J. E. Beringer

plasmids and Host-range in Rhizobium leguminosarum and Rhizobium phaseoli

Summary: Rhizobium phaseoli strain 1233 forms nitrogen-fixing nodules on Phaseolus vulgaris and produces a brown pigment (probably melanin) in plate culture. This strain contains two plasmids of molecular weight about 200 × 106. Spontaneous deletions in the smaller plasmid abolished pigment production and the ability to nodulate Phaseolus beans, suggesting that genes involved in the determination of both these phenotypes are on this plasmid. The conjugative Rhizobium leguminosarum plasmid pJB5JI, which carries genes that determine the ability to nodulate peas, was transferred from R. leguminosarum strain T3 into R. phaseoli strain 1233. The majority (about 97%) of transconjugants produced pigment and nodulated both peas and Phaseolus beans poorly. The others (about 3%) had received not only pJB5JI but also an extra plasmid present in strain T3 and they had lost the seller of the two plasmids of R. phaseoli strain 1233, suggesting incompatibility between the plasmid transferred from T3 and that lost from the recipient. These strains did not produce pigment nor did they form nitrogen-fixing nodules on Phaseolus beans, but they nodulated peas as well as did R. leguminosarum strain T3. Most (about 96%) of the bacteria isolated from nodules following inoculation of peas by the pigmented transconjugants failed to produce pigment. These strains contained the larger plasmid from strain 1233, and pJB5JI, but not the smaller plasmid of strain 1233; they behaved like R. leguminosarum in inducing profuse nitrogen-fixing nodules on peas. The few pigment-producing bacteria isolated from pea nodules had suffered a deletion in the smaller plasmid of strain 1233; they nodulated peas poorly but could not form nitrogen-fixing nodules on Phaseolus beans.

Linkage Mapping in Rhizobium leguminosarum by means of R Plasmid-mediated Recombination

SUMMARY: Wild-type R plasmids of the P1 incompatibility group mediated the transfer of chromosomal genes in Rhizobium leguminosarum, but only at very low frequencies. Two P1 R plasmids, which had originally been selected in Pseudomonas aeruginosa for enhanced donor properties, promoted much higher levels of gene transfer in R. leguminosarum. One of these, R68.45, was used for linkage mapping. All markers tested mapped on a single circular linkage group. Segments of donor chromosome up to at least one-seventh of its total length were transferred and integrated into the recipient chromosome.

Isolation of symbiotically defective mutants in Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid

SummarySelection was made for the transposition of the kanamycin resistance transposon Tn5 from a location on the chromosome of R. leguminosarum into a transmissible, bacteriocinogenic plasmid that also carries genes required for the induction of nitrogen-fixing nodules on peas.One hundred and sixty independent insertions into transmissible plasmids were isolated. When these plasmids were transferred by conjugation into a non-nodulating strain, which carries a deletion in one of its resident plasmids, of the 160 isolates tested 14 yielded transconjugants that formed nodules that did not fix nitrogen (Fix-) and in a further 15 cases the transconjugants were unable to form nodules (were Nod-). When transferred to a symbiotically proficient strain (i.e. Nod+ Fix+) none of the transconjugants was symbiotically defective; thus the mutations were not dominant.When kan was transduced from the clones that generated Fix- transconjugants into a Fix+ recipient the majority of transductants inherited Fix- indicating that the insertion of Tn5 had induced the symbiotic mutations. Transduction of kan, from the clones that failed to donate Nod+ by conjugation to strain 6015, occurred at barely detectable frequencies and it was not possible to demonstrate transduction of Nod-. kan was co-transduced with Nod+ from some of the clones and some of these transductants also inherited the ability to produce medium bacteriocin and to transfer at high frequency by conjugation. Thus the genes for all these characters are closely linked.

The Rhizobium--legume symbiosis.

physiological changes involved in the differentiation of bacteroids within the nodule. Certainly the addition of nodules to fixed nitrogen in the form of NH+4 or NO-3 does repress N2-ase, although the concentrations required are greater than for repression in free-living nitrogen-fixing bacteria (Klamberger 1977). Various hypotheses have been put forward to account for the repression of N2-ase activity in the nodule. One hypothesis was that in the presence of exogenous nitrate the plant’s nitrate reductase competed for photosynthate, thereby rendering N2-ase inactive because it was starved of an energy supply (Oghoghorie & Pate 1971). Evidence against this was the finding (Chen & Phillips 1976) that addition of NO-3 to pea leaves did not inhibit N2-ase, and that, if the plants were exposed to an atmosphere enriched with CO2, the extent of N2-ase repression when NO-3 was applied to the roots was the same as in a normal atmosphere despite the increase in the plants’ photosynthetic capability. Thus, if there is any competition for photosynthate between N2-ase and nitrate reductase, it must be localized. This model does not explain why NH+4 is also efficient as a repressor of N2-ase. Recently Bisseling, van den Bos & van Kamman (1978) showed that under conditions where NH4NO3 repressed N2-ase activity in pea nodules, it did not affect synthesis of the enzyme. They proposed that the inhibition of N2-ase in the nodule was indirect, being mediated by an inhibition of the synthesis of leg-haemoglobin.

