JOURNAL OF BACTERIOLOGY, Jan. 1980, p. 121-1280021-9193/80/01-0121/08$02.00/0

Vol. 141, No. 1

Isolation and Characterization of an R-Prime Plasmid fromRhizobium meliloti

GYORGY B. KISS,* KATALIN DOBO,' ILONA DUSHA,1 AGNES BREZNOVITS,2 LASZLO OROSZ,2EVA VINCZE, AND ADAM KONDOROSI'

Institute of Genetics, Biological Research Center, Hungarian Academy ofSciences, 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 ofplasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41chromosome. The chromosomal segment carried on this plasmid (pGY1) includesthe markers cys-24+, cys-46, and att,s6. Plasmid pGY1 mobilized the chromo-some in a polarized way starting from the region ofhomology, but cannot promotechromosome transfer from other sites. The attlsC3 site on pGY1 allowed theintegration ofphage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltonswas formed. This vector (pGY2) is suitable for the introduction of Rhizobiumbacteriophage 16-3 into other gram-negative bacteria.

Transmissible plasmids carrying segments ofbacterial chromosome are a useful tool for thestudy of bacterial genetics. Generally transmis-sible plasmid primes have been constructed byusing either Hfr donors or donors that carry anautonomously replicating plasmid.By using Hfr donors of Escherichia coli K-12,

F-prime plasmids have been isolated in the fol-lowing three ways: (i) selecting for the earlytransfer of a distal marker (28), (ii) using recAsprains (6, 27, 35) as recipients, or (iii) usingunrelated bacteria (2) as recipients.By using donors carrying autonomously rep-

licating R plasmids, primes have also been iso-lated with recA strains (14, 32) or strains of adifferent species (12, 13, 20) as recipients.

In this paper we describe an R-prime plasmidisolated by a different procedure. This R-primeis a derivative of R68.45 that carries a segmentof the Rhizobium meliloti 41 chromosome con-taining the cy8-24F, cys-46' markers and thebacteriophage 16-3 attachment site. Evidencethat this R-prime plasmid promotes polarizedtransfer of the R. meliloti 41 chromosome froma preferred site is presented.

(Part of this work has been presented previ-ously [Kondorosi et al., Abstr. Int. Congr. Gen.,part I, p. 13, 1978.])

MNATERIALS AND METHODSStrains. The microorganisms, plasmids, and bac-

teriophages used in this study are listed in Table 1.Media. Complete medium (GTA) and minimal me-

dium (GTS supplemented with 1 mg of NH4Cl per ml)were described previously by Kiss et al. (22) and were

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

used for culturing both R. meliloti and E. coli strains.Amino acids and bases were added to final concentra-tion of 50 and 20 ,ug/ml, respectively.

Antibiotics. The concentration and source of an-tibiotics used were: kanamycin sulfate (Medexport,Moscow, U.S.S.R.), 200 ,ug/ml; streptomycin sulfate(Biogal, Debrecen, Hungary), 250 plg/ml; tetracycline-hydrochloride (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, 250pg/ml; tetracycline-hydrochloride, 15 pg/ml; and rifa-mycin, 100 pLg/ml.Growth conditions. R. meliloti strains were cul-

tured at 34°C, unless lysogenic. These lysogenic deriv-atives were grown at 28°C. E. coli strains were cul-tured at 370C.

Matings. These were performed on agar surfacesas described before (9, 24), except that the modifiedmedia described by Kiss et al. (22) were used. Matingswith lysogenized strains were carried out at 28°C.Plasmid elimination. Strains to be cured of a

plamid were inoculated into GTA medium and grownat 3-4C to a density of 1 x 107 to 2 x 107 bacteria perml. The cultures were then shifted to 39.5 to 40.0°C.Growth continued but with a doubling time abouttwofold longer than that at 340C. After 24 to 48 h ofincubation, the bacteria were plated onto GTA me-dium. After 2 days the presence of plasmid markers inthe clones was tested by replicating the colonies ontoselective plates. Generally 10 to 20% of the colonieswere cured of R68.45, RP4, or other derivatives. Thecuring of plasmid was checked for several isolates byagarose gel electrophoresis of plasmid DNA isolatedby using the rapid method described by Eckhardt (10).The use of this curing technique in Rhizobium will bepublished elsewhere.Phage techniques. Isolation of lysogenic strains,

heat induction, and identification of ti3 and c' alleleswere as described by Orosz and Sik (33) and Orosz et

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TABLE 1. Bacterial strains, plasmids, and phages used in this studya

Strains/ Strains/plasmids/ Genotype References plasmids/ Genotype Referencesphages phages

R. meliloti AK495 cys-46 gly-1 phe-15 Cured derivative of41(Rm4l) Wild K. Szende str-I EV79GY75 narA168 met- (22) AK532 cys-46 gly-I pyr-29 str- Cured derivative of

