Use of plasmid R68.45 for constructing a circular linkage map of the Rhizobium trifolii chromosome

Vol. 149, No. 1 JOURNAL OF BACTERIOLOGY, Jan. 1982, p. 59-64 0021-9193/82/010059-06$02.00/0 Use of Plasmid R68.45 for Constructing a Circular Linkage Map of the Rhizobium trifolii Chromosome MANUEL MEGIAS,1 MIGUEL A. CAVIEDES,' ANTONIO J. PALOMARES,l* AND JULIO PEREZSILVA2 Departamento de Microbiologia, Facultad de Farmacia,' and Departamento de Microbiologia, Facultad de Biologia,2 Universidad de Sevilla, Sevilla, Spain Received 10 March 1981/Accepted 29 June 1981 Plasmid R68.45 was used to promote conjugal transfer of chromosomal markers in Rhizobium trifolii RS55. Analysis of two-factor and three-factor crosses among R. trifolii strains enabled construction of a circular linkage map of the R. trifolii chromosome, containing 17 nutritional and resistance markers. The economic advantages that may accrue from the improvement of nitrogen-fixing systems have become increasingly evident during the past decade. One of the most interesting nitrogen-fixing systems is the Rhizobium-legume symbiosis because of its high agricultural importance. The past several years have witnessed important developments in Rhizobium genetics (3, 7), and a number of suitable procedures for the genetic manipulation of rhizobia have recently been developed (4-6, 8, 9, 17, 18, 20, 23, 24). For many years, one of the major limitations for genetic analysis of rhizobia was the lack of known indigenous chromosome-mobilizing plasmids that would allow gene transfer studies. This difficulty has been overcome by transferring broad-host-range plasmids with chromosome-mobilizing ability (Cma) to Rhizobium spp. To date, the Pseudomonas aeruginosa drugresistance plasmids R68.45 (13) and RP4 (12) have been the plasmids of choice for obtaining gene transfer in Rhizobium. R68.45 is very efficient for mobilizing chromosomal markers in R. meliloti (10, 21) and R. leguminosarum (5) as well as in other gram-negative bacteria (14, 26). In both R. meliloti and R. leguminosarum, R68.45 promotes nonpolarized chromosomal transfer from a number of origins (4, 5, 10, 21). In those species of Rhizobium so far examined, R68.45 can transfer fragments of chromosome long enough to enable accurate mapping of a variety of markers and to establish chromosome circularity (5, 10, 21). RP4 is a less efficient sex factor (15). However, it has been successfully used for mediating transfer of chromosomal markers in R. meliloti (22). To date, the only report on linkage mapping studies in R. trifolii was an attempt at using comutation techniques (29). As an alternative approach, we describe here the use of plasmid R68.45 for promoting chromosomal transfer in R. trifolii. Linkage mapping experiments reported here have led to the construction of a circular linkage map of R. trifolii containing 17 nutritional and resistance markers. MATERIALS AND METHODS Bacterial sains. The strains used are listed in Table 1. Plasmid. R68.45, belonging to the P1 incompatibility group and mediating resistance to ampicillin, kanamycin, and tetracycline, was used (13). Media and growth conditions. Escherichia coli was cultured on nutrient broth (Oxoid); when required in solidified form, agar (Oxoid) was added at 10 g/liter. For R. trifolii strains complete medium was YT (2) and minimal medium was that of Vogel and Bonner (27) supplemented with 19 g of glucose per liter and 12 ,ug of biotin per ml. When required, amino acid and base supplements were used at a final concentration of 1 mM. E. coli was always grown at 35°C. R. trifolii was always grown at 28(C. Antibiotics. Except for rifampin, which was dissolved in methanol and added unfiltered, antibiotics were added to agar media as filter-sterilized aqueous solutions. Concentrations used were as follows: streptomycin sulfate (Sigma), 1 mg/ml; kanamycin sulfate (Sigma), 25 pg/ml; oxytetracycline (Sigma), 10 t.g/ml; rifampin (Lepetit), 20 ,ug/ml; ampicillin (Sigma), 10 Ag/ ml, and canavanine sulfate (Sigma), 80 ,ug/ml. Matings. Matings were performed on Sartorius filters (0.45-,m pore size) as described by Jacob et al. (16). One milliliter of log-phase donor and 1 ml of stationary-phase recipient (both containing at least 108 cells per ml) were mated on the membrane. Membranes were placed on the surface of nutrient agar plates and incubated for about 20 h at 28°C. Then mating mixtures were resuspended in S ml of Tris-salts buffer, pH 7.2, diluted, and plated on selective media. Linkage mapping of the R. Ifoii chromosome. Recombinant colonies for a selected marker were picked onto selective media to determine coheritance of unselected markers. Each pair of markers was analyzed by scoring 150 to 300 colonies. It was assumed that linkage values were inversely related to 59 TABLE 1. Bacterial strainsa Strain Relevant characteristics Reference or source E. coli 1230 ProMet- (R68.45) J. E. Beringer R. trifolii RS55 Wild-type Isolated from clover root nodules RS176 str-l This paper RS225 his-6 str-l This paper RS235 his-6 met-36 str-l This paper RS238 his-6 phe-18 str-l This paper RS241 rif-6 ade-17 This paper RS246 ri4f-6 ade-29 This paper RS271 str-l leu-23 This paper RS288 his-6 tyr-28 str-l This paper RS290 his-6 thr-14 str-l This paper RS292 his-6 pdx-3 str-l This paper RS293 rif-6 ade-29 tyr-13 This paper RS294 rif-6 ade-29 thr-27 This paper RS295 rif-6 ade-29 arg-19 This paper RS296 his-6 met-31 str-I can-3 This paper RS230 (R68.45) his-6 str-l Cross 1230 x RS225 RS278 (R68.45) rif-6 ade-17 Cross 1230 x RS241 I Allelle numbers are arbitrary. Gene symbols are those of Bachmann and Low (1). All the R. trifolii strains are derived from the wild-type RS55, isolated in our laboratory from Trifolium repens L. nodules. Auxotrophic mutations were induced by either N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methane sulfonate mutagenesis. Nitrosoguanidtne treatments were carried out as follows: a log culture containing about 10' cells per ml was cooled, washed twice with Tris-maleate buffer, pH 7.5, and resuspended in 500 mg of nitrosoguanidine (Sigma) per liter for 30 min. Then the cells were harvested and carefully washed with buffer before plating on YT agar. For ethyl methane sulfonate mutagenesis, a culture containing about 4 x 10' cells per ml was washed twice with phosphate buffer, pH 8.0, and suspended in 0.1 M ethyl methane sulfonate (Sigma). After incubation at 30°C for 45 min, 6% (wt/vol) Na2S203 was added. Cells were harvested, washed, and plated on YT. Drug-resistance mutations were spontaneous. physical distances between genes. Linkage values were transformed into additive map distances by using the equation derived by Kemper (19): C = (1 - t) + t(Qn t) where C is the linkage frequency value and t is the map distance. RESULTS Transer of plasd R68.45 from E. coli to R. &fol. Plasmid R68.45 was easily transferred from E. coli 1230 to R. trifolii RS176 in membrane matings. Transconjugants were selected on minimal medium with antibiotics. When the recipient strain was one of the auxotrophic derivatives of RS176 described above, selective medium was supplemented with the requirements of the recipient strain. In these crosses the frequency of plasmid transfer was about 10per donor bacterium. This frequency is slightly lower than those reTABLE 2. R68.45 transfer and chromosome mobilization in R. trifolii RS176 Donor Recipient Frequency of Selected Frequency of R transfer0 phenotype recombinants' RS230 RS241 2.8 x 10-1 Ade+ 2.7 x 10-4 Strr 8.9 x 1o-4 RS230 RS294 4.3 x 10-1 Thr+ 1.1 X 1O-4 Ade+ 1.1xi10-5 RS278 RS235 4.2 x 10-1 Met+ 3.0 x 1O-5 His+ 9.2 x 10-4 RS278 RS288 8.3 x 10-1 His+ 1.1 x 10-4 Tyr+ 1.6 x 1O-4 Rifr 6.0 x 10-5 RS278 RS271 5.8 x 10-1 Leu+ 1.1 x 1O-4 Rifr 7.0 x 1O-4 RS278 RS292 3.3 x 10-1 Pdx+ 1.7 x 1O-4 a Per donor bacterium. (O