Transfer of a genetic marker from a megaplasmid of Anabaena sp. strain PCC 7120 to a megaplasmid of a different Anabaena strain

JOURNAL OF BAC ERIOLOGY, Feb. 1994, p. 1093-1098 Vol. 176, No. 4 0021-9193/94/$04.00 + 0 Copy igh © 1994, Ame ican Socie y o Mic obiology T ans e o a Gene ic Ma ke om a Megaplasmid o Anabaena sp. S ain PCC 7120 o a Megaplasmid o a Di e en Anabaena S ain ALICIA M. MURO-PASTOR,' TANYA KURITZ,2 ENRIQUE FLORES,l* ANTONIA HERRERO,' AND C. PETER WOLK2 Ins i u o de Bioquimica Vege al y Fo os n esis, Uni e sidad de Se illa-CSIC, Facul ad de Biologia, Apa ado 1113, E-41080 Se ille, Spain,' and MSU-DOE Plan Resea ch Labo a o y and Cen e o Mic obial Ecology, Michigan S a e Uni e si y, Eas Lansing, Michigan 488242 Recei ed 4 Augus 1993/Accep ed 7 Decembe 1993 The 410-kb a megaplasmid o he he e ocys - o ming cyanobac e ium Anabaena sp. s ain PCC 7120 was ound o bea he nucA gene ha encodes a suga -nonspeci ic nuclease. Tha gene was mu a ed by inse ion o a casse e ha con e s esis ance o neomycin. The esul ing s ain, AMP2, was ma ed wi h a s ep omycin- esis an de i a i e o Anabaena sp. s ain PCC 7118, a s ain ha does no o m he e ocys s. Cells esis an o bo h neomycin and s ep omycin ha we e de i ed om such ma ings we e ound o bea he neomycin esis ance casse e o he dono s ain in a la ge megaplasmid cha ac e is ic o he ecipien s ain and did no o m he e ocys s. This is he i s example o ans e o a gene ic ma ke di ec ly be ween s ains o cyanobac e ia in which incon o e ible physical e idence o ans e has been ob ained. DNA sequences homologous o he nucA gene we e p esen in 13 he e ocys - o ming cyanobac e ia ha we e es ed bu in none o six di e se unicellula s ains ha we e examined. The cyanobac e ia a e pho o ophic eubac e ia ha can ca y ou oxygenic (plan - ype) pho osyn hesis. Many ilamen- ous cyanobac e ia a e able o ix dini ogen in specialized cells called he e ocys s ha de elop a semi egula in e als along he ilamen s. We ha e ecen ly shown ha Anabaena sp. s ain PCC 7120, a he e ocys - o ming cyanobac e ium, p oduces an exocy oplasmic suga -nonspeci ic nuclease ha is able o de- g ade linea o co alen ly closed ci cula double-s anded DNA as well as single-s anded DNA o RNA (16). The gene, nucA, encoding his enzyme has been cloned and sequenced (16). In his epo , we show ha nucA is loca ed on he o megaplasmid, whose p esence in s ain PCC 7120 had been epo ed p e iously (4). A casse e con e ing esis ance o neomycin (NM), when in oduced in o he nucA gene o s ain PCC 7120, could he eupon be ans e ed o a la ge megaplasmid o a s ep omycin (SM)- esis an de i a i e o Anabaena sp. s ain PCC 7118. We ha e also ound ha he nucA gene is p esen in nume ous he e ocys - o ming cya- nobac e ia. MATERIALS AND METHODS O ganisms and cul u e condi ions. Cyanobac e ial s ains we e g own pho oau o ophically a 30°C in BG11 medium (18) wi h shaking. Whe e speci ied, BG11 medium was solid- i ied by he addi ion o 1% sepa a ely au ocla ed Bac o Aga (Di co). Fo es ing g ow h on dini ogen, BG11 lacking NaNO3 was used. Fo g ow h o de i a i e AMP2 o Anabaena sp. s ain PCC 7120, liquid BG11 medium was supplemen ed wi h 5 jig o NM pe ml and solidi ied BG11 medium was supplemen ed wi h 25 jig o NM pe ml. De i a i e NW18 o Anabaena sp. s ain PCC 7118 was g own in