168(R68.45) I EV81GY81 narBI5phe- (22) AK746 cys-10 rif-10 Derivative of Rm41

15(R68.45) AK766 pyr-24 str-6 Tn5 insertion mutantGY83 narBl5pur- (22) of Rm4l

15(R68.45) EV79 cys-46 gly-l phe-15 leu-4+ phe-15+ recom-GY102 cys-46 leu-4 str-1 cml- Derivative of GY133 str-l(R68.45) binant of GY81 and

1 AK184GY115 cys-46 leu-4 str-1 rif-2 Derivative of GY133 EV81 cys-46 gly-l pyr-29 str- leu-4+ pyr-29 recombi-GY116 cys-46(pGY1) Derivative of AK80 1(R68.45) nant of AK209 andGY117 cys-46 leu-4 str-l cml- Derivative of GY102 AK184

I(pGY1) ZS1 pur-I str-3 (24)GY133 cys-46 leu-4 str-1 gly-l + revertant of 2011 str (29)

AK184 E. coliGY493 cys-46phe-15 str-l gly-l+ revertant of C600 hsdR hsdM A. Kiss

AK495 HB101 hsdR hsdMpro leu A. KissGY564 cys-46phe-15 str-1(16- Derivative of GY493 thi gal lacY recA

3) strAK74 his-i str-i (24) GY439 HB101 rif Derivative of HB101AK80 cys-46 (24) GY588 HB101(pGY2) Derivative of HB101AK172 pur-l met-13 str-3 Derivative of ZS1 GY611 HB101 rif(pGY1) Derivative of GY439AK173 pur-1 pyr-29 str-3 (24) AK658 HB101(R68.45) Derivative of HB101AK175 pur-I cys-24 str-I (24) PlasmidsAK180 cys-46pur-3 str-1 (24) R68.45 Km Tc Cb Cma+ (11, 24)AK183 cys-46pur-3 his-2 str-l Derivative of AK180 IncPlAK184 cys-46gly-l leu-4 str-1 (24) pGY1 R68.45 Q 1 [Rm4I cys- This studyAK206 cys-46 str-1 rif-2 Derivative of AK80 24-attl6-3]AK208 his-i str-l(R68.45) (24) pGY2 pGY1::16-3 ti3 This studyAK209 pur-l pyr-29 str- Derivative of AK173 Phages

3(R68.45) 16-3 Wild K. SzendeAK351 narBI5pur-15 str-17 (22) 16-3 ti3 cti3 (33)

'Antibiotic resistance mutants were isolated without mutagenesis. Auxotrophic mutants were isolated after mutagenesiswithNTG (N-methyl-N'-nitroso-N-nitroguanidine, Koch-Light Laboratories, Colinbrook, England.) or with Tn5by the methodsof Kondorosi et al. (23) and Beringer et al. (4), respectively. Genotypic abbreviations for chromosomal markers: nar, genesdetermining and regulating nitrate reduction (22); rif, genes determining response to rifamycin (sensitivity, resistance); othersymbols 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).

al. (34). Phage lysates were prepared by the methodof Svab et al. (37).

Isolation of plasmid DNA. Plasmids were pre-pared from bacterial cultures grown in complete me-dium. Cells were harvested by centrifugation at 6,000x g for 20 min. The pellet was then washed threetimes with 50 mM Tris-hydrochloride (pH 8.0) con-taining 20 mM EDTA. Cleared lysates were preparedby the method of Casse et al. (7). Before ultracentrif-ugation, cleared lysates were tested for the presenceof plasmid DNA by using gel electrophoresis. Plasmidpreparations were further purified by cesium chloride(Merck and Co., Inc., Rahway, N.J.)-ethidium bromidedensity gradient centrifugation at 38,000 rpm for 72 hin a Sorvall T-865.1 type rotor at 10°C. Plasmid iso-lates were then extracted with isoamylalcohol to re-move ethidium bromide and dialyzed against 50 mMTris-hydrochloride containing 5 mM EDTA (pH 8.0).

Restriction endonuclease digestion. Digestionswere carried out in a volume of 25 id containing 1 to 2Ag of pamid DNA, 15 mM Tris-hydrochloride (pH9.0), 10mM MgC12, 15mM KCI, 7mM 2-mercaptoeth-

anol, and an excess amount of SmaI enzyme. After 2h at 30°C the reaction was stopped by the addition of5 Al of 0.2 M EDTA (pH 8.05), containing 50% sac-charose and 0.18% bromophenol blue. Restriction en-donuclease SmaI was kindly provided by G. Dallmann.

