Gibberella fujikuroi mutants obtained with UV radiation and N-methyl-N'-nitro-N-nitrosoguanidine

Vol. 49, No. 1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1985, p. 187-191 0099-2240/851010187-05$02.00/0 Copy igh C 1985, Ame ican Socie y o Mic obiology Gibbe ella ujiku oi Mu an s Ob ained wi h UV Radia ion and N- Me hyl-N' -Ni o-N-Ni osoguanidine J. AVALOS, J. CASADESUS, AND E. CERDA OLMEDO* Depa amen o de Gene ica, Facul ad de Biologia, Uni e sidad de Se illa, Se illa, Spain Recei ed 9 Ma ch 1984/Accep ed 16 Oc obe 1984 N-me hyl-N'-ni o-N-ni osoguanidine (ni osoguanidine) and o a lesse ex en UV adia ion a e e y mu agenic o Gibbe ella mic oconidia. The ecommended ni osoguanidine doses lead o much highe equencies o mu an s han a e ound in o he mic oo ganisms. The equency o mu an s among he su i o s inc eases linea ly wi h he ni osoguanidine dose (mola concen a ion x ime); he absolu e numbe o iable mu an s in a gi en popula ion eaches a maximum o a dose o ca. 0.7 M * s. The mic oconidia a e uninuclea e. The onse o ge mina ion b ings abou inc eased le hali y o ni osoguanidine, bu i does no modi y he ac ion o UV adia ion. Mycelia a e mo e esis an han spo es o bo h agen s. Visible illumina ion e ec i ely p e en s le hali y when gi en immedia ely a e UV i adia ion. Auxo ophs and colo mu an s a e e y easily ob ained. Pink adenine auxo ophs and se e al classes o colo mu an s a e a ec ed in he biosyn hesis o he ca o enoid pigmen , neu ospo axan hin. The indus ial in e es in he ungus Gibbe ella ujiku oi (as an impe ec ungus, known also as Fusa ium monili- o me) s ems om i s abundan p oduc ion o gibbe ellins, plan ho mones widely used in ho icul u e and b ewing (15). The o ganism may also ha e a u u e in he indus ial p oduc ion o p o ein and o ca o enoids. Mo phological and biochemical mu an s o G. ujiku oi ha e been isola ed a e ea men wi h di e en chemical and physical mu agens (1, 10, 11, 18, 24). No su p isingly, hese e o s ha e been concen a ed la gely on mu an s a ec ing gibbe ellin p oduc ion. N-me hyl-N'-ni o-N-ni osoguanidine (ni osoguanidine) has been ex ensi ely used o he induc ion o mu a ions in many o ganisms (12). The e ec s o he d ug, including he equency o mu an s and hei dis ibu ion o e he genome, a y om one o ganism o ano he . In yeas s (8), as in bac e ia (5), he mu a ions a e p e e en ially induced in DNA egions being eplica ed a he ime o mu agen expo- su e. In bac e ia (13), bu no in yeas s (2), his esul s in ela i ely high a es o closely linked double mu an s. De ailed s udies o he applica ion o ni osoguanidine o he ungi Saccha omyces ce e isiae and Phycomyces spp. ha e ecen ly been published by Calde on and Ce da'- Olmedo (2) and by Ronce o e al. (21), espec i ely. We in es iga ed he e ec s o ni osoguanidine on G. ujiku oi and compa ed hem wi h hose o UV adia ion. MATERIALS AND METHODS S ain, media, and cul u e condi ions. G. ujiku oi IM158289 was ob ained om he Commonweal h Mycological Ins i- u e, Kew, Su ey, Englapd. Minimal medium con ained (pe li e ) glucose, 30 g; NaNO3, 3 g; KH2PO4, 1 g; MgSO4 * 7H20, 0.5 g; KCl, 0.5 g; HBO4, 10 ,ug; CUSO4, 100 ,ug; FeC13, 200 ,ug; MnCl2, 20 ,ug; MoO4Na, 20 Rxg; and ZnSO4, 2 mg. Nu ien b o h was p epa ed by adding 4 g o yeas ex ac (Di co Labo a o ies, De oi , Mich.) and 8 g o pep one (Bac o-T yp one; Di co) o he minimal medium. Solid medium con ained addi ion- * Co esponding au ho . P esen add ess: Depa men o Biology, Uni e si y o U ah, Sal Lake Ci y, UT 84112. ally 16 g o aga (Analema, Vo quimica, Vigo, Spain). Cul u es we e incuba