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 o Plasmid R68.45 o Cons uc ing a Ci cula Linkage Map o he Rhizobium i olii Ch omosome MANUEL MEGIAS,1 MIGUEL A. CAVIEDES,' ANTONIO J. PALOMARES,l* AND JULIO PEREZ- SILVA2 Depa amen o de Mic obiologia, Facul ad de Fa macia,' and Depa amen o de Mic obiologia, Facul ad de Biologia,2 Uni e sidad de Se illa, Se illa, Spain Recei ed 10 Ma ch 1981/Accep ed 29 June 1981 Plasmid R68.45 was used o p omo e conjugal ans e o ch omosomal ma ke s in Rhizobium i olii RS55. Analysis o wo- ac o and h ee- ac o c osses among R. i olii s ains enabled cons uc ion o a ci cula linkage map o he R. i olii ch omosome, con aining 17 nu i ional and esis ance ma ke s. The economic ad an ages ha may acc ue om he imp o emen o ni ogen- ixing sys- ems ha e become inc easingly e iden du ing he pas decade. One o he mos in e es ing ni ogen- ixing sys ems is he Rhizobium-legume symbiosis because o i s high ag icul u al impo - ance. The pas se e al yea s ha e wi nessed impo an de elopmen s in Rhizobium gene ics (3, 7), and a numbe o sui able p ocedu es o he gene ic manipula ion o hizobia ha e e- cen ly been de eloped (4-6, 8, 9, 17, 18, 20, 23, 24). Fo many yea s, one o he majo limi a ions o gene ic analysis o hizobia was he lack o known indigenous ch omosome-mobilizing plas- mids ha would allow gene ans e s udies. This di icul y has been o e come by ans e - ing b oad-hos - ange plasmids wi h ch omo- some-mobilizing abili y (Cma) o Rhizobium spp. To da e, he Pseudomonas ae uginosa d ug- esis ance plasmids R68.45 (13) and RP4 (12) ha e been he plasmids o choice o ob aining gene ans e in Rhizobium. R68.45 is e y e i- cien o mobilizing ch omosomal ma ke s in R. melilo i (10, 21) and R. leguminosa um (5) as well as in o he g am-nega i e bac e ia (14, 26). In bo h R. melilo i and R. leguminosa um, R68.45 p omo es nonpola ized ch omosomal ans e om a numbe o o igins (4, 5, 10, 21). In hose species o Rhizobium so a examined, R68.45 can ans e agmen s o ch omosome long enough o enable accu a e mapping o a a ie y o ma ke s and o es ablish ch omosome ci cula i y (5, 10, 21). RP4 is a less e icien sex ac o (15). Howe - e , i has been success ully used o media ing ans e o ch omosomal ma ke s in R. melilo i (22). To da e, he only epo on linkage mapping s udies in R. i olii was an a emp a using co- mu a ion echniques (29). As an al e na i e ap- p oach, we desc ibe he e he use o plasmid R68.45 o p omo ing ch omosomal ans e in R. i olii. Linkage mapping expe imen s epo - ed he e ha e led o he cons uc ion o a ci cula linkage map o R. i olii con aining 17 nu i ion- al and esis ance ma ke s. MATERIALS AND METHODS Bac e ial sains. The s ains used a e lis ed in Table 1. Plasmid. R68.45, belonging o he P1 incompa ibili y g oup and media ing esis ance o ampicillin, kanamy- cin, and e acycline, was used (13). Media and g ow h condi ions. Esche ichia coli was cul u ed on nu ien b o h (Oxoid); when equi ed in solidi ied o m, aga (Oxoid) was added a 10 g/li e . Fo R. i olii s ains comple e medium was YT (2) and minimal medium was ha o Vogel and Bonne (27) supplemen ed wi h 19 g o glucose pe li e and 12 ,ug o bio in pe ml. When equi ed, amino acid and base supplemen s we e used a a inal concen a ion o 1 mM. E. coli was always g own a 35°C. R. i olii was always g own a 28(C. An ibio ics. Excep o i ampin, which was dis- sol ed in me hanol and added un il e ed, an ibio ics we e added o aga media as il e -s e ilized aqueous solu ions. Concen a ions used we e as ollows: s ep- omycin sul a e (Sigma), 1 mg/ml; kanamycin sul a e (Sigma), 25 pg/ml; oxy e acycline (Sigma), 10 .g/ml; i ampin (Lepe i ), 20 ,ug/ml; ampicillin (Sigma), 10 Ag/ ml, and