Nitrosoguanidine assay of episomal integration in Escherichia coli

JOURNAL OF BACTERIOLOGY, No . 1973, p. 527-530 Copy igh i 1973 Ame ican Socie y o Mic obiology Vol. 116, No. 2 P in ed in U.S.A. Ni osoguanidine Assay o Episomal In eg a ion in Esche ichia coli CRISTINA P. LLOVERES AND E. CERDA-OLMEDO Depa amen o de Gene ica, Facul ad de Ciencias, Uni e sidad de Se illa, Spain Recei ed o publica ion 10 July 1973 Mu a ions a ec ing u iliza ion o lac ose and esis ance o he male-speci ic phages l, 2, and Q8 end o occu simul aneously mo e o en han expec ed by chance in H s ains whose o igin o ans e is close o he genes o lac ose u iliza ion, bu no in F+ s ains. S ains de i ed om he H , bu exhibi ing poo abili y o ans e ea ly ch omosomal genes, may o may no show his comu a ion phenomenon. These esul s suppo he concep ha he F ac o is in eg a ed in o he H ch omosome du ing ege a i e g ow h, bu is au onomous in he F+ s ains and could se e as an assay o episomal localiza ion. N-me hyl-N'-ni o-N-ni osoguanidine is an e ec i e mu agen in Esche ichia,coli (2, 11) and in many o he o ganisms. The inac i a ion o he F ac o by ni osoguanidine, as we will abb e ia e i s name, has been desc ibed (13), and many mu a ions induced by his agen ha e been used in he gene ic analysis o F unc ions ( e iew in e e ence 1). Al hough all genes a e suscep ible o mu a ion by ni osoguanidine, he mu a ions in any pa icula cell a e no andomly dis ibu ed o e he ch omosome bu c owded oge he in small egions, ex ending o e less han 2 min o he E. coli ch omosome (6), and placed a he posi ions occupied by he eplica ion poin s a he ime o he ea men (5). Gene ic si es close han 2 min exhibi comu a ion, ha is, hey end o mu a e simul- aneously mo e o en han expec ed by chance. Comu a ion may be a use ul ool in some p oblems o gene ic mapping, such as he ecog- ni ion o he in eg a ed and au onomous s a es o episomes. We ha e used he F ac o as a model sys em, de ining i as an episome con e - ing sensi i i y o male-speci ic phages, such as he ibonucleic acid (RNA) phages Q,B and 2 and he deoxy ibonucleic acid (DNA) phage l. MATERIALS AND METHODS Bac e ia and phages. X15 is a p o o ophic F+ s ain o Esche ichia coli, de i ed om he F+ s ain W1485; X493 is a p o o ophic H s ain, de i ed om X15 and ans e ing i s ch omosome in he o de O-p oB-p oA-leu- h -... -lac-F. Bo h we e ecei ed om R. Cu iss (3). DF73 is an F- s ain ca ying he ma ke s s A, hyA, h , leu, hi, p o, a g, lac, gal, a a, xyl, m l, de i ed om he s ain AB1157 and ecei ed om K. B ooks. The wild- ype RNA phages Q and 2 we e ecei ed om A. J. Cla k, and he wild- ype DNA phage l was om N. D. Zinde . Cul u e condi ions. Bac e ia we e g own a 37 C in is(hyd oxyme hyl)aminome hane (T is)-sal s mini- mal medium (10), wi h D-glucose a 2 g/li e as ca bon and ene gy sou ce. Solid media we e p epa ed in he same way wi h 25 g o aga (Di co) pe li e . When app op ia e, amino acids and hymine we e added a 20 gg/ml, hiamine a 1 zg/ml, and s ep omycin a 100 ug/ml, and ,-lac ose was subs i u ed o glucose a 2 g/li e . Ni osoguanidine ea men . Cells om exponen- ially g owing cul u es we e ea ed wi h 100 yg o ni osoguanidine (Se a, Heidelbe g, Ge many) pe ml o T is-malea e bu e , pH 7.5, a 37 C o 30 min (4); hey we e washed wice by cen i uga ion bo h be o e and a e he ea men . Tes o phage esis ance. Phage s ocks we e p oduced in b o h-g own E. coli s ain X493. A d op o phage s ock, con aining o e 109 plaque- o ming uni s o he indica ed phage o phage mix u e, was ex- ended in a diame ical band on an aga pla e wi h a wi e loop. Bac e ia om single colonies we e s eaked ac oss he phage-con aining band wi h a s e ile oo h- pick. A colony was conside ed esis an when bac e- ial g ow h was he same on bo h sides o he phage-con aining band, as obse ed a e o e nigh incuba ion a 37 C. RESULTS New s ains. Two Lac- de i a i es o he H s ain x493, unable o u ilize lac ose, we e isola ed a e ni osoguanidine ea men and called SE12 and SE13. To his end, exponen- ially-g owing cells o s ain x493 we e ea ed wi h ni osoguanidine, allowed o g ow in glu- cose liquid medium o 3 h, and pla ed on glucose solid