An RP4-prime containing a 285 kb fragment of rhizobium meliloti pSym megaplasmid: Structural characterization and utilization for genetic studies of symbiotic functions controlled by pSym

Summary We integrated the RP4 plasmid into a selected region of the pSym megaplasmid of Rhizobium meliloti 2011 by homologous recombination between pSym and a cloned fragment of pSym present in the RP4. This cointegrate was used to mobilize into Escherichia coli a Tn5 transposon located on pSym in the vicinity of the site of integration of the RP4. By this technique we obtained a series of RP4-primes that contained large fragments of the pSym megaplasmid and that were most probably generated by IS8 promoted deletions in the RP4-pSym cointegrate. One of them, pGMI42, which carries nitrogenase genes nifD and H as well as nodulation genes, was used for mutagenesis of the corresponding region of pSym after insertion of the Mu prophage into the tet gene. When various (pGMI-42:: Mu)::Tn7 were introduced into R. meliloti 2011 by conjugation, homologous recombination allowed insertion of Tn7 into pSym whereas the pGMI42::Mu was lost due to the suicide effect of Mu. In this way we obtained several symbiotic mutants deficient in either nodulation (Nod^-) or nitrogen fixation (Fix^-) in association with the host plant Medicago sativa.This paper is affectionately dedicated to the memory of Jean-Simon Julliot who initiated and inspired this work and who was killed by an avalanche on February 21, 1982     

Transfer of Rhizobium meliloti pSym genes into Agrobacterium tumefaciens: host-specific nodulation by atypical infection

The pSym megaplasmid of Rhizobium meliloti 2011 mobilized by plasmid RP4, or plasmid pGMI42, an RP4-prime derivative which carries a 290-kilobase pSym fragment including nitrogenase and nod genes, was introduced into Agrobacterium tumefaciens. The resulting transconjugants induced root deformations specifically on the homologous hosts Medicago sativa and Melilotus alba and not on the heterologous hosts Trifolium pratense and Trifolium repens. The root deformations were shown to be genuine nodules by physiological and cytological studies. Thus, host specificity nodulation genes are located on the pSym megaplasmid. Host nodulation specificity did not seem to require recognition at the root hair level since no infection threads could be detected in the root hairs. Cytological observations indicated that bacteria penetrated only the superficial layers of the host root tissue by an atypical infection process. The submeristematic zone and the central tissue of the nodules were bacteria free. Thus, nodule organogenesis was probably triggered from a distance by the bacteria. Agrobacterium transconjugants carrying pSym induced the formation of more numerous and larger nodules than those carrying the RP4-prime plasmid pGMI42, suggesting that some genes influencing nodule organogenesis are located in a pSym region(s) outside that which has been cloned into pGMI42.     

Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation.

In Rhizobium meliloti 2011 nodulation genes (nod) required to nodulate specifically alfalfa are located on a pSym megaplasmid. Nod- derivatives carrying large pSym deletions were isolated. By complementation of these strains with in vivo- and in vitro-constructed episomes containing pSym of sequences and introduction of these episomes into Agrobacterium tumefaciens, we show (i) that from a region of pSym of about 360 kilobases, genes required for specific alfalfa nodulation are clustered in a DNA fragment of less than 30 kilobases and (ii) that a nod region located between nifHDK and the common nod genes is absolutely required for alfalfa nodulation and controls the specificity of root hair curling and nodule organogenesis initiation.     

The presence of repeated DNA sequences and a partial restriction map of the pSym of Rhizobium fredii USDA193

The large, 350-kb Sym (symbiotic) plasmid pRjaUSDA193 of Rhizobium fredii was examined to determine the frequency of repeated sequences present and to produce a physical and genetic map of a large region of the plasmid. A novel hybridization method, the Southern Cross, revealed that the plasmid pRjaUSDA193 contained many repeated sequences and assisted in restriction enzyme mapping of a 100-kb region containing nod genes. A cosmid clone bank was prepared with the broad-host-range cosmid pVK102. The restriction enzymes HindIII, HpaI, and KpnI were used to construct a physical map of overlapping clones. Labeled nod gene sequences were used to determine their location in the mapped region.     

Genetic analysis of a region of the Rhizobium meliloti pSym plasmid specifying catabolism of trigonelline, a secondary metabolite present in legumes.