BG11 medium * Co esponding au ho . Telephone: 34-5-455.70.86. Fax: 34-5- 462.01.54. Elec onic mail add ess: [email p o ec ed]. P esen add ess: Depa men o Mic obiology, Uni e si y o Illi- nois a U bana-Champaign, U bana, IL 61801. supplemen ed wi h 20 jig o SM pe ml. Nm Sm de i a i es o s ain NW18 we e g own in BG11 medium supplemen ed wi h a combina ion o 20 jig o SM pe ml and NM ei he a 5 jig/ml in liquid medium o 25 ,ug/ml in solid medium. Chlo ophyll a was de e mined in me hanolic ex ac s (13). Esche ichia coli DH5o , used o all plasmid cons uc ions, was g own in Lu ia b o h as desc ibed p e iously (14). P epa a ion and manipula ion o DNA. To al DNA om cyanobac e ia was isola ed as desc ibed by Cai and Wolk (6). DNA o high molecula weigh om Anabaena sp. s ains PCC 7120 and PCC 7118 and hei de i a i es was p epa ed and cu wi h es ic ion endonucleases, and he esul ing plasmids we e subjec ed o pulsed homogeneous o hogonal ield gel elec o- pho esis (PHOGE) (2) as desc ibed by Ku i z e al. (11). Bands wi h a size o less han 50 kb we e measu ed by using HindIll diges s o coliphage lambda DNA as s anda ds in pulsed- ield gels (11). Plasmid isola ion om E. coli, ans o ma ion o E. coli, es ic ion, and liga ion wi h T4 DNA ligase we e pe o med by s anda d p ocedu es (1, 14). DNA agmen s we e pu i ied om aga ose gels wi h he GeneClean ki (Bio 101, Inc.). The nucA-con aining hyb idiza ion p obes we e ei he plasmid pRLA1, which ca ies 12 kb o s ain PCC 7120 genomic DNA including he nucA gene and lanking sequences, o a 473-bp HindIII-Bs NI agmen in e nal o nucA, isola ed om pCSAM29 (16). The neomycin phospho ans e ase (np ) p obe used was casse e C.K3 (7) excised wi h BamHI. DNA p obes we e 32P labeled wi h [oL-32P]dCTP by using a nick ansla ion ki (Boeh inge Mannheim) o wi h [a_-32P]dATP by using a andomly p imed DNA labeling sys em ki (Be hesda Re- sea ch Labo a o ies) o we e digoxigenin labeled wi h a Ge- nius II ki and he signal was enhanced by Lumi-Phos 530 (Boeh inge Mannheim). Sou he n blo ing o o al DNA om cyanobac e ia o Hybond-N+ memb anes (Ame sham) was pe o med unde alkaline condi ions (14). Hyb idiza ion was ca ied ou unde low-s ingency condi ions a 55°C as de- sc ibed p e iously (1) o unde high-s ingency condi ions a 1093 on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om 1094 MURO-PASTOR ET AL. J. BAC ERIOL. 65°C as desc ibed in he ecommenda ions o he manu ac u e o Hybond-N+ memb anes by using 5 mM KHP04 bu e (pH 7.2) con aining 1 mM EDTA and 0.2% sodium dodecyl sul a e (SDS) o wash he il e s. PHOGE- esol ed agmen s o DNA we e blo ed o GeneSc een memb anes (DuPon ), and hy- b idiza ion was ca ied ou as desc ibed p e iously (4, 11). Plasmid RP-4 was diges ed wi h EcoRV be o e labeling wi h 32p. Hyb idiza ion o 2P-labeled RP-4 wi h HindIII o EcoRV diges s o genomic DNA om s ains AMP2 and PCC 7120 was ca ied ou unde high-s ingency condi ions. Ma ings be ween cyanobac e ia. Cells o s ain NW18 and o a dono s ain ca ying he np gene in eg a ed in i s genome we e ha es ed and washed wi h BG11 medium by low-speed cen i uga ion, mixed, and sp ead on Nuclepo e REC-85 il e s se a op pla es o solid BG11 medium. The dono s ain used in mos expe imen s was AMP2 (16), bu s ain EFI 16(pRL52) (9) was also ied. An amoun o cells con aining