MEGIAS ET AL. J. BACTERIOL. CIRCULAR LINKAGE MAP OF R. TRIFOLII TABLE 3. Detection of R68.45 markers in transconjugants that had received chromosomal markers Cross ~~Selected No. ofR R%R Cross phenotype clones testeda % R RS230 x RS293 Ade+ 204 198 6 3 RS278 x RS288 Tyr' 163 160 3 2 RS278 x RS238 His+ 113 112 1 1 RS230 x RS271 Strr 199 197 2 1 a Replica plated on antibiotic medium. ported for E. coli x R. leguminosarum and E. R68.45 was able to promote chromosome transcoli x R. meliloti crosses (5, 10). fer at a variety of origin sites as previously From these crosses R' transconjugants of R. reported for other rhizobia (4, 5, 10, 21). trifolii were isolated to be used as further donors When the presence of plasmid R68.45 was in intraspecific crosses. All isolates were puriinvestigated among the recombinants, most of fied two or three times before being stored. them proved to be R' at least for resistance Intraspecific matings: transfer of plasmid markers (see Table 3). This suggested that chroR68.45 and mobilization of chromosomal markers mosomal transfer mediated by plasmid R68.45 in R. trifol i. As shown in Table 2, the frequency was F'-like rather than Hfr-like. The same pheof plasmid transfer in intraspecific matings was nomenon has been described in R. leguminohigher than in intergeneric crosses. Transfer sarum and R. meliloti (4, 10, 21). frequencies described here are similar to those Linkage analysis. The rationale for linkage reported for other Rhizobium species, such as R. studies based upon R68.45-mediated gene transmeliloti (9, 21), R. leguminosarum (4, 5), and fer can be summarized as follows: since any interspecific crosses of R. leguminosarum x R. marker has a relatively defined frequency of meliloti (17). transfer, the chance of a pair of markers being Mobilization of single chromosomal markers cotransferred in the same cross will depend on occurred at frequencies ranging from about 5 x the distance between them. 10-5 to 5 x 10-3 per donor cell. Absence of Table 4 shows the results obtained in a series great differences among the frequencies of mobiof two-factor crosses. In each mating initial lization of different markers indicated that selection was carried out for a single marker. TABLE 4. Linkage analysis of several marker pairs of R. trifolii RS176 Donor Recipient Selected Markers Linkage Distance phenotype pairs (C) (t) RS230 RS246 jffr rif-6 ade-29 0.41 0.24 rif-6 str-l 0.21 0.42 Ade+ ade-29 str-1 0.04 0.73 RS230 RS293 ade-29 tyr-13 0.39 0.26 RifF rif-6 tyr-13 0.79 0.05 Tyr' tyr-13 str-l 0.17 0.47 RS230 RS294 Thr+ thr-18 ade-29 0.75 0.06 thr-18 rif-6 0.69 0.09 RS230 RS295 Ade+ ade-29 arg-19 0.65 0.10 RMr rif-6 arg-19 0.25 0.38 RS278 RS235 His+ his-6 met-36 0.40 0.25 Met+ met-36 rjf-6 0.03 0.76 RS278 RS238 Phe+ phe-18 his-6 0.04 0.73 phe-18 rif-6 0.16 0.49 RS278 RS271 Leu+ leu-23 rif-6 0.04 0.73 Ade+ ade-29 leu-23 0.47 0.20 RS278 RS290 Thr+ thr-27 his-6 0.42 0.23 Strr str-l thr-27 0.09 0.60 RifTr nf-6 thr-27 0.70 0.08 RS278 RS292 Pdx+ pdx-3 str-l 0.27 0.36 pdx-3 his-6 0.03 0.76 RS278 RS296 Canr can-3 his-6 0.16 0.49 can-3 met-31 0.54 0.16 His+ his-6 met-31 0.35 0.29 Met+ met-31 rif-6 0.12 0.55 61 VOL. 149, 1982 TABLE 5. Three-factor crosses between R. trifolii strains Donor Recipient Selected Markers Linkage Distance phenotype pairs (C) (t) RS278 RS288 His' his-6 tyr-28 0.43 0.23 his-6ade-17 0.06 0.67 Tyr' tyr-28 his-6 0.46 0.21 tyr-28 ade-17 0.28 0.35 RS278 RS238 His' his-6 phe-18 0.05 0.70 his-6 ade-17 0.07 0.65 Phe+ phe-18 his-6 0.06 0.67 RS278 RS271 Strr str-l ade-17 0.19 0.45 Leu+ leu-23 ade-17 0.03 0.76 RS230 RS293 Tyr' tyr-13 ade-29 0.28 0.35 tyr-13 his-6 0.23 0.40 Ade+ ade-29 tyr-13 0.26 0.37 ade-29 his-6 0.03 0.76 Cotransfer of unselected markers was detected RP1, RP4, Rldrdl9, and R68.45 (M. Meglas, by replica plating on appropriate selective mePh.D. thesis, University of Sevilla, Sevilla, dia. Linkage frequencies lower