome apolarly. With the aid of this R plasmid the linkage between 26 markers was determined, from which a circular genetic map of the A. tumefaciens chromosome could be constructed. The recombinants obtained were stable i.e. they did not segregate strains, with the parental phenotype upon purification. A system for the polar transfer of chromosomal material from a fixed origin was developed for A. tumefaciens. It was found that R plasmid pRL189, which carries a copy of transposon Tn5, is able to mobilize the chromosome polarly from chromosomal Tn5-insertion sites. A. tumefaciens phe-1 and trp-22 auxotrophs became prototrophic after the introduction of R primes pAJ21JI and pAJ73JI, respectively, which carry the corresponding phe and trp genes of Rhizobium meliloti. This result enabled a preliminary comparison of the gene orders in A. tumefaciens and Rhizobium spp. which suggested that the chromosome maps of these organisms are quite similar.     

Rough and fine linkage mapping of the Rhizobium meliloti chromosome.

Summary A circular linkage map of the Rhizobium meliloti chromosome, obtained from R68.45-mediated crosses, has been revised by cotransductional analysis, after general transduction by DF2 phage.Three short chromosomal regions have been mapped by cotransduction. Comparison between conjugal and cotransductional data suggests that R68.45-mediated linkage measures are indeed rough. Cotransduction seems to be a useful tool for improving the linkage map of R. meliloti.     

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.     

Use of RP4-prime plasmids constructed in vitro to promote a polarized transfer of the chromosome in Escherichia coli and Rhizobium meliloti.

Summary RP4-prime plasmids containing chromosomal fragments of either Escherichia coli or Rhizobium meliloti were constructed in vitro. When introduced into E. coli or R. meliloti respectively, they promoted a polarized transfer of the chromosome as demonstrated either by the gradient of transfer of various markers or by the study of the genetic constitution of recombinants. In E. coli, mobilization was shown to be dependent upon the presence of a functional rec A system. Inheritance of markers was due to their integration into the chromosome of the recipient as shown by the need for a functional rec A system in the recipient E. coli or by mobilization of recessive markers in R. meliloti. The system described could be applied to genetic mapping in any Gram negative bacteria.     

Detection of loci in theleu region ofRhizobium meliloti chromosome

A multi-marked strain ofRhizobium meliloti was developed by the co-mutation method and employed to contribute to the genetic map ofR. meliloti chromosome. Seven loci were placed at 5 sites in theleu region in the orderman-aba, fix, leu-cro-azt, ost-thi.     

Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium

Summary The indigenous megaplasmid pRme41b of Rhizobium meliloti 41 was made susceptible to mobilization with the P-1 type plasmid pJB3JI by inserting the mobilization (mob) region of RP4 into it. First the mob region together with a kanamycin resistance marker was inserted in vitro into a fragment of pRme41b cloned into pBR322. The recombinant plasmids so formed (pAK11 and pAK12) were then mobilized into R. meliloti. Since these recombinant plasmids were unable to replicate in R. meliloti, selection for kanamycin resistant derivatives allowed the isolation of pRme41b::pAK11 or pRme41b::pAK12 cointegrates. It was shown that in the majority of these recombinants, pAK11 or pAK12 was integrated into the homologous fragment of pRme41b. The pRme41b cointegrates were transferred into nod-nif deletion mutants of R. meliloti 41 where it was shown that both Nod⁺ and Fix⁺ phenotypes could be restored. The pRme41b cointegrates were also transferred into two other Rhizobium strains and into Agrobacterium tumefaciens. The Rhizobium strains and A. tumefaciens carrying pRme41b formed nodules of variable size on Medicago sativa roots, indicating that at least the early steps of nodulation of M. sativa are coded by pRme41b and are expressed in these bacteria.     

The complete genome sequence of the dominant Sinorhizobium meliloti field isolate SM11 extends the S. meliloti pan-genome

Isolates of the symbiotic nitrogen-fixing species Sinorhizobium meliloti usually contain a chromosome and two large megaplasmids encoding functions that are absolutely required for the specific interaction of the microsymbiont with corresponding host plants leading to an effective symbiosis. The complete genome sequence, including the megaplasmids pSmeSM11c (related to pSymA) and pSmeSM11d (related to pSymB), was established for the dominant, indigenous S. meliloti strain SM11 that had been isolated during a long-term field release experiment with genetically modified S. meliloti strains. The chromosome, the largest replicon of S. meliloti SM11, is 3,908,022 bp in size and codes for 3785 predicted protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319 bp and it contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is 1,632,395 bp in size and comprises 1548 predicted coding sequences. The gene content of the SM11 chromosome is quite similar to that of the reference strain S. meliloti Rm1021. Comparison of pSmeSM11c to pSymA of the reference strain revealed that many gene regions of these replicons are variable, supporting the assessment that pSymA is a major hot-spot for intra-specific differentiation. Plasmids pSymA and pSmeSM11c both encode unique genes. Large gene regions of pSmeSM11c are closely related to corresponding parts of Sinorhizobium medicae WSM419 plasmids. Moreover, pSmeSM11c encodes further novel gene regions, e.g. additional plasmid survival genes (partition, mobilisation and conjugative transfer genes), acdS encoding 1-aminocyclopropane-1-carboxylate deaminase involved in modulation of the phytohormone ethylene level and genes having predicted functions in degradative capabilities, stress response, amino acid metabolism and associated pathways. In contrast to Rm1021 pSymA and pSmeSM11c, megaplasmid pSymB of strain Rm1021 and pSmeSM11d are highly conserved showing extensive synteny with only few rearrangements. Most remarkably, pSmeSM11b contains a new gene cluster predicted to be involved in polysaccharide biosynthesis. Compilation of the S. meliloti SM11 genome sequence contributes to an extension of the S. meliloti pan-genome.     

Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti

Summary Large plasmids of molecular weight varying from 90 to around 200×10⁶ have earlier been detected in most Rhizobium meliloti strains using an alkaline denaturation-phenol extraction procedure. With a less destructive method (Eckhardt 1978) it was possible additionally to detect one plasmid of molecular weight clearly greater than 300×10⁶ (=megaplasmid) in all of twenty-seven R. meliloti strains of various geographical origins and nodulation groupings investigated. Four strains (RCR 2011, A145, S26 and CC2013) were found to carry one megaplasmid and no smaller plasmids. Hybridization experiments with Klebsiella pneumoniae and R. meliloti cloned nitrogenase structural genes D and H showed that these genes are located on the megaplasmid and not on the smaller plasmids.All of the ten independent spontaneous non-nodulating derivatives of three strains of R. meliloti were shown to have suffered a deletion in the nif DH region of the megaplasmid. These results indicate that a gene controlling an early step in nodule formation is located in the nif DH region of the megaplasmid. This indicates that the same replicon carries genes controlling early and late functions in symbiosis.     

Accumulation of ppGpp in symbiotic and free-living nitrogen-fixing bacteria following amino acid starvation

Following amino acid or ammonium starvation, ppGpp is accumulated by Rhizobium meliloti strain 1021 but not by R. meliloti strain 41 or Rhizobium tropici. Azorhizobium caulinodans ORS571 produced ppGpp following amino acid deprivation; however, the free-living nitrogen-fixing bacteria Azotobacter vinelandii and Azomonas agilis did not produce ppGpp. Western blot analysis using anti-RelA antibody demonstrated that R. meliloti strain 1021, Azotobacter vinelandii and Azorhizobium caulinodans cross-reacted under conditions that detected RelA in Escherichia coli CF1648. Cross-reaction was not observed in R. meliloti strain 41, R. tropici, or Azomonas agilis. All strains that accumulated ppGpp also produced high intracellular levels of ATP. Received: 28 August 1998 / Accepted: 11 November 1998     

Novel DNA Sequences from Natural Strains of the Nitrogen-Fixing Symbiotic Bacterium Sinorhizobium meliloti

Variation in genome size and content is common among bacterial strains. Identifying these naturally occurring differences can accelerate our understanding of bacterial attributes, such as ecological specialization and genome evolution. In this study, we used representational difference analysis to identify potentially novel sequences not present in the sequenced laboratory strain Rm1021 of the nitrogen-fixing bacterium Sinorhizobium meliloti. Using strain Rm1021 as the driver and the type strain of S. meliloti ATCC 9930, which has a genome size ~370 kilobases bigger than that of strain Rm1021, as the tester, we identified several groups of sequences in the ATCC 9930 genome not present in strain Rm1021. Among the 85 novel DNA fragments examined, 55 showed no obvious homologs anywhere in the public databases. Of the remaining 30 sequences, 24 contained homologs to the Rm1021 genome as well as unique segments not found in Rm1021, 3 contained sequences homologous to those published for another S. meliloti strain but absent in Rm1021, 2 contained sequences homologous to other symbiotic nitrogen-fixing bacteria (Rhizobium etli and Bradyrhizobium japonicum), and 1 contained a sequence homologous to a gene in a non-nitrogen-fixing species, Pseudomonas sp. NK87. Using PCR, we assayed the distribution of 12 of the above 85 novel sequences in a collection of 59 natural S. meliloti strains. The distribution varied widely among the 12 novel DNA fragments, from 1.7% to 72.9%. No apparent correlation was found between the distribution of these novel DNA sequences and their genotypes obtained using multilocus enzyme electrophoresis. Our results suggest potentially high rates of gene gain and loss in S. meliloti genomes.     

DNA sequence homology in Rhizobium meliloti plasmids

Summary Plasmids were recovered by an alkaline procedure from six symbiotically effective strains of Rhizobium meliloti of diverse geographical origin, reported to harbour only one middle-size large plasmid (ranging from 89 to 143 Megadaltons). Each purified plasmid was digested with eight restriction endonucleases; cleavage patterns were very complex: only KpnI and XbaI gave a limited number of bands. Fingerprints were very different, whatever the restriction enzyme or the geographical origin of the strains. However, Southern DNA-DNA hybridizations revealed that the plasmids showed homologous sequences having a high thermal stability. We gave evidence that some of these sequences are common to all the plasmids of R. meliloti. The biological function of these common sequences is unknown. Hybridization with cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae had demonstrated that nif genes were not located on the middle — size plasmids of R. meliloti studied in this paper.     