Genes controlling the catabolism of trigonelline, a secondary metabolite that is often present in legumes, are located on the pSym megaplasmid of Rhizobium meliloti. To investigate the role of bacterial trigonelline catabolism in the Rhizobium-legume symbiosis, we identified and characterized the R. meliloti RCR2011 genetic loci (trc) controlling trigonelline catabolism. Tn5-B20 mutagenesis showed that the trc region is a continuous DNA segment of 9 kb located 4 kb downstream of the nifAB and fdxN genes. Trc mutants fell into two classes according to their phenotype and location: (i) mutants carrying Tn5-B20 insertions in the right-hand part of the trc region were incapable of growing on trigonelline as the sole carbon and/or nitrogen source, and (ii) insertions in the left-hand part of the trc region resulted in delayed growth on trigonelline as the sole carbon and/or nitrogen source. No significant defect in nodule formation or nitrogen fixation was detected for mutants of either class. Screening of a set of R. meliloti strains from various geographical origins showed that all of these strains are able to catabolize trigonelline and show sequence homology between their megaplasmids and a trc probe.     

A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus.

A 290-kilobase (kb) region of the Rhizobium meliloti 2011 pSym megaplasmid, which contains nodulation genes (nod) as well as genes involved in nitrogen fixation (nif and fix), was shown to carry at least six sequences repeated elsewhere in the genome. One of these reiterated sequences, about 5 kb in size, had previously been identified as part of a cluster of fix genes located 220 kb downstream of the nifHDK promoter. Deletion of the reiterated part of this fix cluster does not alter the symbiotic phenotype. Deletion of the second copy of this reiterated sequence, which maps on pSym 40 kb upstream of the nifHDK promoter, also has no effect. Deletion of both of these copies however leads to a Fix- phenotype, indicating that both sequences carry functionally reiterated fix gene(s). The fix copy 40 kb upstream of nifHDK is part of a symbiotic cluster which also carries a nod locus, the deletion of which produces a marked delay in nodulation.     

Plasmid pSym1-32 of Rhizobium leguminosarumbv. viceae Controlling Nitrogen Fixation Activity,Effectiveness of Symbiosis,Competitiveness, and Acid Tolerance

The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarumbv. viceae1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mobinto the plasmidless Agrobacterium tumefaciensGm1-9023 strain. Plasmid pSym1-32 was transferred intoR. leguminosarumbv. viceaestrains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen). Transconjugants formed Fix⁺nodules on roots of V. villosaand had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains. Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected. Moreover, the donor strain R. leguminosarumbv. viceae1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain. Both these properties were transmitted upon transfer of pSym1-32 into the recipient. Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R. leguminosarumbv. viceae.     

Plasmid pSym1-32 from Rhizobium leguminosarum bv. viceae, controlling nitrogen-fixing activity, effectiveness of symbiosis, competitiveness, and acid tolerance

The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarum bv. viceae 1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mob into the plasmidless Agrobacterium tumefaciens Gm1-9023 strain. Plasmid pSym1-32 was transferred into R. leguminosarum bv. viceae strains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen). Transconjugants formed Fix+ nodules on roots of V. villosa and had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains. Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected. Moreover, the donor strain R. leguminosarum bv. viceae 1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain. Both these properties were transmitted upon transfer of pSym1-32 into the recipient. Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R. leguminosarum bv. viceae.     

Localization of a symbiotic fix region on Rhizobium meliloti pSym megaplasmid more than 200 kilobases from the nod-nif region

Summary We have established the HindIII physical map of a cloned 290 kilobase fragment of the Rhizobium meliloti 2011 pSym megaplasmid. The cloned fragment, which contains nodulation genes as well as the nitrogenase structural genes (nifHDK), has been shown to be colinear with the corresponding genomic region. Using transposon mutagenesis we have demonstrated that a region which is located more than 200 kb from the nifHDK operon on pSym is essential for symbiotic nitrogen fixation.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Betaine use by rhizosphere bacteria: genes essential for trigonelline, stachydrine, and carnitine catabolism in Rhizobium meliloti are located on pSym in the symbiotic region.

Rhizobium meliloti is known to use betaines synthesized by its host, Medicago sativa, as osmoprotectants and sources of energy. It is shown in the present report that the symbiotic megaplasmid (pSym) of R. meliloti RCR2011 encodes functions essential to the catabolism of three betaines, trigonelline (nicotinic acid N-methylbetaine), stachydrine (proline betaine or dimethylproline), and carnitine (gamma-trimethyl-beta-hydroxybutyrobetaine). Preliminary evidence is presented showing that functions on pSym also influence the catabolism of choline and its oxidative product, glycine betaine. Genes implicated in betaine catabolism are found in the symbiotic region of pSym. Trigonelline catabolism functions lie between two clusters of symbiotic genes, nifKDH and nok/fixVI'. Stachydrine and carnitine functions lie to the right of trigonelline catabolism functions, immediately to the right of fixVI'. Information necessary to choline and glycine betaine catabolism is probably encoded to the right of stachydrine catabolism functions.     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.