Isolation and Characterization of an R-Prime Plasmid from Rhizobium ...

JOURNAL OF BACTERIOLOGY, Jan. 1980, p. 121-128 0021-9193/80/01-0121/08$02.00/0

Vol. 141, No. 1

Isolation and Characterization of an R-Prime Plasmid from Rhizobium meliloti GYORGY B. KISS,* KATALIN DOBO,' ILONA DUSHA,1 AGNES BREZNOVITS,2 LASZLO OROSZ,2

EVA VINCZE, AND ADAM KONDOROSI' Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary,' and Department of Genetics, Attila Jozsef University, H-6726 Szeged, Kozepfasor 52, Hungary2

Using a simple enrichment procedure, we isolated an R-prime derivative of plasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41 chromosome. The chromosomal segment carried on this plasmid (pGY1) includes the markers cys-24+, cys-46, and att,s6. Plasmid pGY1 mobilized the chromosome in a polarized way starting from the region of homology, but cannot promote chromosome transfer from other sites. The attlsC3 site on pGY1 allowed the integration of phage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltons was formed. This vector (pGY2) is suitable for the introduction of Rhizobium bacteriophage 16-3 into other gram-negative bacteria.

Transmissible plasmids carrying segments of bacterial chromosome are a useful tool for the study of bacterial genetics. Generally transmissible plasmid primes have been constructed by using either Hfr donors or donors that carry an autonomously replicating plasmid. By using Hfr donors of Escherichia coli K-12, F-prime plasmids have been isolated in the following three ways: (i) selecting for the early transfer of a distal marker (28), (ii) using recA

(6, 27, 35) as recipients, or (iii) using sprains unrelated bacteria (2) as recipients. By using donors carrying autonomously replicating R plasmids, primes have also been isolated with recA strains (14, 32) or strains of a different species (12, 13, 20) as recipients. In this paper we describe an R-prime plasmid isolated by a different procedure. This R-prime is a derivative of R68.45 that carries a segment of the Rhizobium meliloti 41 chromosome containing the cy8-24F, cys-46' markers and the bacteriophage 16-3 attachment site. Evidence that this R-prime plasmid promotes polarized transfer of the R. meliloti 41 chromosome from a preferred site is presented. (Part of this work has been presented previously [Kondorosi et al., Abstr. Int. Congr. Gen., part I, p. 13, 1978.]) MNATERIALS AND METHODS Strains. The microorganisms, plasmids, and bacteriophages used in this study are listed in Table 1. Media. Complete medium (GTA) and minimal medium (GTS supplemented with 1 mg of NH4Cl per ml) were described previously by Kiss et al. (22) and were

used for culturing both R. meliloti and E. coli strains. Amino acids and bases were added to final concentration of 50 and 20 ,ug/ml, respectively. Antibiotics. The concentration and source of antibiotics used were: kanamycin sulfate (Medexport, Moscow, U.S.S.R.), 200 ,ug/ml; streptomycin sulfate (Biogal, Debrecen, Hungary), 250 plg/ml; tetracyclinehydrochloride (Sigma Chemical Co., St. Louis, Mo.), 15 pg/ml; rifamycin (Tubocin; Pharmachim, Sofia, Bulgaria), 100 pg/ml; chloramphenicol (Sigma), 30 pg/ ml. For E. coli the following concentrations were used: kanamycin sulfate, 30 ,ug/ml; streptomycin sulfate, 250 pg/ml; tetracycline-hydrochloride, 15 pg/ml; and rifamycin, 100 pLg/ml. Growth conditions. R. meliloti strains were cultured at 34°C, unless lysogenic. These lysogenic derivatives were grown at 28°C. E. coli strains were cultured at 370C. Matings. These were performed on agar surfaces as described before (9, 24), except that the modified media described by Kiss et al. (22) were used. Matings with lysogenized strains were carried out at 28°C. Plasmid elimination. Strains to be cured of a plamid were inoculated into GTA medium and grown at 3-4C to a density of 1 x 107 to 2 x 107 bacteria per ml. The cultures were then shifted to 39.5 to 40.0°C. Growth continued but with a doubling time about twofold longer than that at 340C. After 24 to 48 h of incubation, the bacteria were plated onto GTA medium. After 2 days the presence of plasmid markers in the clones was tested by replicating the colonies onto selective plates. Generally 10 to 20% of the colonies were cured of R68.45, RP4, or other derivatives. The curing of plasmid was checked for several isolates by agarose gel electrophoresis of plasmid DNA isolated by using the rapid method described by Eckhardt (10). The use of this curing technique in Rhizobium will be published elsewhere. Phage techniques. Isolation of lysogenic strains, heat induction, and identification of ti3 and c' alleles were as described by Orosz and Sik (33) and Orosz et

t Present address: Department of Biology, York University, Downsview, Ontario, Canada M3J 1P3. 121