RESULTSIsolation of an R-prime carrying the cys-

46' marker. Previous studies on the mappingof the R. meliloti 41 (Rm41) chromosome re-vealed that plasmid R68.45 promoted genetransfer from many chromosomal sites of Rm4land recombinants for various markers appearedessentially at the same frequency (24). It wasfound, however, in the course of mapping morethan 30 markers that the cys-46' recombinantsappeared consistently at a frequency 1.5 to 3.0times higher than the other markers. Recombi-nation frequencies for the cys-46' marker andseveral other markers, obtained in matings with

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R-PRIME PLASMID FROM RHIZOBIUM 123

R68.45, are shown in Table 2. This suggestedthat plasmid R68.45 interacted more easily withthe chromosome around the cys-46+ region;therefore, the formation of R-primes carryingthis chromosomal section should be more prob-able. An R-prime derivative of the plasmidR68.45 (pGYl) that carries the cys-46+ region ofthe bacterial chromosome was isolated as fol-lows. Bacterial strain GY75 was mated to a cys-46 str-1 recipient (AK184) on agar surface. Thecrossed bacterial population was suspended andplated out in appropriate dilution on selectiveplates (Table 3, cross 1). Approximately 104 cys-46+str-1 colonies were scraped off from a singleplate and used as donors in a second mating witha cys-46 rif-2 recipient (AK206). About 104 cys-46+ rif-2 recombinant colonies were pooled andused in a third mating by using a cys-46 cml-lstrain (GY102) as recipients. Three out of sixcys-46+ cml-l colonies, obtained from the thirdmating, transferred the cys-46+ marker at a veryhigh frequency when mated with the cys-46 re-cipient, AK80. One prototrophic colony was pu-rified by restreaking twice on selective plates.This isolate was designated GY116 and furtheranalyzed. The plasmid derivative in strainGY116 was denoted pGY1 according to the no-menclature rules for plasmids recently proposedby Novick et al. (31).Transfer of the cys-46+ marker by pGY1.

TABLE 2. Transfer frequency of variouschromosomal markers by R68.45 andpGYl

Recipient Selected Transfer frequencya by:strain marker R68.45 pGYl

GY493 cys-46+ 1.0 X 10-3 1.0AK175 cys-24+ 1.0 X 10-3 1.0AK172 pur-1+ 1.2 x 10-3 1.9 X 10-3AK183 pur-3+ 1.0 X 10-3 3.6 x 10-4AK766 pyr-24+ 1.0 X 10-3 3.0 x 10-4AK245 strl 4.2 x 10-4 1.9 x io-5AK746 cys-10+ 6.2 x 10-4 8.0 x 10-6AK351 pur-15+ 7.3 x 10-4 6.6 x 10-6GY133 leu-4+ 4.7 x 10-4 <1.0 X 10-7AK74 his-l+ 5.8 x 10-4 <1.0 X 1o-6AK183 his-2+ 4.2 x 10-4 <1.9 X 10-5GY493 phe-15+ 3.3 x 10-4 <1.5 x 10-5AK532 pyr-29+ 5.7 x 10-4 <1.3 X 10-6AK172 met-13 + 8.3 x 10-4 <5.0 X 10-6a Transfer frequencies were related to the number

of recipients which acquired plasmids R68.45 andpGY1, respectively. In matings with R68.45, AK208 orGY83 was used as donor and counterselection wasdone for his-1 or pur-15, respectively. In matings withpGY1, GY116 or GY117 was used as donor and coun-terselection was done for streptomycin or leu-4marker, respectively. We chose the donors in such away that the counterselection should not influence therecombination frequency.

Chromosomal and plasmid markers were se-lected 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 couldbe detected. Plasmid pGY1 was transferred intoE. coli HB101 by selecting for kanamycin resist-ance (Table 3, cross 3). Three independent kan-amycin-resistant colonies were purified twice byrestreaking for single colonies. These three col-onies were mated with a derivative of Rm4l(GY115) followed by selection for kanamycin-resistant transconjugants. Ten ofthese transcon-jugants were analyzed for their cys-46 and leu-4markers (Table 3, cross 4). At least 50% of thetransconjugants inherited the cys-46+ marker,but all were phenotypically Leu-. These resultsstrongly suggested that the plasmid pGY1 con-tained the cys-46+ region of the Rm4l chromo-some.

Stability of pGY1. Instability is a commonproperty of plasmid primes (18, 20). To test thestability of pGY1, we grew strain GY116 undernonselective conditions (complete medium) forabout 20 generations. This culture was thenplated for single colonies on complete mediaplates. A total of 960 colonies were tested forkanamycin resistance, tetracycline resistance,and Cys+ prototrophy. All of the colonies testedretained these three markers, indicating thatpGY1 was fairly stable and the homogenizationof cys-46+ marker to cys-46 was about 10-3 orless. The fact that this R-prime could be satis-factorily maintained in Rm4l was surprising inlight ofthe fact that most plasmid primes readilylost their chromosomal portion in a rec+ host (9,28, 30).

