Mutagenesis of Anacystis nidulans by N-methyl-N′-nitro-N-nitrosoguanidine and UV irradiation

Mutation Research Elsevier P u b l i s h i n g C o m p a n y , A m s t e r d a m P r i n t e d in T h e N e t h e r l a n d s

531

M U T A G E N E S I S OF A N A C Y S T I S N I D U L A N S BY N - M E T H Y L - N ' - N I T R O N - N I T R O S O G U A N I D I N E AND UV I R R A D I A T I O N * YUKIO

A S A T O * * AND C L A I R E. F O L S O M E

Department of Microbiology, University of Hawaii, Honolulu, Hawaii (U.S.A.) (Received A u g u s t i 2 t h , 1969)

SUMMARY

Inactivation of the blue-green alga, Anacystis nidulans, with nitrosoguanidine was concentration-dependent. Nitrosoguanidine-indueed mutation frequencies were: 1.8'IO -3, 2.98'1o -3, 3.7"1o -3, I.O.IO -3 and 4.8-1o 4 for yellow and blue, minutecolony-forming, filamentous or snake, and polymixin-B-resistant mutants, respectively. UV-irradiated damage within the experimental conditions utilized was completely photoreactivable by visible light. When a UV-irradiated culture was incubated in the dark for 9 h, a survival curve with slope k = --0.066 (where k = in S/So.t -~) was observed. A small shoulder was evident, and the extrapolation of the asymptote of the curve to the ordinate showed an inactivation number of 2. Minute-colonyforming and snake mutants were also induced by UV irradiation if photoreactivation was prevented. The inability to isolate auxotrophic mutants of Anacystis nidulans was considered to be due to intrinsic properties and therefore presents a major problem in blue-green algal genetics.

INTRODUCTION

The genetics of blue-green algae to date have been hampered by two major difficulties. First, a sexual system appears at best to be most inefficient, although very low levels of reeombinants have been reporteda,s,n, ia. Second, appropriate auxotrophic mutants are not obtainable in these phototrophs; thus the present array of genetic markers is quite limitedg, 11. Most of these difficulties of the experimental system can be met. A method of temporal genetic mapping has recently been devised and employed with Anacvstis nidulaus2; this method promises about the same resolving power as the E. coli K-I2 conjugation system. Methods permitting high efficiency of agar plating exist2,a,12,16. The object of this report is to demonstrate that some mutations can be induced in Anacystis nidulans with high efficiency by UV or NG, to discuss the nature of * This r e p o r t is p a r t of a d i s s e r t a t i o n s u b m i t t e d to t h e G r a d u a t e School of t h e U n i v e r s i t y of H a w a i i in p a r t i a l fulfillment of r e q u i r e m e n t s for t h e P h . D . degree. ** P r e s e n t a d d r e s s : N a t i o n a l A e r o n a u t i c s a n d Space A d m i n i s t r a t i o n , A m e s R e s e a r c h Center, Moffett Field, Calif. 94o35 (U.S.A.). A b b r e v i a t i o n s : mcf, m i n u t e - c o l o n y - f o r m i n g ; NBG-DIn, D m a g a r s u p p l e n l e n t e d w i t h n u t r i e n t b r o t h (i g / I o o ml) a n d glucose (o. 5 g / i o o ml); NG, N-nlethyl-N'-nitro-N-nitrosoguanidine; \VT, wild-type.

Mutation Res., 8 (1969) 531-536

532

Y[ KI() :kSAI(), ( 1.\IR I~. I.()ks~.\II

those induced m u t a n t s , Ctnd to sho\x tlmt a u x o t r o p h i c m u t a n t s \vcrt' n~t lCC~,vcr¢.,l from v e r y large s a m p l e s of m u t a g e n - t r e a t e d cells. MATF.I¢.IALSANI) METHOI)S