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Strains/ plasmids/

TABLE 1. Bacterial strains, plasmids, and phages used in this studya Strains/ Genotype References Genotype plasmids/

phages R. meliloti 41(Rm4l) Wild narA168 metGY75 168(R68.45)

GY81 GY83

GY102 GY115 GY116 GY117

J. BACTERIOL.

narBI5phe-

References

phages AK495

K. Szende (22)

AK532

(22)

AK746 AK766

15(R68.45) (22) narBl5pur15(R68.45) cys-46 leu-4 str-1 cml- Derivative of GY133 1 cys-46 leu-4 str-1 rif-2 Derivative of GY133 Derivative of AK80 cys-46(pGY1) cys-46 leu-4 str-l cml- Derivative of GY102 I(pGY1) gly-l + revertant of cys-46 leu-4 str-1 AK184 gly-l+ revertant of cys-46phe-15 str-l AK495 cys-46phe-15 str-1(16- Derivative of GY493 3) (24) his-i str-i (24) cys-46 Derivative of ZS1 pur-l met-13 str-3 (24) pur-1 pyr-29 str-3 (24) pur-I cys-24 str-I (24) cys-46pur-3 str-1 Derivative of AK180 cys-46pur-3 his-2 str-l cys-46gly-l leu-4 str-1 (24) Derivative of AK80 cys-46 str-1 rif-2 (24) his-i str-l(R68.45) Derivative of AK173 pur-l pyr-29 str-

EV79

EV81

Cured derivative of cys-46 gly-1 phe-15 EV79 str-I cys-46 gly-I pyr-29 str- Cured derivative of EV81 I Derivative of Rm41 cys-10 rif-10 Tn5 insertion mutant pyr-24 str-6 of Rm4l leu-4+ phe-15+ recomcys-46 gly-l phe-15 binant of GY81 and str-l(R68.45) AK184 cys-46 gly-l pyr-29 str- leu-4+ pyr-29 recombinant of AK209 and 1(R68.45) AK184 pur-I str-3 (24) str (29)

ZS1 2011 E. coli A. Kiss C600 hsdR hsdM GY493 A. Kiss hsdR hsdM pro leu HB101 thi gal lacY recA GY564 str GY439 Derivative of HB101 HB101 rif AK74 Derivative of HB101 GY588 HB101(pGY2) AK80 Derivative of GY439 GY611 HB101 rif(pGY1) AK172 Derivative of HB101 AK658 HB101(R68.45) AK173 Plasmids AK175 Km Tc Cb Cma+ R68.45 (11, 24) AK180 IncPl AK183 R68.45 Q 1 [Rm4I cys- This study pGY1 AK184 24-attl6-3] AK206 This study pGY1::16-3 ti3 pGY2 AK208 Phages AK209 K. Szende Wild 16-3 3(R68.45) (33) 16-3 ti3 cti3 AK351 narBI5pur-15 str-17 (22) 'Antibiotic resistance mutants were isolated without mutagenesis. Auxotrophic mutants were isolated after mutagenesis with NTG (N-methyl-N'-nitroso-N-nitroguanidine, Koch-Light Laboratories, Colinbrook, England.) or with Tn5by the methods of Kondorosi et al. (23) and Beringer et al. (4), respectively. Genotypic abbreviations for chromosomal markers: nar, genes determining and regulating nitrate reduction (22); rif, genes determining response to rifamycin (sensitivity, resistance); other symbols are according to Demerec et al. (8) and Bachmann et al. (1). Phenotypic abbreviations for plasmid markers: Cma, chromosome mobilization ability; other symbols are according to Novick et al. (31). Abbreviation for phage marker: cti3, gene(s) determining and regulating inducibility (thermoinducible).