Incorporation of the chromosomal segment ofthe cys-46+ marker into R68.45 was also sup-ported by the analysis of Rm41 strains fromwhich the plasmid pGY1 had been eliminated.When strain GY116 was grown in complete me-dium at 400C (see above) for about 10 genera-tions and then tested for kanamycin, tetracy-cline, and cys-46i markers, 90% ofthe cells tested(636 colonies) lost all three markers and theremaining 10% were also phenotypically Cys-but retained the two antibiotic resistancemarkers. This indicated that the chromosomalsegment of the R-prime segregated more quicklythan the other markers. These data showed thatthe elimination of pGY1 was strongly correlatedwith the loss of the cys-46+ marker and indicatedthat R68.45 carried the cys-46+ region of theRm4l chromosome.Chromosome mobilization by pGY1. The

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TABLE 3. Isolation and transfer properties ofpGYI andpGY2

Crossn.DCounterse- Selected Transfer fre- Coinheritance of unse-Crossno.Donor Recipients lected marker marker quency' lected markers1 Rm4l(R68.45) Rm4l

GY75 AK184 str-1 cys-46+ 7.7 x 10-4str-l leu-4+ 2.8 x 10-4str-l gly-lI + 1.8 x 10-4

2 Rm4l(pGY1) Rm4lGY116 GY133 str-l Km 1.5 x 10-' 10/10 cys-46+ leu-4

str-l cys-46+ 1.5 x 10-' 10/10 Km leu-4str-l leu-4+ <1.4 x 10-8

3 Rm4l(pGY1) E. coliGY116 HB101 str Km 4.3 x 10-6

4 E. coli(pGY1) Rm4lclone 1 GY115 rif Km 4.3 x 10-4 10/10 cys-46+ leu-4clone 2 GYllS rif Km 1.8 x 10-4 5/10 cys-46+ leu-4clone 3 GY115 rif Km 3.1 x 10-6 7/10 cys-46+ leu-4

5 E. Coli(pGY2) Rm4lGY588 GY493 pro leu thi Km 1.2 x 10-5

cys-46+ 5.8 x 10-5 1/3 Km Tc 16-3ti3phe-15+ <2.3 x 10-7Km cys-46+ 1.0 x 10-5 17/34 16-3ti3

6 E. coli(pGY2) Rm41(16-3) pro leu thiGY588 GY564 Km 2.2 x 10-5

cys-46+ 9.2 x 10-5 1/3 Km Tc 16-3ti3Km cys-46+ 1.5 x 10-5 15/31 16-3ti3

a Transfer frequency was related to the number of recipients. Mating experiments and selection for transcon-jugants were carried out as described in the text except that crosses 5 and 6 were done at 28°C.

kanamycin resistance marker ofthe plasmid andthe chromosomal marker cys-46+ were coinher-ited at the same frequency (Table 3, cross 2). Onthe other hand, no recombinants were detectedfor the leu-4 marker, although the originalR68.45 promoted chromosomal transfer for var-ious markers at a frequency of 10-3 to 10-4(Table 2). The above data suggested that theCma+ property of pGY1 was different from thatof R68.45. To demonstrate these differences, wedetermined the recombination frequencies for 14different chromosomal markers in matings byusing pGY1. The map position of markers usedwere determined previously and are shown inFig. 1. Recombinants were obtained at variousfrequencies for markers located counter-clock-wise from the cys-46+ marker. Furthermore, agradient of transmission of markers was ob-served. Very few recombinant colonies appearedfor markers located clockwise from cys-46+.These data indicated that pGYl promoted chro-mosome transfer from a preferred origin (cys-46+ region) in a polarized manner.

It was previously published that another cysmarker, cys-24, mapped close to the cys-46marker on the Rm4l chromosome (24). Table 2shows that by using pGY1 as a sex factor, thecys-24+ marker was coinherited with the kana-mycin resistance marker, indicating that the cys-24+ marker was also located on pGY1.

his-2/eu-4 his-1

FIG. 1. Location of markers used in this study onthe R. meliloti 41 chromosome. Map location ofmarkers were determined previously (22, 24, 37), ex-cept for phe-15, cys-10, and pyr-24. These markerswere mapped in three-factor crosses as describedbefore (24). Distances between markers are in addi-tive map distance values d (24).

Integration of 16-3 into pGYl. Specializedtransduction of the cys-46' marker by phage 16-3 of Rm4l was recently described. It was alsoshown that the cys-46' marker and the ti3

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R-PRIME PLASMID FROM RHIZOBIUM 125

marker of the prophage 16-3 could be cotrans-ferred at a frequency of 0.9 (37). To demonstratethe presence of the attachment site for phage16-3 on plasmid pGY1, we lysogenized strainGY116 with a thermoinducible mutant of phage16-3, carrying the ti3 allele. (The thermoinduci-ble ti3 mutant of 16-3 produces turbid plaquesat 28°C and clear ones at 360C.) Seven inde-pendent lysogenic clones were isolated from tur-bid plaques. These clones were mated with E.coli HB101 or with its rifamycin-resistant deriv-ative followed by selection for kanamycin-resist-ant transconjugants. From each cross, transcon-jugants were isolated by purifying on selectiveplates and mated with Rm4l followed by testingfor mature phage particles in the chloroform-treated supernatants. It was found that threeout of seven HB101 isolates were able to donatethe prophage 16-3 ti3 to Rm4l. When recipientslysogenic for wild-type 16-3 were used in thesame manner, 10 to 40% of the plaques were ofthe ti3 type. These results suggested that theprophage 16-3 ti3 could integrate into pGY1 andbe maintained in E. coli.One E. coli clone (GY588) which donated