cana anine sul a e (Sigma), 80 ,ug/ml. Ma ings. Ma ings we e pe o med on Sa o ius il- e s (0.45-,m po e size) as desc ibed by Jacob e al. (16). One millili e o log-phase dono and 1 ml o s a iona y-phase ecipien (bo h con aining a leas 108 cells pe ml) we e ma ed on he memb ane. Mem- b anes we e placed on he su ace o nu ien aga pla es and incuba ed o abou 20 h a 28°C. Then ma ing mix u es we e esuspended in S ml o T is-sal s bu e , pH 7.2, dilu ed, and pla ed on selec i e media. Linkage mapping o he R. I oii ch omosome. Recombinan colonies o a selec ed ma ke we e picked on o selec i e media o de e mine cohe i ance o unselec ed ma ke s. Each pai o ma ke s was analyzed by sco ing 150 o 300 colonies. I was as- sumed ha linkage alues we e in e sely ela ed o 59 TABLE 1. Bac e ial s ainsa S ain Rele an cha ac e is ics Re e ence o sou ce E. coli 1230 P o- Me - (R68.45) J. E. Be inge R. i olii RS55 Wild- ype Isola ed om clo e oo nodules RS176 s -l This pape RS225 his-6 s -l This pape RS235 his-6 me -36 s -l This pape RS238 his-6 phe-18 s -l This pape RS241 i -6 ade-17 This pape RS246 i4 -6 ade-29 This pape RS271 s -l leu-23 This pape RS288 his-6 y -28 s -l This pape RS290 his-6 h -14 s -l This pape RS292 his-6 pdx-3 s -l This pape RS293 i -6 ade-29 y -13 This pape RS294 i -6 ade-29 h -27 This pape RS295 i -6 ade-29 a g-19 This pape RS296 his-6 me -31 s -I can-3 This pape RS230 (R68.45) his-6 s -l C oss 1230 x RS225 RS278 (R68.45) i -6 ade-17 C oss 1230 x RS241 I Allelle numbe s a e a bi a y. Gene symbols a e hose o Bachmann and Low (1). All he R. i olii s ains a e de i ed om he wild- ype RS55, isola ed in ou labo a o y om T i olium epens L. nodules. Auxo ophic mu a ions we e induced by ei he N-me hyl-N'-ni o-N-ni osoguanidine o e hyl me hane sul ona e mu agene- sis. Ni osoguanid ne ea men s we e ca ied ou as ollows: a log cul u e con aining abou 10' cells pe ml was cooled, washed wice wi h T is-malea e bu e , pH 7.5, and esuspended in 500 mg o ni osoguanidine (Sigma) pe li e o 30 min. Then he cells we e ha es ed and ca e ully washed wi h bu e be o e pla ing on YT aga . Fo e hyl me hane sul ona e mu agenesis, a cul u e con aining abou 4 x 10' cells pe ml was washed wice wi h phospha e bu e , pH 8.0, and suspended in 0.1 M e hyl me hane sul ona e (Sigma). A e incuba ion a 30°C o 45 min, 6% (w / ol) Na2S203 was added. Cells we e ha es ed, washed, and pla ed on YT. D ug- esis ance mu a ions we e spon aneous. physical dis ances be ween genes. Linkage alues we e ans o med in o addi i e map dis ances by using he equa ion de i ed by Kempe (19): C = (1 - ) + (Qn ) whe e C is he linkage equency alue and is he map dis ance. RESULTS T anse o plasd R68.45 om E. coli o R. & ol. Plasmid R68.45 was easily ans e ed om E. coli 1230 o R. i olii RS176 in mem- b ane ma ings. T ansconjugan s we e selec ed on minimal medium wi h an ibio ics. When he ecipien s ain was one o he auxo ophic de- i a i es o RS176 desc ibed abo e, selec i e medium was supplemen ed wi h he equi e- men s o he ecipien s ain. In hese c osses he equency o plasmid ans e was abou 10- pe dono bac e ium. This equency is sligh ly lowe han hose e- TABLE 2. R68.45 ans e and ch omosome mobiliza ion in R. i olii RS176 Dono Recipien F equency o Selec ed F equency o R ans e 0 pheno ype ecombinan s' RS230 RS241 2.8 x 10-1 Ade+ 2.7 x 10-4 S 8.9 x 1o-4 RS230 RS294 4.3 x 10-1 Th + 1.1 X 1O-4 Ade+ 1.1- xi10-5 RS278 RS235 4.2 x 10-1 Me + 3.0 x 1O-5 His+ 9.2 x 10-4 RS278 RS288 8.3 x 10-1 His+ 1.1 x 10-4 Ty + 1.6 x 1O-4 Ri 6.0 x 10-5 RS278 RS271 5.8 x 10-1 Leu+ 1.1 x 1O-4 Ri 7.0 x 1O-4 RS278 RS292 3.3 x 10-1 Pdx+ 1.7 x 1O-4 a Pe dono bac e ium. (O MEGIAS ET AL. J. BACTERIOL. CIRCULAR LINKAGE MAP OF