abou 4 ,ug o chlo ophy!l o he dono s ain and 8 ,ug o chlo ophyll o s ain NW18 was inocula ed pe pla e (1 ,ug o chlo ophyll co e- sponds o ca. 3.3 x 106 cells o hese cyanobac e ia). A e 48 h o incuba ion unde g ow h condi ions, he il e s we e ans e ed o pla es o BG11 medium supplemen ed wi h 25 ,ug o NM pe ml and 20 p,g o SM pe ml and u he incuba ed o 2 o 4 weeks. Nuclease assays. Nuclease assays in DNA-con aining SDS- polyac ylamide gel elec opho esis (SDS-PAGE) gels we e pe o med essen ially as desc ibed p e iously (19) bu wi h modi ica ions (16). Samples we e subjec ed o elec opho esis in gels con aining 15 ,ug o cal hymus DNA ( ype IV; Sigma) pe ml. A e elec opho esis, p o eins we e ena u ed by gen le agi a ion o he gels in nuclease bu e , and gels we e incuba ed o e nigh in he same bu e o allow enzyma ic deg ada ion o embedded nucleic acids. The gels we e hen s ained wi h e hidium b omide and pho og aphed unde UV illumina ion. Fo p epa a ion o cell ex ac s o cyanobac e ia, cells (ca. 200 ,ug o chlo ophyll) we e suspended in 0.25 ml o 10 mM T is-HCl bu e (pH 7.5) con aining 1 mM phenylme hylsul o- nyl luo ide and hen subjec ed o eezing in liquid ai in a p echilled mo a and g inding. The homogena e was cen i- uged a 10,000 x g o 10 min. The esul ing supe na an solu ion cons i u ed he cell ex ac . P o ein concen a ion in cell ex ac s was es ima ed as desc ibed p e iously (5). P o ein concen a ion in cell suspensions was es ima ed by he me hod o Ma kwell e al. (15). RESULTS AND DISCUSSION Loca ion o nucA in he a megaplasmid. S ain AMP2 is a de i a i e o Anabaena sp. s ain PCC 7120 in which he nucA gene had been inac i a ed by he inse ion o C.K3, a casse e bea ing he np gene om Tn5 (16). DNA o high molecula weigh was isola ed om PCC 7120 and AMP2 and subjec ed o diges ion wi h Ps I o Sall, and he es ic ion agmen s hus gene a ed we e esol ed as desc ibed p e iously (2). The pa e n o bands (4, 11) ob ained in each o he wo diges ions was iden ical o PCC 7120 and AMP2, excep ha he band co esponding o he o megaplasmid (Fig. 1, a ow a), a plasmid o 410 kb p esen in Anabaena sp. s ain PCC 7120 ha bea s a single si e o Sall (3, 4), was eplaced by bands o ca. 390 (Fig. 1, a ow c) and 19 (no seen in Fig. 1) kb in he Sall diges o AMP2 because o he p esence o a Sall si e nea he end o he np -con aining casse e inse ed in nucA in AMP2. In addi ion, he a megaplasmid om AMP2 is linea - ized by Ps I (Fig. 1, a ow b) because o he in oduc ion o a Ps I es ic ion si e on he inse ed casse e. These esul s 1 2 3 4 b c -500 *- a -400 -300 -200 100 FIG. 1. Al e a ion o Anabaena sp. s ain PCC 7120 a megaplasmid by he inse ion o casse e C.K3 in o he nucA gene encoding a suga -nonspeci ic nuclease. DNA o high molecula weigh om s ains AMP2 (lanes 1 and 2) o PCC 7120 (lanes 3 and 4) was subjec ed o diges ion wi h Ps I (lanes 1 and 3) o Sall (lanes 2 and 4) and esol ed by he PHOGE sys em. A ow a poin s o plasmid ao (410 kb) in Sall-diges ed s ain PCC 7120 DNA, whe eas a ow c poin s o a diges ion p oduc o his plasmid in Sall-diges ed DNA o s ain AMP2. A ow b poin s o plasmid a in Ps I-diges ed DNA o s ain AMP2. The posi ions o se e al size ma ke s (in kilobases), as deduced om a 48.5-kb lambda ladde , a e shown o he igh o he igu e. The e ec o DNA concen a ion on he