ed a 30°C in he da k. To ha es mic oconidia, cul u es we e g own on a special, ca bon-lim- i ed medium con aining (pe li e ) yeas ex ac (Di co), 1 g; NH4NO3, 1 g; KH2PO4, 1 g; MgSO4 * 7H20, 0.5 g; and aga , 16 g. The pla es we e g own o 5 days a 30°C 10 cm away om a ba e y o 40-W luo escen lamps (Syl ania Li eline Dayligh F40 V/D), yielding ca. 1.25 W o whi e ligh m Mic oconidia we e ha es ed by washing he spo ula ed mycelia wi h s e ile dis illed wa e and cleaned by passage h ough il e pape and low-speed cen i uga ion. Conidial suspensions we e s o ed a 4°C and i a ed by mic oscopic obse a ion wi h a Pe o -Hause chambe . Viable coun s we e de i ed om colony- o ming abili y on nu ien aga . Fo nuclea s aining, mi h amycin (a kind gi o P ize , Inc., G o on, Conn.) was dissol ed a 10 ,ug ml-' in a mix u e o equal olumes o dime hyl sul oxide and wa e . Ni osoguanidine mu agenesis. Ni osoguanidine (Sigma Chemical Co., S . Louis, Mo.) was dissol ed in dis illed wa e a 1 mg ml-', kep ozen un il use, and ne e e ozen. The sa e y ules p oposed by Eh enbe g and Wach meis e (9) we e adop ed. F eshly collec ed suspensions o ca. 106 conidia pe ml o dis illed wa e we e exposed o he d ug. Se e al doses o ni osoguanidine we e used. We de ine he dose as he p oduc o he mola concen a ion o he d ug and he exposu e ime in seconds. Any dose can he e o e be ob- ained by using di e en combina ions o concen a ion and exposu e. T ea men s we e s opped by dilu ing and wash- ing. UV mu agenesis. F eshly collec ed mic oconidia we e in- ocula ed on o nu ien -aga pla es, placed 49 cm away om a Syl ania G15T8 lamp (UV lux, 0.35 W m-2), and hen incuba ed in he da k. Fo pho o eac i a ion, UV exposu e was ollowed by a 5-h incuba ion in he ligh , as desc ibed abo e, be o e incuba ion in he da k. Cha ac e iza ion o mu an s. G ow h equi emen s o auxo- ophic mu an s we e de e mined ollowing he me hod o Holliday (14). Ca o enoids we e ex ac ed wi h ace one om lyophilized, weighed, and g ound mycelial samples and de e mined wi h a Bausch & Lomb Spec onic 2000 eco d- ing spec opho ome e . 187 on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om 188 AVALOS, CASADESUS, AND CERDA-OLMEDO 'I FIG. 1. Spo es o G. ujiku oi IM158289 s ained wi h mi h- amycin and pho og aphed wi h a luo oscence mic oscope. Ba , 10 ,um. RESULTS Mic oconidia. Ou Gibbe ella s ain g ows igo ously bu conidia es poo ly unde usual labo a o y condi ions. Less han 103 conidia pe pla e we e ound a e 5 days o g ow h in he da k o on nu ien medium in he ligh . Simila cul u es g own on minimal aga in he ligh p oduced ca. 2.4 x 104 conidia pe pla e. Abundan spo es (5.1 x 106 pe pla e) we e p oduced in cul u es g own o 5 days on an DOSE ( M x s) FIG. 2. Su i al o Gibbe ella spo es exposed o ni osoguani- dine. Open symbols, es ing spo es (kep in wa e ); closed symbols, spo es p eincuba ed o 2 h in nu ien b o h. The doses indica ed in he abscissa we e ob ained by exposu es o 15 ( iangles), 30 (squa es), 45 (ci cles), 60 (diamonds), o 75 (s a s) min o he app op ia e ni osoguanidine concen a ions. o I I I I I I I I I *1I .01 1 10 8 16 GERMINATION TIME ( h ) FIG. 3. E ec o ge mina ion on he su i al o Gibbe ella spo es mu agenized wi h ni osoguanidine. The spo es we e p ein- cuba ed o a ious pe iods in nu ien b o h and hen exposed o ni osoguanidine a 0.234 M * s (100 ,ug ml-' o 30 min; iangles) o 0.351 M - s (100 ,ug ml-' o 45 min; ci cles). especially poo aga (conidia ion aga ) unde he same b igh illumina ion. The spo es a e mic oconidia, ha is, unicellula , ellipsoid- al