R. TRIFOLII TABLE 3. De ec ion o R68.45 ma ke s in ansconjugan s ha had ecei ed ch omosomal ma ke s C oss ~~Selec ed No. o R R%R C oss pheno ype clones es eda % R RS230 x RS293 Ade+ 204 198 6 3 RS278 x RS288 Ty ' 163 160 3 2 RS278 x RS238 His+ 113 112 1 1 RS230 x RS271 S 199 197 2 1 a Replica pla ed on an ibio ic medium. po ed o E. coli x R. leguminosa um and E. R68.45 was able o p omo e ch omosome ans- coli x R. melilo i c osses (5, 10). e a a a ie y o o igin si es as p e iously F om hese c osses R' ansconjugan s o R. epo ed o o he hizobia (4, 5, 10, 21). i olii we e isola ed o be used as u he dono s When he p esence o plasmid R68.45 was in in aspeci ic c osses. All isola es we e pu i- in es iga ed among he ecombinan s, mos o ied wo o h ee imes be o e being s o ed. hem p o ed o be R' a leas o esis ance In aspeci ic ma ings: ans e o plasmid ma ke s (see Table 3). This sugges ed ha ch o- R68.45 and mobiliza ion o ch omosomal ma ke s mosomal ans e media ed by plasmid R68.45 in R. i ol i. As shown in Table 2, he equency was F'-like a he han H -like. The same phe- o plasmid ans e in in aspeci ic ma ings was nomenon has been desc ibed in R. legumino- highe han in in e gene ic c osses. T ans e sa um and R. melilo i (4, 10, 21). equencies desc ibed he e a e simila o hose Linkage analysis. The a ionale o linkage epo ed o o he Rhizobium species, such as R. s udies based upon R68.45-media ed gene ans- melilo i (9, 21), R. leguminosa um (4, 5), and e can be summa ized as ollows: since any in e speci ic c osses o R. leguminosa um x R. ma ke has a ela i ely de ined equency o melilo i (17). ans e , he chance o a pai o ma ke s being Mobiliza ion o single ch omosomal ma ke s co ans e ed in he same c oss will depend on occu ed a equencies anging om abou 5 x he dis ance be ween hem. 10-5 o 5 x 10-3 pe dono cell. Absence o Table 4 shows he esul s ob ained in a se ies g ea di e ences among he equencies o mobi- o wo- ac o c osses. In each ma ing ini ial liza ion o di e en ma ke s indica ed ha selec ion was ca ied ou o a single ma ke . TABLE 4. Linkage analysis o se e al ma ke pai s o R. i olii RS176 Dono Recipien Selec ed Ma ke s Linkage Dis ance pheno ype pai s (C) ( ) RS230 RS246 j i -6 ade-29 0.41 0.24 i -6 s -l 0.21 0.42 Ade+ ade-29 s -1 0.04 0.73 RS230 RS293 ade-29 y -13 0.39 0.26 Ri F i -6 y -13 0.79 0.05 Ty ' y -13 s -l 0.17 0.47 RS230 RS294 Th + h -18 ade-29 0.75 0.06 h -18 i -6 0.69 0.09 RS230 RS295 Ade+ ade-29 a g-19 0.65 0.10 RM i -6 a g-19 0.25 0.38 RS278 RS235 His+ his-6 me -36 0.40 0.25 Me + me -36 j -6 0.03 0.76 RS278 RS238 Phe+ phe-18 his-6 0.04 0.73 phe-18 i -6 0.16 0.49 RS278 RS271 Leu+ leu-23 i -6 0.04 0.73 Ade+ ade-29 leu-23 0.47 0.20 RS278 RS290 Th + h -27 his-6 0.42 0.23 S s -l h -27 0.09 0.60 Ri T n -6 h -27 0.70 0.08 RS278 RS292 Pdx+ pdx-3 s -l 0.27 0.36 pdx-3 his-6 0.03 0.76 RS278 RS296 Can can-3 his-6 0.16 0.49 can-3 me -31 0.54 0.16 His+ his-6 me -31 0.35 0.29 Me + me -31 i -6 0.12 0.55 61 VOL. 149, 1982 TABLE 5. Th ee- ac o c osses be ween R. i olii s ains Dono Recipien Selec ed Ma ke s Linkage Dis ance pheno ype pai s (C) ( ) RS278 RS288 His' his-6 y -28 0.43 0.23 his-6ade-17 0.06 0.67 Ty ' y -28 his-6 0.46 0.21 y -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 S s -l ade-17 0.19 0.45 Leu+ leu-23 ade-17 0.03 0.76 RS230 RS293 Ty ' y -13 ade-29 0.28 0.35 y -13 his-6 0.23 0.40 Ade+ ade-29 y -13 0.26 0.37 ade-29 his-6 0.03 0.76 Co ans e o unselec ed ma ke s was de ec ed RP1, RP4, Rld dl9, and R68.45 (M. Meglas, by eplica pla ing on app op ia e selec i e me- Ph.D. hesis, Uni e si y o Se illa, Se illa, dia. Linkage equencies lowe han 0.03 we e Spain, 1981). Howe e , as in o he Rhizobium disca ded. Dis ances we e