dis ance o mig a ion was dis- cussed p e iously (11). sugges ed ha he nucA gene was loca ed in he a megaplas- mid. To es his possibili y u he , labeled plasmid pRLA1, which ca ies he nucA gene and lanking DNA, was used o p obe high-molecula -weigh DNA o s ain PCC 7120 ha had been diges ed wi h BlnI, Ps I, o Sall, esol ed by PHOGE, and blo ed. A s ong hyb idiza ion signal esul ed in he band ha ep esen s he linea ized a megaplasmid (Fig. 2). Megaplasmid a, poo ly seen on he PHOGE gels o ei he Ps I o BlnI diges s o DNA om wild- ype PCC 7120 ( he e a e no si es o Ps I o BlnI in he (x megaplasmid), is isualized in Sou he n hyb idiza ions because some megaplasmid linea ized by nicking en e s he gel. Plasmids anging in size om ca. 1.5 kb o 1 Mb ha e been desc ibed in he cyanobac e ia (10, 17, 26). All cyanobac e ial plasmids he e o o e s udied emain c yp ic; i.e., beyond epli- ca ion unc ions (20, 24-26, 30), he e a e no known unc ions encoded by hem excep ha a no mally silen copy o psbG has been ound o be plasmid localized in Synechocys is sp. s ain PCC 6803 (22). Ou da a show ha nucA ep esen s he i s exp essed ma ke o an indigenous cyanobac e ial plasmid (11). T ans e o nucA::C.K3 be ween cyanobac e ial s ains. Many la ge, bac e ial plasmids a e sel - ansmissible by conju- ga ion (27). Conjugal ans e o plasmid DNA om E. coli o Anabaena sp. p oceeds e icien ly (29), bu gene ic ans e be ween cyanobac e ial cells (see, e.g., e e ence 23) has no been unequi ocally demons a ed p e iously. The nucA gene, and he e o e he nucA ::C.K3 cons uc ion p esen in s ain AMP2, ep esen s a ma ke o he a- megaplasmid o Anabaena sp. s ain PCC 7120. Conjuga ion be ween cya- nobac e ia was sough by using his ma ke . We sough o ans e nucA::C.K3 om s ain AMP2 o NW18, an Sm de i a i e o Anabaena sp. s ain PCC 7118. Like i s pa en s ain PCC 7118, s ain NW18 is unable o on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om GENE TRANSFER BETWEEN CYANOBACTERIA 1095 B 1 2 3 4 5 a a 11.38- 4.4- 2.61- 1.7- FIG. 2. Localiza ion o he nucA gene in he a megaplasmid. (A) High-molecula -weigh DNA om Anabaena sp. s ain PCC 7120 was diges ed wi h BlnI (lane 1), Ps I (lane 2), o Sall (lane 3), and he agmen s we e esol ed by PHOGE. Lane 4 con ains lambda DNA oligome s. (B) Hyb idiza ion o he PHOGE- esol ed agmen s wi h 32P-labeled nucA-con aining plasmid pRLA1 is shown. The posi ion o he linea ized a megaplasmid is indica ed. The posi ions o se e al size ma ke s (in kilobases), as deduced om he 48.5-kb lambda ladde , a e shown o he igh o he igu e. de elop he e ocys s o o ix dini ogen unde ae obic condi- ions, whe eas AMP2, like i s pa en PCC 7120, makes he e o- cys s and can g ow on dini ogen unde ae obic condi ions. Anabaena sp. s ain PCC 7120 and i s de i a i e AMP2 ha e he 410-kb megaplasmid men ioned abo e, whe eas de i a i e NW18 o Anabaena sp. s ain PCC 7118 bea s a 510-kb plasmid, de ec ed by pulsed- ield gel elec opho esis o un e- s ic ed DNA (da a no shown). Cells o s ains AMP2 and NW18 we e mixed and sp ead on il e s se a op pla es o solid cyanobac e ial medium (see Ma e ials and Me hods o de- ails). F om a o al amoun o ca. 6.6 x 108 cells o s ain NW18 and ca. 3.3 x 108 cells o s ain AMP2, in se e al expe imen s, 117 Nm Sm colonies we e ob ained. Se en y-six o hese