s uc u es, 4 o 6 pum long and 2 o 3 ,um wide, each con aining a single nucleus (Fig. 1). Ni osoguanidine le hali y and mu agenesis. The conidia a e qui e sensi i e o he le hal e ec s o ni osoguanidine. Su i al and mu agenesis depended only on he dose o ni osoguanidine and no on he pa icula concen a ions and imes used in each expe imen (Fig. 2). Ni osoguani- dine le hali y was enhanced by p eincuba ion o he mic o- conidia in nu ien b o h, i.e., by he onse o ge mina ion (Fig. 2 and 3). The highes sensi i i y was eached a e a 2-h p eincuba ion, be o e he appea ance o he i s g oss mo phological changes and was main ained h ough he p o usion o ge m ubes. A e 6 h o p eincuba ion, 80% o he spo es exhibi ed ge m ubes; a e 12 h, 99% exhibi ed ge m ubes. Mu agenesis, as judged by he p opo ion o auxo ophs among he su i o s, inc eased linea ly wi h he dose. The numbe o iable mu an s induced by ni osoguanidine in a gi en ini ial cell popula ion eached a maximum a a dose o abou 0.07 M * s (Fig. 4). Res ing spo es su e ed ewe dea hs and mu a ions han p eincuba ed spo es, i he com- pa ison was made a he same dose. I bo h kinds o spo es we e killed o he same su i al le el, hey showed he same mu a ion equencies (Table 1). UV le hali y and mu agenesis. The Gibbe ella spo es we e eadily killed by UV i adia ion a a wa eleng h o 254 nm. The le hal e ec o UV i adia ion was e e sed by immedia e exposu e o whi e ligh (Fig. 5). The le hali y app oxima ely ollowed i s o de kine ics wi h a small shoulde . APPL. ENVIRON. MICROBIOL. on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om GIBBERELLA MUTANTS 0-% C,, :No I-- 0 X C -4 CD a --4 -4 C-, 'm I-e %,, < .1 cn, .01 DOSE (M * s) FIG. 4. Pe cen age o auxo ophs among he su i o s o ni oso- guanidine exposu e (0). The spo es we e p eincuba ed o 2 h in nu ien b o h, exposed o ni osoguanidine, and pla ed on nu ien aga ; he esul ing colonies we e es ed o auxo ophy. The same da a a e eplo ed as he pe cen age o iable auxo ophs ela i e o he o al numbe o cells, li ing o dead (A). UV le hali y was no a ec ed by ge mina ion up o he massi e p oduc ion o ge m ubes; mycelia we e mo e esis an o UV i adia ion han we e conidia (Fig. 6). UV exposu e was mu agenic, bu less so han was ni oso- guanidine ea men , leading o compa able su i al le els (Table 1). Gibbe ella mu an s. The su i o s o ni osoguanidine ea men we e no always iden ical o each o he and o he wild ype. Colony size, su ace ex u e, colo , and o e all mo phology we e o en abe an . The same was ue, o a lesse ex en , o he su i o s o UV i adia ion. Ni osoguanidine-induced auxo ophs showed a wide a- ie y o nu i ional equi emen s: we iden i ied se en a gi- nine, i e me hionine, ou leucine, h ee lysine, wo bio ine, wo nico inic acid, one yp ophan, and one phenylalanine auxo oph. We s udied wo pink mu an s, which con ained he same ca o enoids as he wild ype and an addi ional wa e -soluble TABLE 1. Mu agenic e ec s o ni osoguanidine and UV adia ion on G. ujiku oi" Auxo ophs P eincuba ion Ax oh P icb ion Su i al among he Mu agen Dose in nu ien b o h (h) ) su i o s Ni oso- 0.5 M * s 0 15 2.5 guanidine 0.1 M * s 2 14 3.1 0.05 M * s2 33 1.4 UV adia ion 0.7 J m-2 0 20 0.8 A o al o 1.040 colonies we e es ed o auxo ophy in each case. The auxo oph equency o UV adia ion is signi ican ly lowe han hose o ni osoguanidine, as judged by he es a e he a csin ans o ma ion (23). Fo example. i he mu agenici ies a e assumed o be equal. he p obabili y o a di e ence as la ge as o la ge han ha be ween he lowe wo lines is P < 0.04. 