calcula ed by using species, he mos e icien plasmid wi h ch omo- he Kempe equa ion as desc ibed abo e. some-mobilizing abili y is R68.45 (15; Megias, Resul s ob ained in h ee- ac o c osses a e Ph.D. hesis, 1981). summa ized in Table 5. Usual equencies o mobiliza ion o ch omo- A diag am ha summa izes he linkage ela- somal ma ke s by R68.45 a e highe han e e - ionships among 17 ch omosomal ma ke s is sion a es. Fo ins ance, all he auxo ophic p esen ed in Fig. 1. Numbe s abo e he a ows mu a ions epo ed in his pape had e e sion indica e linkage equencies ound in indepen- equencies lowe han 10-6, whe eas he den expe imen s. This p elimina y linkage map chance o de ec ing single-ma ke ans e in an can be ci cula ized and ep oduced o scale as R68.45-media ed c oss was 10-4 o 10-5 pe shown in Fig. 2. dono cell. Thus, accu a e measu es o ans e DISCUSSION equencies may be eadily ob ained. F equency anges desc ibed in his pape a e simila o Mobiliza ion o he R. i olii ch omosome can hose epo ed o R. melilo i (10, 21) and o he be achie ed by using se e al plasmids, such as o iginal hos o R68.45, P. ae uginosa (13). Iu-23 ad.-17 pdx-3 can-3 me -36 me -31 y -28 s -1 his-6 hi-l y -13 i -0 h -27 h -18 ade-29 . g-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. P elimina y linkage map o he R. i olii RS176 ch omosome. Numbe s beside gene symbols indica e allele numbe s. Numbe s on he a ows indica e conjugal linkage pe cen ages. 62 MEGIAS ET AL. J. BACTERIOL. CIRCULAR LINKAGE MAP OF R. TRIFOLII FIG. 2. Ci cula linkage map o he R. i olii RS176 ch omosome. Dis ances a e abou in e sely p opo ional o he linkage equencies. Minimal link- age alues (3%) would ep esen , a leas , one- ou h o he o al leng h o he ch omosome. Ano he ad an age o using plasmid R68.45 o mapping pu poses in Rhizobium spp. is he la ge size o ch omosomal agmen s mobilized by he plasmid. Subsequen ly, simul aneous ans e o dis an ma ke s may be eadily de- ec ed in app op ia e c osses. Fo ins ance, ma ke pai s showing low linkage equencies, such as his-61phe-18, me -361 i -6, and his-61pdx- 3, may be loca ed as a apa as one- ou h o he o al leng h o he ch omosome. These e- sul s ag ee wi h hose epo ed o o he Rhizo- bium species (4, 10, 21). T ans o ma ion o linkage measu es in o addi- i e map dis ances is easily ob ained by use o o mulas such as ha o Wu (28) o ha o Kempe (19). These equa ions we e i s p o- posed o use in co ansduc ion expe imen s, bu hey a e also sui able o conjugal mapping (21; Meg as, Ph.D. Thesis, 1981). We ha e cho- sen o use Kempe 's equa ion because i does no equi e any assessmen o he leng h o ans e ed ch omosomal segmen s. Unlike R. melilo i (10, 11, 25) and R. legumin- osa um (8) gene al ansduc ion sys ems ha e no ye been desc ibed o R. i olii. Thus, conjugal mapping media ed by plasmid R68.45 is he only me hod now a ailable o ex ensi e mapping s udies in R. i olii. We hope i will be sui able o u he gene ic analysis o ni ogen- ixing clo e symbiosis. ACKNOWLEDGMENTS We a e g a e ul o John Be inge o p o iding plasmid R68.45 and o Josep Casades s o help ul sugges ions du ing he p epa a ion o he manusc ip . LITERATURE CITED 1. Bachmann, B. J., and K. B. Low. 1980. Linkage map o Esche ichia coli K-12, edi ion 6. Mic obiol. Re . 44:1-56. 2. Be inge , J. E. 1974. R- ac o ans e in Rhizobium leguminosa um. J. Gen. Mic obiol. 84:188-198. 3. Be inge , J. E. 1980. The de elopmen o Rhizobium gene ics. J. Gen. Mic obiol. 116:1-7. 4. Be lnge , J. E., S. A. Hoggan, and A. W. B. Johns on. 1978. Linkage mapping in Rhizobium leguminosa um by means o R plasmid-media ed ecombina ion. J. Gen. Mic obiol. 104:201-207. 5. 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