intact prophage 16-3 ti3 to Rm4l was kept forfurther studies. The plasmid derivative ofpGY1present in this clone was denoted pGY2. Thetransfer properties of pGY2 are shown in Table3, crosses 5 and 6. Strain GY588 was used as adonor in matings with a nonlysogenic Rm4l(GY493) and a 16-3 lysogenized Rm4l (GY564).The transfer frequencies were repeatedly abouttwo times higher for both cys-46" and kanamy-cin resistance markers when lysogenic recipientswere used. This could be explained by zygoticinduction of the prophage entering the nonly-sogenic strain. It was also remarkable that thecys-46+ transconjugants were higher in numberthan the kanamycin-resistant ones in both mat-ings. Therefore, three cys-46+ transconjugants ofcrosses 5 and 6 were purified and tested for thecoinheritance of antibiotic resistance markers,phage markers,- and transfer properties. Oneclone out of three from both matings was resist-ant to both kanamycin and tetracycline, pro-duced 16-3 ti3 phage particles, and donated allmarkers at a very high frequency. The othercys+ clones tested did not inherit plasmidmarkers. These could be cys-46* revertants orclones in which the antibiotic resistance markersand prophage segregated. The nature of theseclones is not clear. When selection was made forboth kanamycin resistance and cys-46+ markers,about 50% of the transconjugants lost the pro-phage, both in the nonlysogenic and lysogenicrecipients. Such an instability of the prophageon pGY2 was not observed when pGY2 was

transferred between derivatives of Rm4l.Plasmid pGY2 was transferred into a different

R. meliloti isolate (R. meliloti 2011) which isresistant to phage 16-3. It was found that R.meliloti 2011 carrying pGY2 could liberate 16-3ti3 as effectively as Rm4l(pGY2). Conjugationexperiments with this strain also indicated thestability of plasmid pGY2 in matings betweenstrains of R. meliloti.Physical characterization of plasmids

pGY1 and pGY2. As described by Casse et al.(7), Rm4l contains a large plasmid which mightcause difficulties in plasmid isolation. To avoidthis, we transferred plasmids R68.45, pGY1, andpGY2 from Rm4l into E. coli HB101 and testedfor the presence of all plasmid markers. TheseE. coli transconjugant derivatives, AK658,GY611, and GY588, were used as sources for theisolation of the plasmids.To characterize pGY1 and pGY2, we used

restriction endonuclease digestion. Covalentlyclosed circular plasmidDNA was separated fromthe chromosomal, linear plasmid, and nickedplasmid DNAs by cesium chloride-ethidium bro-mide density gradient centrifugation. The plas-mid DNA was digested with the restriction en-donuclease SmaI. The restriction fragmentswere separated by agarose gel electrophoresis,and molecular weights were determined. Theresults of this analysis are summarized in Table4. As a summary of the fragment molecularmasses, R68.45 had 36.6 megadaltons (md). Thisresult is consistent with the data (36.8 md) ofJohnston et al. (19). Surprisingly, we obtainedan additional 1.5-md fragment, and instead ofthe 10.1-md fragment a 8.4-md fragment oc-curred. We believe the presence of a sixth SmaIcleavage site in the 10.1-md fragment resulted inthe formation of the 8.4-md and 1.5-md frag-ments. These differences might be explained bya molecular rearrangement within R68.45. Insta-bility of R68.45 has been described previously(11,24).The molecular mass of pGY1 was 54.4 md;

therefore, the chromosomal DNA segment in-serted was about 17.8 md. Two fragments ofR68.45 were missing in pGY1. We suppose thatthe chromosomal segment was integrated intothe 4.25-md fragment of R68.45. Interestingly,pGY1 was also missing a 1.5-md fragment pres-ent in R68.45. Although we have no evidencethat this fragment is responsible for the Cma+phenotype of R68.45, it was shown earlier (16)that R68.45 contains an extra piece of DNA ofabout 1.5 md.The molecular mass of pGY2 was 91.8 md. As

a result of the integration of phage 16-3 ti3 DNAinto pGY1, the molecular mass increased by 37.4

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TABLE 4. Molecular weight offragments ofR68.45,pGYl, andpGY2plasmids obtained by digestion

with SmaI restriction endonucleaseMol wt of fragmentsa

R68.45pGY1 pGY2

ab bc

12.5 12.5 12.5 12.510.19.2 9.4 9.4 9.4

8.4 8.4 8.47.86.7

4.9 4.94.5 4.5

4.25 4.23.3

2.8 2.82.7 2.7

2.62.55 2.552.4 2.41.95 1.951.85 1.85

1.781.5 1.6

1.451.39

1.35 1.180.98 0.970.93 0.93

0.830.750.70

0.63 0.640.54 0.55

0.5 0.52 0.52 0.52

Total 36.8 36.6 54.4 91.84

aMolecular weights are means from four to sixseparate experiments. The digested plasmids weresubjected to electrophoresis on a 3 mm thick 1%agarose slab gel. Restriction fragments of wild-typelambda phage DNA cleaved by HindIII restrictionendonuclease were used as molecular weight stand-ards. Although we attempted to digest by SmaI phage16-3 DNA, the digestion was partial. Phage 16-3 wasisolated and purified from Rm4l in contrast to plas-mids prepared from E. coli.