colonies we e es ed o he abili y o g ow on dini ogen unde ae obic condi ions (an unselec ed ma ke ), and all we e ound o be unable o do so, implying ha hey we e Nm de i a i es o s ain NW18 a he han Sm de i a- i es o s ain AMP2. Because we ha e ound ha spon aneous Nm mu an s appea a a equency o less han 10-9 pe cell, Nm de i a i es o s ain NW18 we e expec ed o ha e e- cei ed he C.K3 casse e om s ain AMP2 a he han o ha e a isen om spon aneous mu a ion. Sou he n blo analysis o o al DNA isola ed om se e al Nm Sm de i a i es ha was p obed wi h a 32P-labeled, C.K3-con aining DNA agmen showed ha hose de i a i es con ained he C.K3 casse e (Fig. 3A). In addi ion, a Sou he n blo o o al DNA isola ed om s ains AMP2 and NW18 and i e Nm Sm de i a i es was p obed wi h a 32P-labeled in e nal agmen o he nucA gene (Fig. 3B). The esul s ob ained (see lowe band in each lane) indica e ha he nucA::C.K3 cons uc o iginally p esen in s ain AMP2 ( ha esul s in a Clal band o ca. 5.7 kb) had eplaced he wild- ype nucA gene o s ain NW18 ( ha esul s in a ClaI band o ca. 4.4 kb) in he i e Nm Sm de i a i es ha we e examined. Mo eo e , unlike NW18, hese s ains exhib- i ed no ac i i y o he 29-kDa nuclease in SDS-PAGE assays (Fig. 4). The uppe nucA-hyb idizing bands obse ed in AMP2 FIG. 3. Sou he n analysis o Nm Sm de i a i es. To al DNA om dono s ain AMP2, ecipien s ain NW18, and i e Nm Sm de i a- i es (lanes 1 o 5) was diges ed wi h Clal and hyb idized wi h a 1.1-kb BamHI DNA agmen con aining he TnS np gene in casse e C.K3 (A) o a 473-bp HindIlI-Bs NI DNA agmen in e nal o he nucA gene o Anabaena sp. s ain PCC 7120 (B). Hyb idiza ion was ca ied ou unde condi ions o high s ingency (see Ma e ials and Me hods). The posi ions o se e al size s anda ds (in kilobases) a e shown o he le o he igu e. and NW18 in Fig. 3B do no co espond o a gene ha encodes an ac i e 29-kDa nuclease (16) (Fig. 4). Figu e 5 p esen s a Sou he n blo o PHOGE- esol ed high-molecula -weigh DNA om s ains PCC 7120, AMP2, and NW18, and wo Nm Sm de i a i es, p obed wi h pRLA1 (nucA). The a megaplasmid o Anabaena sp. s ain PCC 7120 has single es ic ion si es o SphI and Sall ha a e abou 105 kb dis an om one ano he (4). In oduc ion o C.K3 in o nucA added a second Sall si e ca. 19 kb dis an om he i s (sizing da a no shown), a Ps I si e a which he plasmid can be linea ized, and a second SphI si e ca. 126 kb dis an om he i s (Fig. 5, lanes 4 and 5; Fig. 1). The 510-kb megaplasmid o s ain NW18 also appea s o ha e single si es o SphI and Sall (no shown) as well as a locus ha hyb idizes wi h pRLA1 (Fig. 0 N co < Z 1 2 3 4 5 29-kDa nuclease FIG. 4. Nuclease ac i i y o cell suspensions om wild- ype Anabaena sp. s ain PCC 7120, dono s ain AMP2, ecipien s ain NW18, and se e al Nm Sm de i a i es (lanes 1 o 5). Cul u e samples con aining 5 o 10 ,ug o p o ein we e used. The loca ion o he 29-kDa nuclease (NucA) is indica ed. A 1 234 A 1 234 ! I: N Co 0z 2 3 4 5 VOL. 176, 1994 1 im. I- 7717. ` 77"77777.. I on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om 1096 MURO-PASTOR ET AL. Sphl Ps l Sall 1 23 4 5 X1 23 4 5 X 1 23 4 5 510 kb ** *** 440 0; i* 0 i * i i ; X;0i- 410kb FIG. 5. Sou he n analysis o PHOGE gel o NM SM de i a i es. Aga ose beads con aining DNA de i ed om NW18 (lanes 1) NM SM de i a i e 2 (lanes 2), NM