A. W. B. Johnston

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.

Plasmid determined nodulation and nitrogen fixation abilities in rhizobium phaseoli

SummaryInRhizobium 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.

Identification of genes and gene products involved in the nodulation of peas by Rhizobium leguminosarum

SummaryMutant strains of Rhizobium leguminosarum have been isolated in which the transposon Tn5 was inserted into the cloned nodulation genes. The mutant strains were examined for their ability to induce nodules and to induce normal root hair curling on peas. Five distinct classes of nod mutations were identified on the basis of the phenotypes of the various mutant strains, and of the mapped locations of the nod:: Tn5 alleles. Three classes of mutants (I, II and III) were defective in nodulation and two classes (IV and V) were delayed in nodule appearance. Mutations of classes I, II and III types affected root hair curling and a subcloned 6.6 kb fragment of DNA containing this region conferred the ability to induce root hair curling upon a Rhizobium strain deleted of its nod genes. Analysis of protein products formed after in vitro transcription and translation of the plasmid DNA corresponding to the class I, II, III and IV regions indicated that proteins of molecular weights 48,000, 45,000, 36,000 and 34,000 were formed. A comparison of protein products formed by equivalent plasmids carrying nod:: Tn5 alleles indicated that the class I mutations blocked the synthesis of the 45,000 molecular weight peptide and that the two products of molecular weights 36,000 and 34,000 were absent from the class II mutants. It is concluded that at least five nod genes are involved in nodulation and the polypeptide products of some of these genes have been identified.

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

SummaryThree 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.

A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli and is corrected by cloned DNA from Rhizobium or the phytopathogen Xanthomonas

SummaryA Tn5-induced mutant strain of R. phaseoli which failed to synthesize exopolysaccharide (EPS) was isolated and was shown to induce normal nitrogen-fixing nodules on Phaseolus beans, the host of this Rhizobium species. The corresponding wild-type Rhizobium DNA was cloned in a wide host-range vector and by isolating Tn5 insertions in this cloned DNA, mutations in a gene termed pss (polysaccharide synthesis) were isolated. These were introduced by marker exchange into near-isogenic strains of R. leguminosarum and R. phaseoli which differed only in the identity of their symbiotic plasmids. Whereas the EPS-deficient mutant strain of R. phaseoli induced normal nitrogen-fixing nodules on Phaseolus beans, the same mutation prevented nodulation of peas by a strain of R. leguminosarum which normally nodulates this host. Further, it was found that DNA cloned from the plant pathogen Xanthomonas campestris pathover campestris could correct the defect in EPS synthesis in R. leguminosarum and R. phaseoli and also restored the ability to nodulate peas to the pss::Tn5 mutant strain of R. leguminosarum.

Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli

SummaryThe ability to identify genes that specify nitrogenase (nif genes) in Rhizobium depends on the close homology between then and the corresponding nif genes of Klebsiella pneumoniae (Nuti et al. 1979; Ruvkun and Ausubel 1980). Rhizobium plasmids of high molecular weight (>100 Md) were separated on agarose gels, transferred to nitrocellulose filters and tested for their ability to hybridise with radioactively labelled pSA30, containing the nifKDH region of K. pneumoniae. Five large plasmids, each present in different strains of R. leguminosarum or R. phaseoli, were found to hybridise. Each of these plasmids had previously been shown to determine other symbiotic functions such as nodulation ability. The nif genes on three different plasmids appeared to be in conserved DNA regions since they were within an EcoRI restriction fragment of the same size.

Identification of nodX, a gene that allows Rhizobium leguminosarum biovar viciae strain TOM to nodulate Afghanistan peas.

SummaryGene(s) conferring the ability of Rhizobium leguminosarum biovar viciae strain TOM to nodulate primitive peas (cultivar Afghanistan) had been located in a 2.0 kb region of its sym plasmid, pRL5JI. In this DNA, a single open reading frame of 1101 bp, corresponding to a gene, nodX was found. nodX is downstream of nodJ which is present in strain TOM and also in the sym plasmid of a typical strain of this biovar. nodX specifies a hydrophobic protein (of Mr 41 036) with no clear similarity to other proteins in data bases. Mutations in nodX abolished nodulation of Afghanistan peas but not nodulation of commercial peas. nodX-lacZ fusions were used to show that transcription of nodX was activated by root exudates from both commercial and Afghanistan peas and by defined flavonoids. Exudate from Afghanistan peas activated nod genes of typical strains of R. leguminosarum biovar viciae which fail to nodulate these peas; thus, their failure to nodulate these primitive peas is not due to a lack of activation of their nod genes by exudate from Afghanistan peas. A homologue of nodX exists in R. leguminosarum biovar trifolii (which nodulates clover) but not in typical strains of R. leguminosarum biovar viciae.

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.

Recombinant Nodulation Plasmids in Rhizobium leguminosarum

SUMMARY: Plasmids pRL 1JI and pRL6JI, which carry determinants essential for symbiosis in Rhizobium leguminosarum field isolates 248 and 128C53, respectively, were both incompatible with two other transmissible plasmids that did not carry symbiotic determinants. When derivatives of these two plasmids (pRL3JI or pRL4JI) were introduced into a strain which already contained pRL 1JI or pRL6JI, recombinant replicons were often obtained: these were of uniform size for each pair of incompatible plasmids. Recombinant nodulation plasmids were also observed following infrequent interactions with pRL10JI, a natural nodulation plasmid that does not belong to the same incompatibility group as pRL3JI or pRL4JI. By the formation of a recombinant nodulation plasmid, it has been possible to transfer the determinants for nodulation ability (Nod+), nitrogen fixation (Fix+) and hydrogen uptake (Hup+) from pRL6JI, a plasmid that was not self-transmissible, on to a replicon that was transmissible at high frequency and carried a selectable drug resistance marker. In the case of pRL10JI, which is also a non-transmissible nodulation plasmid, the formation of a recombinant nodulation plasmid transferred the symbiotic determinants to a transmissible replicon belonging to a different incompatibility group from pRL10JI itself.

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.