medium; indi idual colonies we e hen es ed o g ow h on lac ose solid medium. The conjuga ional abili y and he o de o gene 527 LLOVERES AND CERDA-OLMEDO ans e we e checked in hese s ains by in e - up ed ma ing (9) wi h DF73, and hey we e ound o be he same as in he o iginal s ain. Two Lac- de i a i es o he F+ s ain X15 we e isola ed by he same p ocedu e as o he H , and called SE20 and SE21. They we e checked o be s ill F+ by obse ing he appea - ance o esis ance o he male-speci ic phages l, 2, and QB a e p olonged incuba ion wi h ac idine o ange (7). S ains exhibi ing poo conjuga ional abili y we e isola ed om he Lac- H . Bac e ia om SE12 colonies we e spo ed on o pla es co e ed wi h some 109 cells o s ain DF73 and con ain- ing adequa e supplemen s o he selec ion o Leu+ Th + S + ecombinan s ( ha is, con ain- ing s ep omycin, hiamine, hymine, p oline, and a ginine). In mos cases a hick g ow h o ecombinan s appea ed, bu some colonies, con- s i u ing abou 1% o he o al, did no p oduce any ecombinan s. A e eisola ion and e es - ing, ou o hem we e called SE18, SE19, SE22, and SE23. The same p ocedu e was applied o he s ain SE13, and s ains SE15, SE16, and SE17 we e isola ed. These s ains we e all ound o be sensi i e o male-speci ic phages. Comu a ion. The lac mu a ions in s ains SE12, SE13, SE20, and SE21 we e ob ained independen ly. The su i al o hese s ains a e ni osoguanidine ea men and he e- quencies o Lac+ e e an s a e indica ed in Table 1, which gi es he esul s o ypical expe imen s in which bac e ial samples, aken jus be o e o jus a e he ea men wi h ni osoguanidine, we e pla ed on glucose and ac ose media. Highe su i al a es can be ob ained by dec easing he pH a he ime o he ea men o 5.5. I can be no ed ha ni- osoguanidine usually inc eases he equency o e e an s mo e han 1,000- old. The s ains de i ed om SE12 and SE13 beha e like hei pa en s in hese espec s. When un ea ed samples we e pla ed on ei he glucose o lac ose, he esul ing colonies we e always phage sensi i e. No a single phage- esis an colony was ound a e es ing a o al o 8,000 colonies belonging o all he s ains ob ained in he p e ious sec ion. A e he mu agenic ea men , a sizable p opo ion o phage- esis an colonies was ound bo h among he gene al popula ion (glu- cose pla es) and among he Lac+ e e an s (lac ose pla es). Table 2 gi es he incidence o esis ance o phages QB and 2, and Table 3 gi es he incidence o esis ance o phage l. The colonies esis an o l we e ound o be esis an o Q, and 2, bu he e e se was o en un ue. As a consequence, he p opo ions in Table 2 end o be highe han hose in Table 3. TABLE 1. Su i al and e e sion o Lac+ a e ni osoguanidine ea men F equency o Lac+ e e an s S ain S ing (pe colony- o ming uni ) Su i ing cin Be o e he A e he ea men ea men SE12 (H ) 0.15 2.2 x 10' 2.7 x 10-' SE13 (H ) 0.050 7.5 x 10-7 9.4 x 10-4 SE20 (F+) 0.096 < 10-8 4.1 x 10-4 SE21 (F+) 0.072 < 10-8 2.8 x 10- In he H s ains he mu a ion o esis ance o male-speci ic phages was always mo e e- quen among he Lac+ e e an s han among he gene al popula ion; hus, bo h kinds o mu a ions ended o occu simul aneously in he same cells. No such associa ion was ound in he F+ s ains. whe e bo h kinds o mu a ions occu ed as independen e en s. One o he se en s ains isola ed om he H because o hei poo abili y o ans e ea ly ch omosomal genes showed comu a ion be ween phage esis - ance and Lac+ e e sion, bu he o he s did no . DISCUSSION The wo kinds o mu a ions in es iga ed he e we e chosen because one is p esumed o depend on ch omosomal genes and he o he is p e- sumed o depend on F- ac o genes. The expe i- men al esul is ha in he H he wo kinds o mu a ions occu simul aneously mo e o en han expec ed by chance a e ni osoguanidine ea men , whe eas his does no happen in F+ s ains. This esul indica es ha he genes e- sponsible o he wo mu a ions eplica e a abou he same ime in he H bu no in he F+. This is in excellen ag eemen wi h he hy- po hesis (8) ha he F ac o o he H is in- eg a ed wi h he ch omosome and eplica es as a pa o i , e en du ing ege a i e g ow h un ela ed o conjuga ion. The s ains isola ed om he H because o hei poo abili y o ans e ea ly ch omosomal