Sm de i a i e (lanes 3), AMP2 (lanes 4), and wild- ype PCC 7120 (lanes 5) we e subjec ed o PHOGE analysis as desc ibed p e iously (11) a e diges ion wi h Sphl (pa ial diges [ 1 1]), Ps l, o Sall. A Sou he n blo o he gel was p obed wi h pRLAL. X, beads con aining conca eme s o DNA om coliphage lambda; *, size o his agmen measu ed sepa a ely. 5, lanes 1). We shall e e o his locus as he nucA gene o NW18. The NM Sm de i a i es con ain plasmids ha a e app oxima ely equal in size o he ca. 510-kb megaplasmid o NW18 (Fig. 5, lanes 1 o 3; Ps l diges ion). Upon pa ial diges ion wi h Sphl, hese plasmids as well as he smalle a. megaplasmid o AMP2 gi e ise o a agmen o abou 126 kb (Fig. 5, lanes 2 o 4; SphI diges ion) ha measu es he dis ance om SphI in CdK3 (in nucA) o he unique SphI o he megaplasmid. SaIl cu s he same megaplasmids and gi es ise o agmen s wi h a size close o 19 kb (sizing da a no shown). (Bands p esen in lanes 3 ha a e absen om lanes 2 a e o unclea o igin.) Acco ding o hese esul s, he Sphl and Sail si es a e as a om nucA in he la ges megaplasmid o he NM SM de i a i es o s ain NW18 as hey a e in he a megaplasmid o PCC 7120. These esul s sugges ed ha , in he wo NM SM de i a i es in es iga ed, he nucA::C.K3 ma ke had been inco po a ed by ecombina ion in he megaplasmid o s ain NW18. The p esence o he CK3 ma ke in he 510-kb plasmid o he NM SM de i a i es o s ain NW18 was con i med by hyb idiza ion using casse e CaK3 as a p obe. When DNA diges ed wi h Sall was es ed, Cu3 was obse ed o hyb idize (unde he condi ions o hyb idiza ion used o he PHOGE gels) only o he 19-kb band (da a no shown). T ans e o he C a3 casse e om s ain AMP2 o s ain NW18 was no a ec ed by he p esence o bo ine panc ea ic DNase (g ade II; Boeh inge Mannheim), which was added a 7.5 s g/ml bo h o he mix u e o cells be o e hey we e sp ead in he solid medium and o he medium in he pla es. The e- o e, he ans e p esumably did no in ol e DNA ee in he medium. T ans e by conjuga ion is possible, al hough o he mechanisms a e no excluded (21). Because he dono s ain used in he ma ings, AMP2, is i sel an exconjugan , de i ed om Anabaena sp. s ain PCC 7120, which o igina ed in an RP-4-p omo ed conjuga ion wi h E. coli, i was possible ha s ain AMP2 bo e he a genes om RP-4 and ha hese genes we e esponsible o he phenom- enon o DNA ans e be ween cyanobac e ia ha we ha e obse ed. The e o e, we es ed by hyb idiza ion he p esence o DNA sequences om RP-4 in s ain AMP2. No e idence o he p esence o RP-4 o RP-4 agmen s in his s ain was ound (da a no shown). To es whe he ans e occu s speci ically o genes in he a megaplasmid, we sough o ans e he np gene om s ain EF116(pRL52) o s ain NW18. EF116 is a mu an o Anabaena sp. s ain PCC 7120 ha is unable o ix dini ogen unde ae obic condi ions, and pRL52 is an np -bea ing shu le plasmid whose inse complemen s he mu a ion in EF116 (9, 28). In ma ings in ol ing, in se e al expe imen s, a o al amoun o ca. 2.3 x 108 cells o s ain NW18 and ca. 1.15 x 108 cells o s ain EF116(pRL52), no Nm Sm colonies we e ob ained. These nega i e esul s p o ide no e idence ha genes loca ed ou side he ao megaplasmid can be ans e ed. Widesp ead occu ence o nucA in he e ocys - o ming cya- nobac e ia. I was o in e es o es how widesp ead he nucA gene is in cyanobac e ia o he han