Orgamsm. Anacystis nidula~ls No. (>25, a unicellular, obligate photoautotr<@jr, was o b t a i n e d from the algal culture collection of I n d i a n a U n i v e r s i t \ ''~. Media and growth conditions. D m m i n i m a l b r o t h a n d a g a r was m a d e according to VAN BAAI.EN 1~. A culture of Anacvstis nidulans was grown in l)m broth in an e r l e n m e y e r flask with a v o l u m e r a t i o of I : I o . IAght was p r o v i d e d by linear b a n k s of 2 cool white a n d 2 d a y l i g h t fluorescent t u b e s (Westinghous, 4 ° \V) with an i n t e n s i t y of 35o ft.-candles. L i q u i d cultures were i n c u b a t e d at 320 in a shaking w a t e > b a t h . Cultures on agar plates were i n c u b a t e d at room t e m p e r a t u r e u n d e r fluorescent lamps. To p r e v e n t excessive d e h y d r a t i o n of the agar, fluorescent l a m p s were placed u n d e r the shelves where the plates lay. Visible colonies were observed within 3 days. Inactivation with NG and UV. NG (Aldrich Chemical Co., Milwaukee, \Visc.) was dissolved in dist. H~,O or in o.2 :lI KH.,P04 (pH 6.o) a n d filter sterilized. The m u t a g e n was used within 5 h after p r e p a r a t i o n . Y o u n g e x p o n e n t i a l liquid cultures of A n a c y s t i s were t r e a t e d with NG in final c o n c e n t r a t i o n s of 25, 5o a n d I o o / , ' g / m l at p H 8.o. Samples were t a k e n at 5 rain intervals, diluted, and o.I ml aliquots were s p r e a d on agar plates. Surviving colonies were c o u n t e d on the 6th day. E x p e r i m e n t s were also carried out at p H 6.o in o.~ M KH2P04 buffer. UV i r r a d i a t i o n was p e r f o r m e d by e m p l o y i n g a 15 \ r g e r m i c i d a l l a m p ((;I5T8 , General Electric) at a d i s t a n c e of 4 ° cm. 15 lnl of a culture in D m m e d i u m (absorbance, o.37 at 254 nm) containing 2. ~o 7 cells per ml were placed in a glass petri dish. Cultures were g e n t l y swirled b y h a n d during i r r a d i a t i o n in a room with dim reflected light. Two sets of samples (o.2-ml aliquots) were t a k e n at various time intervals. One set was d i l u t e d x : i o in D m m i n i m a l b r o t h a n d k e p t in the d a r k fl~r 0 h. The other set was p l a t e d a n d i m m e d i a t e l y i n c u b a t e d in the light. Mutagcnesis. Cultures of A n a c v s t i s were t r e a t e d with N(i to o b t a i n o.I -x.¢/!,, survivors. The cells were washed free of the m u t a g e n b y eentrifugation, inoculated in D m m e d i u m a n d i n c u b a t e d for <> 0 generations. Morphological a n d p i g m e n t m u t a n t s were a s s a y e d b y diluting a n d s p o t t i n g o.oI ml on agar plates. A b o u t 3 ° spots can be a c c o m m o d a t e d on a p l a t e each spot c o n t a i n e d a b o u t 3oo 4oo cohmics. P o l y m i x i n - B - r e s i s t a n t n m t a n t s were a s s a y e d b y s p r e a d i n g o . i - m l aliquots of diluted cells on d u p l i c a t e plates c o n t a i n i n g I5O/.'g p o l y m i x i n B per ml. I n a t t e i n p t s to isolate a u x o t r o p h i c m u t a n t s , cultures <>f A.uacvstis nid~daJ~s were s t r e a k e d on N B G - I ) m . A single large cohmy was t r a n s f e r r e d to N B G - D m broth. A f t e r the culture h a d grown for 4 days, an aliquot was centrifuged and resuspended in D m mediuna. NG was a d d e d to a final e<>ncentrati h) the penicillin was r e m o v e d b y centrifugation, a n d the cultures were inoculated and i n c u b a t e d in N B G - D m broth for 2 4 days. S u b s e q u e n t l y , replica plates were made. :llutation l&s., 8 (I969) 53 ~-53 l'

MUTAGENESlS

OF

A. nidulans BY NG a~b UV

533

RESULTS AND DISCUSSION

Inactivation studies. Inactivation with NG was concentration-dependent (Fig. I). Lowering the pH to 6.0 with NG in a final concentration of 50/:g/ml decreased the slope of the survival curve (Fig. 2). Incubation of NG-treated culture in the dark for up to 9 h had no effect on the slope of the inactivation curve. Mcf mutants increased with the time of NG treatment, reaching a maximal frequency of 2.0' IO-3 at o.2°0 survival levels. NG inactivation, pH 6.0 100

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Time (rain)

I;ig. I. S u r v i v a l c u r v e of Anacystis nidulans t r e a t e d w i t h N G a t p H 8.0. Fig. 2. S u r v i v a l c u r v e of Anacystis nidulans t r e a t e d w i t h NG a t p H 6.o.