Host Restriction and Transduction in Rhizobium meliloti

A host restriction difference exists between Rhizobium meliloti Rm41 and SU47 exists as indicated by the reduce plating efficiency of transducing phage ΦM12h1. Restriction can be attenuated by incubating cells at 42°C for 3 h; this procedure overcomes a block to transduction from SU47 to Rm41.     

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     

Some physiological characteristics ofRhizobium meliloti andR. trifolii in relation to efficiency of symbiosis with lucerne and Egyptian clover

Summary Several strains ofRhizobium meliloti andR. trifolii were screened for efficiency of symbiosis with their respective hosts, lucerne (Medicago sativa L.) and Egyptian clover or berseem (Trifolium alexandrinum L.) under bacteriologically controlled conditions on agar slopes. Based on the dry weight of plants which was taken as the parameter of symbiotic efficiency, the strains of the two species of rhizobia were grouped into different categories with the aim of correlating some physiological properties of bacteria with their symbiotic efficiency. The physiological properties taken into account were the abilities of bacteria to produce total Salkowski positive compounds (SPC) in terms of indole acetic acid (IAA) equivalents on a tryptophane containing substrate, to catabolize added IAA, to reduce nitrate, to utilize nitrite and to reduce triphenyl tetrazolium chloride (TTC).No positive and significant correlations could be established between symbiotic efficiency and the physiological properties of bacteria. Nevertheless, significant inverse relationships between formation of SPC from tryptophane and IAA destruction (in bothR. meliloti andR. trifolii), between nitrite formation and nitrite utilization (only inR. trifolii) and between symbiotic efficiency and nitrite utilization (only inR. meliloti) were apparent.     

Generalized transduction in Rhizobium meliloti

Summary The phage 11 of R. meliloti performs generalized transduction. This was confirmed by the variety of single markers transferred and by separating transducing particles containing BUdR-labelled bacterial DNA. The transduction frequencies depended on the marker. Linked alleles were mapped by cotransduction on fragments of bacterial DNA equal in size to the phage DNA. With crosses between antibiotic resistancy and auxotrophic markers a partial map was constructed with str, cml, pur-19, and leu-44 sites. With a few multi-auxotrophic mutants linkage data of conjugation were compared with the linkage by cotransduction.     

Site specific transposition of Tn7 into a Rhizobium meliloti megaplasmid

Summary Transposon Tn7 was shown to insert specifically into the megaplasmid of different Rhizobium meliloti strains. Tn7 transposition could not be detected in other Rhizobium strains such as R. trifolii, R. leguminosarum, R. phaseoli and R. japonicum. In R. meliloti strains, two unique sites in the megaplasmid were observed into which Tn7 can transpose at different frequencies. Only one copy of Tn7 could be detected in the megaplasmid and the insertion sites for Tn7 are outside the nif and nod region. Tn7 transposition in R. meliloti showed a marked preference for sites on plasmid RP4 compared to the megaplasmid sites. Attempts to cure Tn7 from the megaplasmid were unsuccessful. This site specific transposition of Tn7 in R. meliloti provides an additional genetic tool to further manipulate this important plasmid in symbiotic nitrogen fixation.     

Identification of QTL for resistance and susceptibility to <Emphasis Type="Italic">Stagonospora meliloti</Emphasis> in autotetraploid lucerne

In eastern Australia and California, USA, one of the major lethal fungal diseases of lucerne (Medicago sativa) is Stagonospora root and crown rot, caused by Stagonospora meliloti. Quantitative trait loci (QTL) involved in resistance and susceptibility to S. meliloti were identified in an autotetraploid lucerne backcross population of 145 individuals. Using regression analysis and interval mapping, we detected one region each on linkage groups 2, 6 and 7 that were consistently associated with disease reaction to S. meliloti in two separate experiments. The largest QTL on linkage group 7, which is associated with resistance to S. meliloti, contributed up to 17% of the phenotypic variation. The QTL located on linkage group 2, which is potentially a resistance allele in repulsion to the markers for susceptibility to S. meliloti, contributed up to 8% of the phenotypic variation. The QTL located on linkage group 6, which is associated with susceptibility to S. meliloti, contributed up to 16% of the phenotypic variation. A further two unlinked markers contributed 5 and 8% of the phenotypic variation, and were detected in only one experiment. A total of 517 simple sequence repeat (SSR) markers from Medicago truncatula were screened on the parents of the mapping population. Only 27 (6%) SSR markers were polymorphic and could be incorporated into the autotetraploid map of M. sativa. This allowed alignment of our M. sativa linkage map with published M. truncatula maps. The markers linked to the QTL we have reported will be useful for marker assisted selection for partial resistance to S. meliloti in lucerne.