than 0.03 were Spain, 1981). However, as in other Rhizobium discarded. Distances were calculated by using species, the most efficient plasmid with chromothe Kemper equation as described above. some-mobilizing ability is R68.45 (15; Megias, Results obtained in three-factor crosses are Ph.D. thesis, 1981). summarized in Table 5. Usual frequencies of mobilization of chromoA diagram that summarizes the linkage relasomal markers by R68.45 are higher than revertionships among 17 chromosomal markers is sion rates. For instance, all the auxotrophic presented in Fig. 1. Numbers above the arrows mutations reported in this paper had reversion indicate linkage frequencies found in indepenfrequencies lower than 10-6, whereas the dent experiments. This preliminary linkage map chance of detecting single-marker transfer in an can be circularized and reproduced to scale as R68.45-mediated cross was 10-4 to 10-5 per shown in Fig. 2. donor cell. Thus, accurate measures of transfer DISCUSSION frequencies may be readily obtained. Frequency ranges described in this paper are similar to Mobilization of the R. trifolii chromosome can those reported for R. meliloti (10, 21) and for the be achieved by using several plasmids, such as original host of R68.45, P. aeruginosa (13). Iu-23 ad.-17 pdx-3 can-3 met-36 met-31 tyr-28 str-1 his-6 thi-l tyr-13 rif-0 thr-27 thr-18 ade-29 .rg-19 phe-18 leu-23 116 LI S L LII 6~~~~~~~~~~~~~~~~~~~.73 6.76 gg s.11 176 6.66 6.23;6.21 6.37; 6.26 :..35 126 6~~~~~~~~~~~~.36 6.26 6.24 6.55 6.42 6.76; 6.67; 6.73 6.76 6.23 6.76 6.73 FIG. 1. Preliminary linkage map of the R. trifolii RS176 chromosome. Numbers beside gene symbols indicate allele numbers. Numbers on the arrows indicate conjugal linkage percentages. 62 MEGIAS ET AL. J. BACTERIOL. CIRCULAR LINKAGE MAP OF R. TRIFOLII FIG. 2. Circular linkage map of the R. trifolii RS176 chromosome. Distances are about inversely proportional to the linkage frequencies. Minimal linkage values (3%) would represent, at least, one-fourth of the total length of the chromosome. Another advantage of using plasmid R68.45 for mapping purposes in Rhizobium spp. is the large size of chromosomal fragments mobilized by the plasmid. Subsequently, simultaneous transfer of distant markers may be readily detected in appropriate crosses. For instance, marker pairs showing low linkage frequencies, such as his-61phe-18, met-361rif-6, and his-61pdx3, may be located as far apart as one-fourth of the total length of the chromosome. These results agree with those reported for other Rhizobium species (4, 10, 21). Transformation of linkage measures into additive map distances is easily obtained by use of formulas such as that of Wu (28) or that of Kemper (19). These equations were first proposed for use in cotransduction experiments, but they are also suitable for conjugal mapping (21; Megfas, Ph.D. Thesis, 1981). We have chosen to use Kemper's equation because it does not require any assessment of the length of transferred chromosomal segments. Unlike R. meliloti (10, 11, 25) and R. leguminosarum (8) general transduction systems have not yet been described for R. trifolii. Thus, conjugal mapping mediated by plasmid R68.45 is the only method now available for extensive mapping studies in R. trifolii. We hope it will be suitable for further genetic analysis of nitrogenfixing clover symbiosis. ACKNOWLEDGMENTS We are grateful to John Beringer for providing plasmid R68.45 and to Josep Casadesfs for helpful suggestions during the preparation of the manuscript. LITERATURE CITED 1. Bachmann, B. J., and K. B. Low. 1980. Linkage map of Escherichia coli K-12, edition 6. Microbiol. Rev. 44:1-56. 2. Beringer, J. E. 1974. R-factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84:188-198. 3. Beringer, J. E. 1980. The development of Rhizobium genetics. J. Gen. Microbiol. 116:1-7. 4. Berlnger, J. 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