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

Julliot, J. S.; Dusha, Ilona; Renalier, M. H.; Terzaghi, Betty E.; Garnerone, Anne-Marie; Boistard, P.

doi:10.1007/bf00327408

Cited by 33 publications

(24 citation statements)

References 33 publications

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“…In addition, the construction of a physical map of pRmeSU47a (ca. 1,500 kb) is currently under way (3,26), and the resolution of the chromosomal map is continually being increased as transductional linkage data between chromosomal markers and transposon insertions are generated (G. Walker, personal communication). However, we do not have a good estimate of the chromosome size so it is not yet possible to estimate the proportion of the R. meliloti genome represented by megaplasmid DNA.…”

Section: Discussionmentioning

confidence: 99%

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Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b

Charles

Finan

1990

J Bacteriol

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A circular linkage map of the Rhizobium meliloti megaplasmid pRmeSU47b was constructed. The map consists of transposon insertions carrying alternating antibiotic resistance markers linked by 4CM12 transduction. Data from conjugation experiments utilizing donor strains carrying TnS-oriT insertions in the megaplasmid supported the proposed genetic map. In addition, the positions of previously identified Fix, exopolysaccharide synthetic, thia'mine synthetic, and C4-dicarboxylate transport loci on the megaplasmid map were determined. By converting cotran'sduction frequencies to physical distance, we calculated the replicon to be 1,600 kilobases in size, which compares favorably with previous physical estimates.Large extrachromosomal replicons known as megaplasmids (39), ranging in size from 400 kilobases (kb) to well over 1,000 kb, are found in a number of bacteria. Among the phenotypic traits known to be associated with megaplasmids are CO2 fixation, hydrogen oxidation, and denitrification by the lithoautotroph Alcaligenes eutrophus (21,22,29,30,38) and resistance to several antibiotics and heavy metals in Pseudomonas spp. (20,25,37). Megaplasmids are commonly found in plant-associated bacteria. For example, in the phytopathogen Erwinia herbicola, genes required for thiamine prototrophy and pigment production are carried on a ca. 525-kb plasmid (18), and in Pseudomonas solanacearum, acridine-resistant mutants deleted of megaplasmid regions were found to be avirulent and altered in exopolysaccharide, lipopolysaccharide, and methionine biosyntheses (5). The genes responsible for induction of crown gall and hairy root neoplastic diseases by Agrobacterium tumefaciens and Agrobacterium rhizogenes are normally present on plasmids that range in size from 150 to 400 kb. However, recently larger (500-to 900-kb) tumor-inducing megaplasmids have been detected in limited-host-range strains of A. tumefaciens (41).In Rhizobium species, many of the genes involved in nodule formation and nitrogen fixation are found on socalled Sym plasmids. The largest of these, and the largest megaplasmids known at ca. 1,500 kb, are the two plasmids found in many strains of Rhizobium meliloti, the endosymbiont of alfalfa (2, 6-8, 39). The most characterized of these two megaplasmids (pRmeSU47a in strain SU47) carries nodulation (nod and hsn) and nitrogen fixation (nif and fix)genes. In addition, Murphy et al. (36) have recently shown that genes involved in the synthesis and catabolism of a nodule-specific opinelike compound are located on the counterpart of pRmeSU47a in strain L5-30.The second megaplasmid in R. meliloti SU47 (designated pRmeSU47b) carries genes involved in exopolysaccharide synthesis (exo) and dicarboxylate transport (dct), both of which are required for the formation of effective N2-fixing root nodules (11,13,14,23,31,42). In addition, pRmeSU47b carries two loci required for thiamine biosynthesis (thi) (14).Aside from the few markers indicated-above, most of the genes encoded by these and other megaplasmids remain to be identifi...