GY133

al. (34). Phage lysates were prepared by the method of Svab et al. (37). Isolation of plasmid DNA. Plasmids were prepared from bacterial cultures grown in complete medium. Cells were harvested by centrifugation at 6,000 x g for 20 min. The pellet was then washed three times with 50 mM Tris-hydrochloride (pH 8.0) containing 20 mM EDTA. Cleared lysates were prepared by the method of Casse et al. (7). Before ultracentrifugation, cleared lysates were tested for the presence of plasmid DNA by using gel electrophoresis. Plasmid preparations were further purified by cesium chloride (Merck and Co., Inc., Rahway, N.J.)-ethidium bromide density gradient centrifugation at 38,000 rpm for 72 h in a Sorvall T-865.1 type rotor at 10°C. Plasmid isolates were then extracted with isoamylalcohol to remove ethidium bromide and dialyzed against 50 mM Tris-hydrochloride containing 5 mM EDTA (pH 8.0). Restriction endonuclease digestion. Digestions were carried out in a volume of 25 id containing 1 to 2 Ag of pamid DNA, 15 mM Tris-hydrochloride (pH 9.0), 10 mM MgC12, 15 mM KCI, 7 mM 2-mercaptoeth-

anol, and an excess amount of SmaI enzyme. After 2 h at 30°C the reaction was stopped by the addition of 5 Al of 0.2 M EDTA (pH 8.05), containing 50% saccharose and 0.18% bromophenol blue. Restriction endonuclease SmaI was kindly provided by G. Dallmann.

RESULTS Isolation of an R-prime carrying the cys46' marker. Previous studies on the mapping of the R. meliloti 41 (Rm41) chromosome revealed that plasmid R68.45 promoted gene transfer from many chromosomal sites of Rm4l and recombinants for various markers appeared essentially at the same frequency (24). It was found, however, in the course of mapping more than 30 markers that the cys-46' recombinants appeared consistently at a frequency 1.5 to 3.0 times higher than the other markers. Recombination frequencies for the cys-46' marker and several other markers, obtained in matings with

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R68.45, are shown in Table 2. This suggested that plasmid R68.45 interacted more easily with the chromosome around the cys-46+ region; therefore, the formation of R-primes carrying this chromosomal section should be more probable. An R-prime derivative of the plasmid R68.45 (pGYl) that carries the cys-46+ region of the bacterial chromosome was isolated as follows. Bacterial strain GY75 was mated to a cys46 str-1 recipient (AK184) on agar surface. The crossed bacterial population was suspended and plated out in appropriate dilution on selective plates (Table 3, cross 1). Approximately 104 cys46+str-1 colonies were scraped off from a single plate and used as donors in a second mating with a cys-46 rif-2 recipient (AK206). About 104 cys46+ rif-2 recombinant colonies were pooled and used in a third mating by using a cys-46 cml-l strain (GY102) as recipients. Three out of six cys-46+ cml-l colonies, obtained from the third mating, transferred the cys-46+ marker at a very high frequency when mated with the cys-46 recipient, AK80. One prototrophic colony was purified by restreaking twice on selective plates. This isolate was designated GY116 and further analyzed. The plasmid derivative in strain GY116 was denoted pGY1 according to the nomenclature rules for plasmids recently proposed by Novick et al. (31). Transfer of the cys-46+ marker by pGY1.

Chromosomal and plasmid markers were selected after matings between GY116 and GY133. These data are presented in Table 2, cross 1. The kanamycin resistance and the cys-46+ markers were transferred at the same frequency, and analysis showed them to be tightly linked. On the other hand, no leu-4+ recombinants could be detected. Plasmid pGY1 was transferred into E. coli HB101 by selecting for kanamycin resistance (Table 3, cross 3). Three independent kanamycin-resistant colonies were purified twice by restreaking for single colonies. These three colonies were mated with a derivative of Rm4l (GY115) followed by selection for kanamycinresistant transconjugants. Ten ofthese transconjugants were analyzed for their cys-46 and leu-4 markers (Table 3, cross 4). At least 50% of the transconjugants inherited the cys-46+ marker, but all were phenotypically Leu-. These results strongly suggested that the plasmid pGY1 contained the cys-46+ region of the Rm4l chromo-

TABLE 2. Transfer frequency of various chromosomal markers by R68.45 and pGYl Recipient strain

Selected marker

Transfer frequencya by: pGYl

R68.45

1.0 1.0 X 10-3 cys-46+ 1.0 1.0 X 10-3 cys-24+ 1.9 X 10-3 pur-1+ 1.2 x 10-3 3.6 x 10-4 1.0 X 10-3 pur-3+ 1.0 X 10-3 3.0 x 10-4 pyr-24+ 1.9 x io-5 4.2 x 10-4 strl 6.2 x 10-4 8.0 x 10-6 cys-10+ 6.6 x 10-6 pur-15+ 7.3 x 10-4 4.7 x 10-4