UV irradiation inactivated Anacystis if the irradiated cultures were then incubated in the dark for 9 h before exposure to light. A slope k = --o.o66 (where k -- in S/So't -1) was calculated from the linear portion of the curve. A small shoulder was observed; extrapolation of the asymptote to the ordinate indicated a target number of 2. Using larger UV doses, SINGH et al. la reported extrapolation or target numbers of 8o-9o and slope k --o.o55 (in terms of in S/So't 1). Apparently, the Anacvstis nidulans strain utilized in the present experiments lacks an efficient darkrepair system. However, cells irradiated with UV for up to 8o sec showed a complete and striking photorecovery if incubated immediately in the light. Photorecovery of UV damage could very well be due to a photoreactivating enzyme since both photoreactivation TM and photoreactivation enzyme were demonstrated on a filamentous blue-green alga, Plectonema boryanum. Mutagenesis. NG-induced mutants were recovered after exposure of cultures at pH 8.o to a final NG concentration of ~oo t.,g/ml for 12 min. The surviving fractions at these concentrations were between o.1-1.o°%. Mutant phenotypes were: yellow (yel) and blue (blu) pigment, mcf (mcf), filamentous (sna), and polymixin-B-resistant 2~lutation Res., 8 (1969) 531-536

534

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(pmb r) with frequencies

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(p;nbr). The .sna mutants, as previously descril?ed by VAx BA.\L]-:X~", ar~' long lila:m'n tous cells (15 2o microns) with no observable cross septa and are easily detected on agar by their rhizoid appearances. Comparison of mutation frequencies performed at pH 6.o with those done at pH 8.o is not warranted. Experiments at pH 6.o were dora, with a final NG concentration of 5 ° i~g/ml, and cells were plated without prior incubation to permit segregation. UV irradiation induced both filamentous and mcf mutants at frequencies of 2.I. IO-3 and 5.8.IO 2, respectively, at o.0";) survivors. These values are higher than those induced with NG since the irradiated culture was not incubated for segregation. The mutation frequency values, however, suggest that UV irradiation induced mutation at nearly the same efficiency as N(i. Inactivation 200

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130

T i m e (sec)

F i g . 3- U V i n a c t i v a t i o n

curve.

Attempts to isolate auxotrophic mutants were made. For the penicillin selection method an unusually high frequency of mcf mutants was observed. Of approx. 8-IO * mutagen-treated colonies screened, about 25 ° suspected colonies were picked out and tested for nutrient dependence. All suspected colonies were found not to be auxotrophs. Characterization ofyel mutant. The pigment ratios calculated Iron: the absorption spectrum (Table I) show lower amounts of phycocyanin, chlorophyll a and the major carotenoid fraction. The colonies of the yellow mutants began growth on agar as standard type (WT) but upon further incubation turned yellow. These cells were not dead since upon transfer to fresh agar medium, growth resumed and the green to yellow pigment pattern was repeated. The reason for this change in colony pigment became apparent when the mutant was grown in Dm liquid medium at various pH's ranging from 6.0-9. 5 (Table II). The culture turned yellow only above pH 8.0. The pH surrounding the colony on the agar during growth rose and the color of the colony turned from green to yellow. Various inorganic substances such as NO:c and (NH4) +, ,~lulation Res., ~ (1969) 5 3 1 - 5 3 6

MUTAGENESIS OF A . n i d u l a n s

BY N G AND U V

535

TABLE I PIGMENT

RATIOS OF YELLOW

MUTANT

A bsorbance ratios ~

WT

Yellow mutant

A 6so/A 590 A s25/A ~9o A51o/A~9 o

1 .o 1.o8 1.68

0.785 o.875 1.45

a A~80' c h l o r o p h y l l a; Ae25, p h y c o c y a n i n ; A510, c a r o t e n o i d ; A~90, w a v e l e n g t h a t w h i c h g r o w t h y i e l d was m e a s u r e d .