genes may be expec ed a p io i o all in o wo g oups, one lacking comu a ion be ween episo- mal and ch omosomal genes (p esumably due o de achmen om he ch omosome o an F ac o ha does no ca y he ele an ch omo- somal genes) and ano he exhibi ing comu a- ion (de achmen o an F ac o ca ying he ele an ch omosomal genes, o mu a ional loss o conjuga ional abili y wi hou de achmen o acquisi ion o esis ance o male-speci ic phages). Six s ains o he i s g oup and one o he second g oup we e isola ed. Gue ola e al. (6) de ined he comu a ion index as he equency o double mu an s ela- 528 J. BACTERIOL. EPISOMAL INTEGRATION IN E. COLI TABLE 2. Resis ance o phages QpS and 2 a e ni osoguanidine ea men Pla ed on glucose Pla ed on lac ose S ain S a is ical Colonies Pe cen Colonies Pe cen signi icance" es ed esis an es ed esis an H s ains SE12 800 4.6 800 9.2 P < 0.001 SE13 1825 4.8 1825 7.9 P < 0.001 F+ s ains SE20 700 5.0 800 5.1 NS SE21 400 4.7 300 5.3 NS Conjuga ion-de ec i e s ains de i ed om H SE15 400 3.5 400 12.5 P < 0.001 SE16 400 4.7 400 5.0 NS SE17 400 5.2 400 6.7 NS SE18 400 4.0 400 4.0 NS SE19 400 5.0 400 7.0 NS SE22 500 5.0 500 5.6 NS SE23 400 5.0 400 5.4 NS a The p obabili y o a chance de ia ion equal o o la ge han he expe imen al one Was calcula ed a e he a Csin ans o ma ion and he es (12). All "no signi ican " (NS) cases ga e P > 0.2. TABLz 3. Resis ance o phage l a e ni osoguanidine ea men S ain H s ains SE12 SE13 F+ s ains SE21 Conjuga ion-de ec i e s ains de i ed om H SE17 SE18 Pla ed on glucose Colonies es ed 500 375 500 500 500 Pe cen esis an 3.2 2.1 2.6 2.4 1.4 Pla ed on lac ose Colonies es ed 500 375 400 500 500 Pe cen esis an 8.6 7.2 3.0 2.0 1.4 i e o he p oduc o he equencies o he single mu an s; hey ound an index o 15 o genes placed 1 min away in he ch omosome map. The comu a ion index o Lac+ e e sion and l esis ance is abou 3 in he H s ains. By compa ison wi h Gue ola's esul s, he ele- an genes may be placed some 1.5 min away in he H ch omosome. Comu a ion is clea e and he index is sligh ly highe when l is used o assay o esis ance han when Q i and 2 a e used. On he o he hand, bac e ia esis an o l a e usually esis an o Q i and 2, bu no ice e sa. This may be due o mu a ions con e ing esis ance o Q8 and 2, bu no l, loca ed in ch omosomal genes a om lac and he F ac o , and is p ob- ably ela ed o he di e en pa icipa ion o bac- e ial genes in he de elopmen o he wo ypes o phage. Comu a ion s udies ashioned in di e en ways may be used o es o he in eg a ion o an episome in o a ch omosome. Such a es is independen o whe he he episome con e s conjuga ional abili y, o whe he a gene ic sys- em has been de eloped in he bac e ial species unde s udy, and o whe he he episome ca ies any indispensable genes (and hus canno be los upon incuba ion wi h ac idine dyes). Fo example, a mu a ional change in he episome would be selec ed, ei he in he o wa d o S a is ical signi icance P < 0.001 P < 0.001 NS NS NS VOL. 116, 1973 529 530 LLOVERES AND e e se di ec ion, a e ea men wi h ni- osoguanidine, and he esul ing mu an s would be assayed o he p esence o ch omo- somal mu a ions. Auxo ophic mu a ions a e con enien o assay and a e usually sp ead o e he ch omosome. I he episomal mu a ion is equen ly associa ed wi h a pa icula g ow h equi emen , he implica ion would be ha he episome is close o he genes esponsible o ha kind o auxo ophy. In ac comu a ion only means ha he wo gene ic s uc u es eplica e simul aneously. Co- mu a ion migh be ound be ween genes in wo independen eplicons i he e is a p ecise syn- ch oniza ion in hei eplica ion. Au onomous episomes a e known o eplica e a ce ain imes o he cell cycle (14, 15), bu he e is no indica ion ha he synch oniza ion is good enough o show app eciable comu a ion wi h ch omosomal genes. Re e sion o wild- ype pheno ype may some- imes be due mo e o in e genic supp ession han o in agenic e e sion and he comu a ion esul s would hen be misin e p e ed. In ou expe ience such high ela i e incidence o in e - genic supp ession is uncommon and e o s can be p e en ed by he s udy o di e en o iginal mu a ions o he same ype. 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