Transfer of Symbiotic Genes with Bacteriocinogenic Plasmids in Rhizobium leguminosarum

SUMMARY: Transfer of derivatives of the bacteriocinogenic plasmid pRL1JI into eight symbiotically defective strains of R. leguminosarum resulted in suppression of the mutant phenotype in four cases. These included non-infective, ineffective and temperature-sensitive ineffective phenotypes. However, in none of these strains was the defect suppressible after high frequency transfer of derivatives of two other bacteriocinogenic plasmids, pRL3JI or pRL4JI. Nodulation ability was co-transferred at high frequency (> 95%) with bacteriocin production by the plasmid pRL1JI. The other two plasmids, pRL3JI and pRL4JI, also mediated the transfer of nodulation ability but at much lower frequency (10-3 to 10-4 per plasmid transfer). Sometimes, transconjugants that had acquired nodulation ability after the transfer of derivatives of plasmids pRL3JI or pRL4JI acted subsequently as high frequency donors for nodulation ability in a manner that was apparently similar to strains containing pRL1JI.

The genetic analysis of Rhizobium in relation to symbiotic nitrogen fixation

SummaryIn the last few years there has been an increasing interest in the genetics and molecular biology of Rhizobium, the bacterium that is responsible for inducing nitrogen-fixing nodules on the roots of leguminous plants, and in this paper we have reviewed recent work in this field. We have considered, in turn, the development of systems of chromosomal gene transfer and mapping in Rhizobium, the recent studies implicating indigenous plasmids as important determinants of symbiotic functions and, finally, the use of Rhizobium mutants as aids in the biochemical analysis of the symbiosis.

Interspecific Crosses between Rhizobium leguminosarum and R. meliloti: Formation of Haploid Recombinants and of R-primes

SUMMARY: Using R68.45 as a conjugative plasmid in crosses between strains of Rhizobium leguminosarum and R. meliloti, transfer of chromosomal genes occurred. Haploid recombinants were formed in which rif and str alleles were transferred from R. leguminosarum to R. meliloti. In crosses in the reverse direction haploid recombinants arose at barely detectable frequencies and progeny were isolated which were high frequency donors of the selected allele. Genetic evidence showed that such progeny harboured R-primes in which sections of the R. meliloti chromosome were inserted into R68.45. Four different classes of R68.45-prime, each carrying a different prototrophic marker, were obtained.

A comparison of three Rhizobium linkage maps

SummaryLinkage maps of R. meliloti 2011 (Rm2011), R. meliloti 41 (Rm41) and R. leguminosarum 300 (R1300), all constructed by means of P1 plasmid-mediated recombination, were compared. Recombination between the two R. meliloti strains occurred at high frequency but was barely detectable in matings between R1300 and Rm41. When co-inheritance data for the three strains were transformed into additive map distances the arrangement of markers showed striking similarities. Each of eight R68.45-primes, carrying different sections of the Rm2011 chromosome, suppressed only those markers of both R1300 and Rm41 which had the same phenotype and map location. Each of these R-primes promoted polarized chromosome transfer in an anticlockwise direction in Rm41, starting from the region corresponding to that carried on the plasmid.

Tryptophan genes in Rhizobium—Their organization and their transfer to other bacterial genera

SummaryR. leguminosarum trp alleles mapped by R68.45-mediated recombination were located in three distinct chromosomal regions. We isolated three derivatives of R68.45 that carried different trp genes of R. meliloti. Each of the plasmids suppressed all of the R. leguminosarum trp alleles in a particular region. The R-primes were transferred to strains of P. aeruginosa carrying mutations in different trp genes. The plasmid pAJ24JI suppressed trpA, B and F mutants, pAJ73JI suppressed trpC and D and pAJ88JI suppressed a trpE mutant. When the R-primes were transferred to E. coli trp strains they failed to suppress any trp mutants. A derivative of pAJ24JI was isolated which was able to suppress trpA and F mutants of E. coli.

Properties of plasmids constructed by the in vitro insertion of DNA fromRhizobium leguminosarum orProteus mirabilis into RP4

SummaryPlasmids have been constructed by insertion of DNA fromRhizobium leguminosarum orProteus mirabilis into RP4 (an R factor of group P). Such recombinant plasmids retain the wide host range of the parental plasmid, being as efficiently transmissible as the unmodified RP4 and are stably maintained in rapidly growing cultures.The recombinant plasmids, even though each contained a DNA sequence absolutely identical with that of the host strain, are no more efficient at mobilizing the transfer of chromosomal genetic information from that host strain than was unmodified RP4. We therefore conclude that an unknown factor must be essential in the process of chromosome mobilization and rate limiting for that process.