b Results from Johnston et al. (19).'Our data.

md. This value corresponded to the molecularmass of the 16-3 DNA (37.5 md, Dallmann et al.,Mol. Gen. Genet., in press). The prophage prob-ability inserted into the 1.35-md fragment ofpGY1, suggesting that attl63 was on this frag-ment.

DISCUSSIONAn R-prime derivative of R68.45 (pGY1)

carrying the cys-46& region of the Rm4l chro-

mosome was isolated by an enrichment proce-dure where selection for the cys-46+ marker wasmade in three consecutive mating experimentsusing the recombinants as donors. We have bothgenetic and physical evidences that a chromo-somal segment of Rm4l has been inserted intoplasmid R68.45. The genetic evidence is as fol-lows. (i) The antibiotic resistance and cys-46'markers were always transferred together inmatings between derivatives ofRm4l. (ii) WhenpGY1 was introduced into E. coli and thenretransferred into Rm4l, the cys-46' region ofthe Rm4l chromosome was usually transferredwith the antibiotic resistance markers of theplasmid. (iii) When the plsmid was eliminated,the loss of plasmid markers paralleled the lossof prototrophy (cys-46).To obtain physical evidence for the integra-

tion of a part of the Rm41 chromosome intoR68.45, we determined the molecular weights ofR68.45 and pGY1. The values, calculated fromthe molecular weight of SmaI restriction endo-nuclease-generated fragments, were 36.6 and54.4 md, respectively. Consequently the inte-grated chromosomal segment was 17.8 md, andit was probably inserted into the 4.5-md SmaIfragment of R68.45. The restriction pattern ofthree R-primes generated from R68.45 by an invivo procedure was determined (19), and it wasfound that the digestion pattem of one of theplasmids indicated insertion of chromosomalDNA into the same 4.5-md SmaI fragment.As a result of the integration of phage 16-3 ti3

DNA into pGY1, the molecular mass increasedby 37.4 md. It appears that in pGY2 a smallchromosomal fragment present in pGY1 wasmissing (1.35 md). This fragment should containthe phage attachment site.

R68.45-prime formation was demonstrated inother rhizobia as well: R. meliloti 2011 and R.leguminosarum 300 (20). The mechanism ofR68.45-prime formation is not known. In thiscontext one must note that the ability of R68.45to interact with the host chromosome, especiallythe mechanism of chromosome mobilization,mediated by R68.45, is certainly different fromthat of the F sex factor. Unlike the F+ andF-primes, chromosome mobilization by R68.45is not dependent on a functional recA productin E. coli (Z. Banfalvi, unpublished data).R68.45-primes do not stably integrate into thehost chromosome; therefore, chromosome mo-bilization cannot be explained by the formationof Hfr-like derivatives of R68.45 (5, 15, 24). Ef-forts to demonstrate the integration of R68.45 orthe cys-46i marker of pGY1 into the chromo-some were unsuccessfil (data not shown). Sim-ilar negative results on the isolation of Hfr-like

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R-PRIME PLASMID FROM RHIZOBIUM 127

R68.45 derivatives were published by Haas andHolloway (11).

Polarized chromosome transfer from a pre-ferred origin by in vivo-constructed F-primeshas been reported (28, 36). There are conflictingreports regarding the ability of in vitro-con-structed RP4-primes to mobilize the chromo-some. Jacob et al. (17) constructed recombinantplasmids by inserting DNA pieces from R. leg-uminosarum and from Proteus mirabilis intoRP4, but the Cma properties of these plasmidswere unchanged, compared to that of originalRP4. Barth (3) and Julliot and Boistrad (21)reported that in vitro-constructed RP4-primesdid promote chromosome transfer in a polarizedmanner from a preferred origin in both E. coliand R. meliloti 2011. One must note that R.meliloti 2011 is the only rhizobium strain testedso far in which the original RP4 can mobilize thechromosome (21, 29). R68.45-primes carryingsegments of the R. meliloti chromosome weregenerated in vivo by Johnston et al. (20). Wefound that these primes tested also promotedpolarized chromosome transfer in Rm4l (unpub-lished data).The insertion of the cys-46' region of the