s ain PCC 7120. Judging by DNA-DNA eassocia ion expe imen s, Anabaena sp. s ains 010 u C)~~~~~~~~~" . C qj UC a^ qb s . U xu e~ xu xu x Cu 2 0 o0 CQ 11.38 * 10.50' * 4.40- 2.61 - 2.06 - 1.70 - 1.11 - 0.66 - FIG. 6. Hyb idiza ion o DNA om se e al he e ocys - o ming cyanobac e ia wi h he nucA gene om Anabaena sp. s ain PCC 7120. To al DNA om each o he cyanobac e ia was diges ed wi h EcoRV and hyb idized o a 473-bp HindIII-Bs NI agmen in e nal o he nucA gene o Anabaena sp. s ain PCC 7120. Hyb idiza ion was ca ied ou unde condi ions o high s ingency (see Ma e ials and Me hods). The posi ions o size s anda ds (in kilobases) a e indica ed o he le o he igu e. J. BACTERIOL. on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om GENE TRANSFER BETWEEN CYANOBACTERIA 1097 TABLE 1. P esence o absence o nucA gene-hyb idizing DNA and a nuclease o abou 29 kDa in some unicellula and ilamen ous, he e ocys - o ming cyanobac e ia S aina Hyb idi- 29-kDa za ionb nucleasec G oup A Anabaena a iabilis ATCC 29413-FD + + Anabaena sp. s ain PCC 6411 (ATCC 27898) + + Anabaena sp. s ain PCC 7118 (ATCC 27892) + + Anabaena sp. s ain PCC 7119 (ATCC 29151) + + Nos oc sp. s ain PCC 6705 (ATCC 29131) + + Nos oc sp. s ain PCC 6719 (ATCC 29105) + + G oup B Calo h ix sp. s ain PCC 7601 + + Fische ella muscicola UTEX 1829 + Nos oc ellipsospo um B 1453-7d + + Nos oc sp. s ain ATCC 43237 + NDe Nos oc sp. s ain PCC 7107 (ATCC 29150) + Nos oc sp. s ain PCC 7422 (ATCC 29132) + + Nos oc sp. s ain PCC 7413 (ATCC 29106) + + G oup C Gloeobac e sp. s ain PCC 7421 (ATCC 29082) - ND Synechococcus sp. s ain PCC 7202 (ATCC 29140) - - Synechococcus sp. s ain PCC 7425 (ATCC 29141) - - Synechococcus sp. s ain PCC 7942 - - Synechocys is sp. s ain PCC 6308 (ATCC 27150) - - Synechocys is sp. s ain PCC 6803 (ATCC 27184) - - a G oup A includes de i a i e FD o A. a iabilis (6a) and cyanobac e ia ha , as desc ibed by Lachance (12), a e closely ela ed o Anabaena sp. s ain PCC 7120. a G oup B includes he e ocys - o ming cyanobac e ia ha a e no (see oo no e d below) as closely ela ed o Anabaena sp. s ain PCC 7120. G oup C includes unicellula s ains. ATCC, Ame ican Type Cul u e Collec ion; PCC, Pas eu Cul u e Collec ion; UTEX, Uni e si y o Texas Cul u e Collec ion. N. ellipsospo um B 1453-7 was ob ained om he Uni e si y o Go ingen. b Hyb idiza ion was ca ied ou a high s ingency o s ains in g oup A and a low s ingency o s ains in g oups B and C (see Ma e ials and Me hods). c P esence o a nuclease o abou 29 kDa as shown by he SDS-PAGE nuclease assay (see Ma e ials and Me hods) o cell ex ac s (30 ,ug o p o ein o s ains in g oup A and 90 ,ug o p o ein o s ains in g oups B and C). dN. ellipsospo um ga e a pa icula ly s ong hyb idiza ion signal and, as p e iously discussed (8), may be closely ela ed o s ains in g oup A. 