0 1 2 3 DOSE (J*m-2) FIG. 5. Killing o Gibbe ella spo es by UV adia ion and pho o- eac i a ion by isible ligh . A e exposu e o he dose gi en in he abscissa, samples we e exposed (0) o no (0) o isible ligh and hen incuba ed in he da k. pink pigmen . These mu an s we e auxo ophic o adenine. The syn hesis o he pink pigmen was ep essed by adenine: mycelia g own wi h 20 mg o adenine li e -' in he da k we e deeply colo ed; wi h 200 mg li e -', a he aded; and wi h 2 g li e -', whi e. All o hese ea u es a e ypical o some adenine auxo ophs in o he ungi, such as he mu an s o Cn.1 .02 000.0 I I 0 0 0 0 0 ~~~~~0 0 0 1 1 1 8 GERMINATION TIME (h ) 16 FIG. 6. E ec o ge mina ion on he su i al o Gibbe ella spo es exposed o UV adia ion. The spo es we e p eincuba ed in nu ien b o h o he pe iod gi en in he abscissa, exposed o 1 J m-2 UV adia ion, exposed (0) o no (0) o isible ligh , and incuba ed in he da k. 0 0 0 I I I I I I i VOL. 49, 1985 189 on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om 190 AVALOS, CASADESOS, AND CERDA-OLMEDO genes ad] and ad2 in S. ce e isiae (22) and adel and ade2 in Candida albicans (19). O he mu an s we e pu ple when g own in ei he he ligh o he da k. They we e pho o ophic, and hei ca o enoid con en coincided wi h ha o he wild ype. The na u e o he pigmen emains unknown. The es o ou colo mu an s di e ed om he wild ype in neu ospo axan hin con en . The wild ype was o ange when g own in he ligh , con aining 100 o 150 ,ug o neu ospo a- xan hin g-' (d y weigh ) (ppm). In he da k, he syn hesis o neu ospo axan hin was ep essed, and he mycelium was whi e (less han 10 ppm o neu ospo axan hin). The ca o - enoid mu an s can be classi ied in o h ee pheno ypic classes. One o hem is made up o mu an s wi h li le o no pigmen in ei he he ligh o he da k. Ano he class is indis inguish- able om he wild ype in he ligh , bu is o ange in he da k, due o accumula ion o 30 o 100 ppm o neu ospo axan hin. The emaining ca o enoid mu an s a e deep o ange when g own in ei he he ligh o he da k, and hei ca o enoid con en inc eases linea ly wi h ime, up o ca. 2,000 ppm o neu ospo axan hin. DISCUSSION G. ujiku oi is a highly sui able o ganism o he induc ion and isola ion o mu an s. The uninuclea e mic oconidia eadily allow he exp ession o ecessi e mu a ions. To app ecia e he con enience o Gibbe ella mic oconidia, con- side he di icul ies encoun e ed in he isola ion o mu an s in mul inuclea e cells, such as Phycomyces spo es (21). No all s ains o G. ujiku oi a e equally a o o able o hese s udies, because many s ains p oduce mul icellula mac o- conidia (16). Ni osoguanidine and UV adia ion kill Gibbe ella mic o- conidia abou as e ec i ely as hey kill he mic oo ganisms mos o en used in gene ics. The onse o ge mina ion inc eases he sensi i i y o mic oconidia o ni osoguanidine. In a simila way, ac i ely eplica ing bac e ia and yeas and ge mina ing Phycomyces spo es a e mo e eadily killed by ni osoguanidine han he co esponding es ing cells (3, 4, 21). Inc eased cell pe meabili y and changes in nuclea s uc u e and unc ion a e p esumably esponsible o his di e ence. Pe meabili y changes may be he mos impo an , since UV adia ion makes no dis inc ion be ween es ing and ge mina ing conidia. The mycelia, a e he p o usion o he ge m ubes, a e much mo e esis an o bo h le hal agen s han a e spo es. G. ujiku oi shows a mos ac i e pho o eac- i a ion o UV damage, compa able o ha o Phycomyces (6) and Saccha omyces spp. (17). As wi h o he o ganisms, ni osoguanidine is a mo e e ec i e mu agen han UV adia ion o G. ujiku oi. The auxo oph equencies epo ed he