Rm41 chromosome into R68.45 resulted in anR-prime plasmid (pGY1) which promotes polar-ized chromosome transfer from a preferred ori-gin (cys-46 region). The ability of the parentplasmid R68.45 to transfer chromosomalmarkers from many sites at about the samefrequency has been lost (Table 2). R68.45 con-tains an extra piece of DNA of about 1.5 mdcompared to the parent R68 plasmid (16), andprobably this DNA segment is responsible forthe Cma of R68.45 (15). From the restrictionpattern of the plasmids (two restriction frag-ments present in R68.45 are missing from pGY1;Table 4), one possible explanation for the alteredchromosome transfer ofpGYl might be the par-tial or total elimination or inactivation of theoriginal Cma and the presence of a portion ofthe bacterial chromosome. On the basis of thisobservation we believe that by creating homol-ogy between a sex factor and the chromosomeone can establish a polarized gene transfer sys-tem from a preferred origin. However, it is notexcluded that an additional plasmid functionwhich participates in the chromosome mobili-zation from many chromosomal sites at aboutthe same frequency is also required for the po-larized gene transfer of preferred origin, basedon homology. Because RP4 does not mobilizethe chromosome in Rm4l, chromosome mobili-zation experiments with RP4-primes con-structed in vitro and carrying chromosomal seg-ment of Rm4l are in progress to test this hy-pothesis.

The presence ofthe prophage 16-3 attachmentsite on pGY1 allowed the introduction of pro-phage 16-3 into E. coli and other strains ofRhizobium resistant to this phage. Although theintact prophage was present in E. coli, the pro-duction of mature phage was not observed. Bac-teriophage 16-3 has been studied for severalyears in our labs, and the genetic map (34),transfection (25), specialized transduction (37),and physical map for several restriction enzymes(Dallmann et al., Mol. Gen. Genet., in press) areestablished. pGY2 provides a way to eliminatevarious restriction endonuclease sites on phage16-3. By using these derivatives, vectors similarto the EK2 derivatives of bacteriophage lambda(26) could be constructed. The usefulness ofthese vectors for studying the symbiotic nitrogenfixing bacterium R. meliloti is obvious.

ACKNOWLEDGMENTSWe thank all colleagues who provided bacterial strains,

enzymes, and phage DNA. We are also extremely grateful toC. Koncz, A. Udvardy, I. Boros, and R. K. Storms for theirhelp and valuable discussions.

This work was supported by the Hungarian Academy ofSciences and in part by OMFB grant 79978/28/22-2724.

LITERATURE CITED1. Bachmann, B. J., K. B. Low, and A. L Taylor. 1976.

Recalibrated linkage map of Escherichia coli K-12.Bacteriol. Rev. 40:116-167.

2. Baron, L S., P. Gemski, Jr., E. M. Johnson, and J. A.Wohlhieter. 1968. Intergeneric bacterial matings. Bac-teriol. Rev. 32:362-369.

3. Barth, P. T. 1970. Plasmid RP4, with Escherichia coliDNA inserted in vitro, mediates chromosomal transfer.Plasmid 2:130-136.

4. Beringer, J. E., J. L, Beynon, A. V. Buchanon-Wol-laston, and A. W. B. Johnston. 1978. Transfer of thedrug-resistance transposon Tn5 to Rhizobium. Nature(London) 276:633-635.

5. Beringer, J. E., S. A. Hoggan, and A. W. B. Johnston.1978. Linkage mapping in Rhizobium leguminosarumby means ofR plasmid-mediated recombination. J. Gen.Microbiol. 104:201-207.

6. Cannon, F. C., R. A. Dixon, and J. I. Postgate. 1976.Derivation and properties of F-prime factors in Esche-richia coli carrying nitrogen fixation genes from Kleb-siella pneumoniae. J. Gen. Microbiol. 93:111-125.

7. Casse, F., C. Boucher, J. S. Julliot, M. Michel, and J.Denarie. 1979. Identification and characterization oflarge plasmids in Rhizobium meliloti using agarose gelelectrophoresis. J. Gen. Microbiol. 113:229-242.

8. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E.Hartman. 1966. A proposal for a uniform nomenclaturein bacterial genetics. Genetics 54:61-76.

9. Dixon, R. A., F. C. Cannon, and A. Kondorosi. 1976.Construction of a P plasmid carrying nitrogen fixationgenes from Klebsiella pneumoniae. Nature (London)260:268-271.

10. Eckhardt, T. 1978. A rapid method for the identificationof plasmid desoxyribonucleic acid in bacteria. Plasmid1:584-588.

11. Haas, D., and B. W. Holloway. 1976. R factor variantswith enhanced sex factor activity in Pseudomonasaeruginosa. Mol. Gen. Genet. 144:243-251.

12. Hedges, R. W., and A. E. Jacob. 1977. In vivo translo-cation of genes of Pseudomonas aeruginosa onto a

VOL. 141, 1980

on August 8, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

128 KISS ET AL.

promiscuously transmissible plasmid. FEMS Microbiol.Lett. 2:15-19.

13. Hedges, R. W., A. E. Jacob, and L P. Crawford. 1977.Wide ranging plasmid bearing the Pseudomonasaeruginosa tryptophane synthase genes. Nature (Lon-don) 267:283-284.

14. Holloway, B. W. 1978. Isolation and characterization ofan R' plasmid in Pseudomonas aeruginosa. J. Bacte-riol. 133:1078-1082.