'ND, no de e mined. PCC 6411, PCC 7118, and PCC 7119 and Nos oc sp. s ains PCC 6719 and PCC 6705 a e phylogene ically e y closely ela ed o s ain PCC 7120 (12). We ha e in es iga ed, by means o Sou he n analysis, whe he he nucA gene is p esen in hese cyanobac e ia. A 32P-labeled in e nal p obe o he nucA gene (co esponding o amino acids 52 o 210 o he NucA p o ein) was used o hyb idiza ion wi h o al DNA om he cyanobac e ia men ioned abo e unde condi ions o high s ingency. The esul s in Fig. 6 show ha all o hese s ains, as well asA. a iabilis ATCC 29413, ca y DNA sequences ha exhibi homology o nucA. DNA om o he he e ocys - o m- ing cyanobac e ia ha a e no as closely ela ed o s ain PCC 7120 and om six di e se unicellula cyanobac e ia was es ed o hyb idiza ion wi h he nucA p obe om s ain PCC 7120 unde condi ions o low s ingency. All o he ilamen ous, he e ocys - o ming s ains es ed, bu none o he unicellula s ains, showed hyb idiza ion o he nucA p obe (da a no shown). The esul s ob ained a e summa ized in Table 1. Table 1 also shows he esul s o es ing, by means o a DNA- con aining, SDS-PAGE assay (see Ma e ials and Me hods), whe he some o he cyanobac e ial s ains con ain a nuclease o abou 29 kDa, which is he size o he p o ein encoded by he nucA gene o Anabaena sp. s ain PCC 7120. Wi h he excep- ion o Fische ella muscicola and Nos oc sp. s ain PCC 7107, in which no nuclease ac i i y was de ec ed, all o he s ains es ed whose DNA hyb idized wi h he nucA p obe also exhibi ed a nuclease o ca. 29 kDa. I is possible ha he nucA-hyb idizing sequences in F. muscicola and Nos oc sp. s ain PCC 7107 do no encode an ac i e nuclease o ha assay by SDS-PAGE is no sui able o he nuclease om hose pa icula cyanobac- e ia. We conclude ha he nucA gene is commonly p esen in he he e ocys - o ming cyanobac e ia bu no in some o he cyanobac e ia. I he gene ans e ha we obse ed was due o conjuga ion, conjuga ion could accoun o la e al ans e o a megaplasmid, and hus o nucA, be ween s ains. Concluding ema ks. The esul s p esen ed in his wo k show ha he nucA gene is loca ed in he a. megaplasmid o Anabaena sp. s ain PCC 7120 and ha a ma ked e sion o nucA (nucA::np ) can be ans e ed om s ain PCC 7120 di ec ly o ano he s ain o Anabaena sp. Al hough he e iciency o ans e plus ecombina ion appea s low, i may be amenable o inc ease by mu a ion (27). The inding o a na u ally occu ing ans e sys em ep esen s a no el aspec o he biology o cyanobac e ia. Fu he esea ch will be neces- sa y i he p ocess is o be made use ul as a gene ic ool. The ac ha he a megaplasmid om PCC 7120 ecom- bined wi h he megaplasmid om PCC 7118 a he han eplacing i may sugges ha he a megaplasmid is no ans e ed en i ely, al hough o he in e p e a ions a e possi- ble. T ans e by conjuga ion migh be abo ed be o e he plasmid is comple ely ans e ed so ha (i) he equency o ans e o a plasmid-bo ne gene ic ma ke would depend on i s dis ance om he o igin o ans e , and (ii) acquisi ion o a ma ke would depend upon i s inco po a ion in o an endoge- nous eplicon. I ecombina ion is a low- equency e en , as i is upon conjugal ans e om E. coli (ou unpublished obse - a ions), ans e o nucA::np o NW18 may be a much mo e equen e en han we obse ed. ACKNOWLEDGMENTS We hank Nancy Wood o Anabaena sp. s ain NW18 and Ma ia Isabel Mu o-Pas o o help wi h some hyb idiza ions. Wo k in Se ille was suppo ed by he Jun a de Andalucia (g oup no. 3057) and by he Di ecci6n Gene al de In es igaci6n Cien i ica y Tecnica (g an no. PB90-0114), Spain. Wo k in Eas Lansing was suppo ed by he U.S. Depa men o Ene gy g an DE-FG02- 90ER20021 and he Cen e o Mic obial Ecology. REFERENCES 1. Ausubel, F. M., R. B en , R. E. Kings on, D. D. Moo e, J. G. Seidman, J. A. Smi h, and K. S uhl. 1992. 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