e a e among he highes epo ed o any o ganisms. Auxo oph equencies depend o a ce ain ex en on subjec i e assessmen , depending on he c i e ia o inclusion o leaky mu an s. E en so, ou equencies a e much highe han hose usual o Phycomyces spp. (less han 0.1%) o yeas cells (ca. 1%) unde simila condi ions. The ecommended ni osoguanidine dose o p ac ical applica ions depends on he na u e o he desi ed mu an s. Fo a e, nonselec able mu an s, he dose should be high, o imp o e he chances o success a he isk o accumula ing mul iple mu a ions in he same genome. Fo selec able mu an s, pa icula ly hose in ended o physiological s ud- ies, he dose should be as low as possible, o a oid mul iple mu a ions. The s a us o Gibbe ella gene ics does no ye allow a di ec es o whe he a pheno ype is due o one o mo e mu a ions. Ni osoguanidine mu agenesis in Esche ichia coli quickly eaches sa u a ion: u he exposu e inc eases le hali y bu no he equency o mu an s (4). In G. ujiku oi, as in Phycomyces spp. (21), he equency o mu an s inc eases linea ly wi h he dose. This sugges s a undamen al di e - ence in he way ni osoguanidine ac s in he wo ungi and he bac e ium. The whi e and deep-colo ed Gibbe ella mu an s esemble simila mu an s in Phycomyces spp. (7). Mos a ac i e a e he mu an s ha syn hesize neu ospo axa hin in he da k bu ha a e simila o he wild ype in he ligh . These no el mu an s should con ibu e o he s udies o pho oinduc ion o ca o enoids, qui e de eloped in a ela ed ungus, Fusa - ium aquaeduc uum (20). Ou esul s encou age he sea ch o all kinds o Gibbe - ella mu an s, including hose o po en ial indus ial in e es . ACKNOWLEDGMENTS We hank A. Fe nandez Es e ane and D. Sud ez o hei assis - ance and he Comisi6n Aseso a pa a In es igaci6n Cien i ica y Tdcnica o inancial suppo . LITERATURE CITED 1. Bea de , J. R., J. MacMillan, C. M. Wels, M. B. Cha ey, and B. 0. Phinney. 1974. Posi ion o he me abolic block o gibbe - ellin biosn hesis in mu an B1-41a o Gibbe ella ujiku oi. Phy o- chemis y 13:911-917. 2. Calde 6n, I. L., and E. Ce da-Olmedo. 1982. Simul aneous induc ion o mul iple mu a ions by N-me hyl-N'-ni o-N- ni osoguanidine in he yeas Saccha omyces ce e isiae. Cu . Gene . 6:237-243. 3. Calde 6n, I. L., and E. Ce da-Olmedo. 1983. Induc ion by N-me hyl-N'-ni o-N-ni osoguanidine o nuclea and cy oplas- mic mu a ions in Sacha omyces ce e isiae. 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R. Ea on. 1969. Func ional blocks o he adl and ad2 mu an s o Saccha omi ces ce e 'isiae. Biochem. Biophys. Res. Commun. 34:301-305. 23. Sokal, R. R., and F. J. Rohl . 1969. Biome y. W. H. F eeman Publica ions, San F anscisco. 24. Spec o , C., and B. 0. Phinney. 1966. Gibbe ellin p oduc ion: gene ic con ol in he ungus Gibbe ella iujiku oi. Science 153:1397-1398. VOL. 49, 1985 on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om ERRATUM Gibbe ella ujiku oi Mu an s Ob ained wi h UV Radia ion and N-Me hyl-N' -Ni o-N-Ni osoguanidine J. AVALOS, J. CASADESUS, AND E. CERDA-6LMEDO Depa amen o de Gene ica, Facul ad de Biologia, Uni e sidad de Se illa, Se illa, Spain Volume 49, no. 1, p. 187, abs ac , line 5: ". . . eaches a maximum o a dose o ca. 0.7 M s" should ead ". . . eaches a maximum o a dose o ca. 0.1 M. s." Page 187, abs ac , line 9: "Pink adenine auxo ophs and se e al classes o colo mu an s a e a ec ed . . ." should ead "Some adenine auxo ophs a e pink, and se e al classes o colo mu an s a e a ec ed...." Page 187, column 2, lines 2-6: An ing edien should be added o he desc ip ion o he ca bon-limi ed medium: "glucose, 1 g." 735