15. Holloway, B. W. 1979. Plasmids that mobilize bacterialchromosome. Plasmid 2:1-19.

16. Jacob, A. E., J. M. Cresswell, and R. W. Hedges. 1977.Molecular characterization of the P group plasnid R68and variants with enhanced chromosome mobilizingability. FEMS Microbiol. Lett. 2:15-19.

17. Jacob, A. E., J. M. Cresawell, R. W. Hedges, J. N.Coetzee, and J. E. Beringer. 1976. Properties of plas-mids constructed by the in vitro insertion ofDNA fromRhizobium leguinosarum and Proteus mirabilis intoRP4. Mol. Gen. Genet. 147:315-323.

18. Jacob, F., and E. L. Wollmann. i961. Sexuality and thegenetics of bacteria. Academic Press Inc., New York.

19. Johnston, A. W. B., M. J. Bibb, and J. E. Beringer.1978. Tryptophane genes in Rhizobium-their organi-zation and their transfer to other bacterial genera. Mol.Gen. Genet. 165:323-330.

20. Johnton, A. W. B., S. M. Setchell, and J. E. Beringer.1978. Interspecific crosses between Rhizobium legumi-nosarum and R. meliloti: formation of haploid recom-binants and of R-primes. J. Gen. Microbiol. 104:209-218.

21. Julliot, J. S., and P. Boistard. 1979. Use of RP4-primeplasmids constructed in vitro to promote a polarizedtransfer of the chromosome in Escherichia coli andRhizobium meliloti. Mol. Gen. Genet. 173:289-298.

22. Kies, G. B., E. Vincze, Z. Kaiman, T. Forral, and A.Kondorosd. 1979. Genetic and biochemical analysis ofmutants affected in nitrate reduction in Rhizobiummeliloti. J. Gen. Microbiol. 113:105-118.

23. Kondorosi, A., L Barabas, Z. Svab, L Orosz, T. Sik,and R. D. Hotchkiss. 1973. Evidence for commongenetic determinants of nitrogenase and nitrate reduc-tase in Rhizobium meliloti. Nature (tondon) 246:153-154.

24. Kondorosi, A., G. B. Kiss, T. Forrai, E. Vineze, and

Z. Banfalvi. 1977. Circular linkage map of Rhizobiummeliloti chromosome. Nature (London) 268:525-527.

25. Kondorosi, A., L Orosz, Z. Svab, and T. Sik. 1974.Genetic studies on rhizobiophage 16-3. II. Helper-in-duced transfection. Mol. Gen. Genet. 132:153-163.

26. Leder, P., D. Tiemeier, and L Enquist. 1977. EK2derivatives of bacteriophage lambda useful in the clon-ing ofDNA from higher organism: the Agt WES system.Science 196:175-177.

27. Low, K. B. 1968. Formation of merodiploids in matingswith a class of Rec- recipient strains of Escherichia coliK-12. Proc. NatL. Acad. Sci. U.S.A. 60:160-167.

28. Low, K. B. 1972. Escherichia coli K-12 F-prime factors,old and new. Bacteriol. Rev. 36:587-607.

29. Meade, H. M., and E. R. Signer. 1977. Genetic mappingofRhizobium meliloti. Proc. Natl. Acad. Sci. U.S.A. 74:2076-2078.

30. Mergeay, M., and J. Gerits. 1978. F'-plasmid transferfrom Escherichia coli to Pseudomonas fluorescens. J.Bacteriol. 135:18-28.

31. Novick, R. P., R. C. Cloves, S. N. Cohen, R. Curtissm, N. Datta, and S. Falkow. 1976. Uniform nomen-clature for bacterial plasmids: a proposal. Bacteriol.Rev. 40:168-189.

32. Olsen, R. IL, and C. Gonzalez. 1974. Escherichia coligene transfer to unrelated bacteria by histidine operon-RP1 drug resistance plasmid complex. Biochem. Bio-phys. Res. Commun. 59:377-385.

33. Orosz, L, and T. Sik 1970. Genetic mapping of rhizo-biophage 16-3. Acta Microbiol. Acad. Sci. Hung. 17:185-194.

34. Oro.z, L, Z. Svab, A. Kondorosi, and T. Sik. 1973.Genetic studies on rhizobiophage 16-3. I. Genes andfunctions on the chromosome. Mol. Gen. Genet. 125:341-350.

35. Ou, J. T., and T. F. Anderson. 1976. F'-plasmids fromHfrH and HfrC in recA- Escherichia coli. Genetics 83:633-643.

36. Pittard, J., and E. A. Adelberg. 1963. Gene transfer byF' strains of Escherichia coli K-12. II. Interaction be-tween F-merogenote and chromosome during transfer.J. Bactenol. 85:1402-1408.

37. Svab, Z., A. Kondorosi, and L Oros 1978. Specializedtransduction of a cystein marker by Rhizobium melilotiphage 16-3. J. Gen. Microbiol. 106:321-327.

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