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Section: Discussionmentioning

confidence: 99%

“…For megaplasmid pRmeSU47a, a physical map-of a ca. 300-kb region has been constructed (3,26). Many nodulation and N2 fixation genes are located in a 50-kb region, and another cluster carrying genes required for N2 fixation is located more than 200 kb away.…”

Section: Discussionmentioning

confidence: 99%

Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b

Charles

Finan

1990

J Bacteriol

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“…Excellent positive correlation was found between N20 reduction activity and the presence of a nosZlike sequence, thus demonstrating the usefulness of the nosZ probe for screening bacteria for copper-containing N20 pGMI42 is an RP4 prime carrying a 285-kb fragment of the R. meliloti SU47 nod megaplasmid (25). pRWRm67 is a cosmid of R. meliloti JJ1c1O.…”

Section: Discussionmentioning

confidence: 82%

“…mid (pRmeSU47a) of SU47 (6,25,36); cosmid pRWRm67, which carries the homologous region of JJ1c10; and its subclone, pRWRm71. Parity between JJ1c10 and SU47 in this DNA region was demonstrated by juxtaposing the partial physical map of pGMI42 with those of pRWRm67 and pRWRm71 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Detection of a nitrous oxide reductase structural gene in Rhizobium meliloti strains and its location on the nod megaplasmid of JJ1c10 and SU47

Chan¹,

Wheatcroft²

1993

J Bacteriol

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The gene encoding a denitrification enzyme, nitrous oxide reductase (EC 1.7.99.6), in Rhizobium meliloti and other gram-negative bacteria was detected by hybridization to an internal 1.2-kb PstI fragment of the structural gene (nosZ) cloned from Pseudomonas stutzeri Zobell (W. G. Zumft, A. Viebrock-Sambale, and C. Braun, Eur. J. Biochem. 192:591-599, 1990). Homology to the probe was detected in the DNAs of two N2-fixing strains of P. stutzeri, two denitrifying Pseudomonas species, one Alcaligenes eutrophus strain, and 36 of 56 R. meliloi isolates tested. Except for two isolates ofR. meliloti, all showed nitrous oxide reduction activity (Nos').Therefore, at least part of the nosZ sequence appears to be conserved and widely distributed among denitrifiers, which include free-living and symbiotic diazotrophs. By using Agrobacterium tumefaciens transconjugants harboring different megaplasmids ofR. meliloti JJ1c10 and SU47, sequence homology with the nosZ probe was unequivocally located on the nod megaplasmid. A cosmid clone of JJlclO in which nosZ homology was mapped on a 4.2-kb BamHI fragment was selected. This cosmid, which conferred Nos' activity to the R. meliloti wild-type strains ATCC 9930 and Balsac (Nos-and nondenitrifying, respectively) also restored Nos+ activity in the mutants of JJlclO and SU47 in which the 4.2-kb BamHI segment was deleted. Therefore, this segment contains sequences essential for nos gene expression in JJ1c1O and SU47 and thus confirms that the nod megaplasmid in JJlclO and SU47 which carries genes essential for symbiotic dinitrogen fixation also carries genes involved in the antagonistic process of denitrification.Bradyrhizobium and Rhizobium species are bacteria that fix dinitrogen (N2) in symbiosis with legumes (see reference 2 for a review). They are among the seven N2-fixing genera that contain denitrifying strains (53). Denitrifiers are facultative anaerobes capable of growth and energy conservation under 02 limitation. This is achieved by the stepwise dissimilatory reduction of nitrate or nitrite as the terminal electron acceptor coupled to proton translocation with the formation of gaseous N products (40): NO3--*NO2-3NO-N2O-3 N2. Although most denitrifiers possess the four reductases necessary to carry out the complete denitrification pathway, some carry out a truncated one because of their lack of NO3 or N20 reductase (26a). In the latter case, N20 is accumulated as the end product. The basic biochemistry and genetics of denitrification have been well studied with the model organism Pseudomonas stutzeri Zobell (ATCC 14405 [formerly Pseudomonas perfectomarina]; 15, 50), of marine origin, and the results have recently been summarized by Cuypers and Zumft (13a). Its denitrification enzymes and corresponding genes (nar, nir, nor, and nos) have been characterized. With the exception of the nar complex, the structural genes of the dissimilatory reductases are closely linked and transcribed in the same direction. They are arranged in the order nos-nir-nor, spanning a distance of less th...

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“…For example, pRmeSU47a carries the nod genes required for nodule induction and the nifandfix genes which are required for nitrogen fixation (1) while pRmeSU47b carries genes required for exopolysaccharide biosynthesis, dicarboxylic acid transport, and bacteroid development (3). Both genetic and physical maps of the regions of pRmeSU47a carrying the nod, nif, and fix loci have been published (2,9,19). A circular linkage map of pRmeSU47b has recently been prepared (3), as have genetic and physical maps of the regions of the megaplasmid required for exopolysaccharide biosynthesis (8,12).…”

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confidence: 99%