and organic nutrients (1% nutrient broth, o.I°J/o casein hydrolvsates,, o.1% .veast extract, glucose, acetate, etc.), were tried but they failed to reverse the phenotypic color change. The frequency of reversion to WT was approx, lO-7. The blue pigment m u t a n t was neither reparable by organic or inorganic nutrients nor by pH changes. No further characterization was done. Reasons for the absence of auxotrophs m a y be technical, intrinsic or related to genetic structuring. LI et al. 1° noted that, in general, nutritional nmtants of photoautotrophs occur in lower frequencies than those of heterotrophs. The authors concluded that the lower frequencies of nutritional mutants reported were not due to technical aspects but to inherent differences between these organisms. The inability to isolate auxotrophs of Anacystis could be due to the following reasons: (z) Chemical mutagens do not penetrate the cell, or physical nmtagenic agents such as UV irradiation are absorbed by carotenoids, etc., which protect the DNA from damage; (2) Anacystis has an efficient DNA repair mechanism; (3) The occurrence of intrinsic genetic duplication ; (4) Intermediate metabolites or pathways are coupled to, or are solely dependent on, photosynthesis. The present report demonstrates relatively high mutation frequencies of pigment and morphological mutants, thus ruling out (z) and (2). Intrinsic genetic duplication (3) provides a possible answer. Duplication of cistrons can occur in tandem fashion, or alternatively, multiple replicons 6 could be fused together by a replicator thus forming a "diploid" replicon structure which leads to duplication of cistrons in a non-tandem mode. Subsequent evolutionary events could lead to a heterodiploid segregating unit. Hence, all cistrons need not now be duplicated. Cistrons coding for pigment and morphological phenotypes which occur at frequencies of 3' IO a to I" IO-a could be unique in such a postulated genetic structure; however, cistrons coding for TABLE II I'2FFECT OF

pH

6.0 6. 5 7.o 7-5 8.0 8-5 9.o 9-5

pH

ON PIGMENT

MUTANTS

W i l d - t y p e control

Yellow mutant

Blue mutant

Growth

Growth

(;rowth

Cc)-lor

2 F 4+ 4q 4~ 4+ 4+

blue blue bl ue blue blue blue

Color

Color

-. 2+ 4+ 4 4 44+ 4+

.

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green green green green green green

.

2 + 4 } 4 ? 4+ 4 ~ 4 --

.

green green ye l l ow yellow ye l l ow yellow

---. 2 + , 4 + s t a n d for none, fair a n d good g r o w t h , r e s p e c t i v e l y . 3 I u t a t i o n R e s . , 8 (1969) 5 3 1 - 5 3 6

530

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s y n t h e s i s of l n e t a b o l i t e s c o u l d l~e d u l d i c a t e d , t h u s r e q u i r i n g m u t a t i o n f r e q u e n c i e s less t h a n 8 " i o L T h e s t r i c t d e p e n d e n c y u p o n p h o t o s y n t h e s i s f~)i- all i n t e r m e d i a t e l n e t a b o l i t e s i> d i f f i c u l t t o e x p l a i n . A n a c v s t i s is a n o b l i g a t e p h o t o a u t o t r o p h 7 ; in t h e d a r k t h e r e is n<, u p t a k e of r a d i o a c t i v e l y l a b e l e d a c e t a t e a n d g l u c o s e 1', a n d d e p r e s s e d m a c r o m o l e c u l a r s y n t h e s e s 2 s u p p o r t t h i s n o t i o n . H o w e v e r , IH( a c e t a t e is i n c o r p o r a t e d in a m o u n t s ~f IO 18°'o of t h e t o t a l c a r b o n c o m p o u n d s of tile cell in t h e light"',l~; g l u c o s e a n d o t h e r amino acids are incorporated to a lesser extent. These results suggest that a t)ermca b i l i t y b a r r i e r is n o t a n l a j o r p r o b l e m a n d t h a t e x o g e n o u s l y s u p p l i e d o r g a n i c m e t a b o l i t e s c a n s e r v e as a l t e r n a t i v e a n d s u f f i c i e n t s o u r c e s of m e t a b o l i t e s . T h u s n m t a t i o n t o a u x o t r o p h y s h o u l d b e p o s s i b l e s i n c e t h e o n l y r e q u i r e m e n t is a n i n a c t i v a t i o n of o n e ~r " d u p l i c a t e d " c i s t r o n s . F u r t h e r e x p e r i m e n t s a r e n o w in p r o g r e s s t o i n d u c e a u x o t r o p h i c n m t a n t s of A n a c v s t i s n i d u l a n s b a s e d o n g e n e t i c d u p l i c a t i o n as d i s c u s s e d a b o v e . TM

ACKNOWL1r.I)GEMENTS

T h i s w o r k w a s s u p p o r t e d in p a r t by' Life I n s u r a n c e M e d i c a l R e s e a r c h F o u n d a t i o n No. G-67-34. D u r i n g t h e p e r i o d of t h e i n v e s t i g a t i o n , Y.A. h e l d a p r e d o c t o r a l traineeship given by National Aeronautics and Space Administration. REFERENCES I ALLEN, ~l. B., The cultivation of Myxophyceae, Arch. Mihrobiol., ~7 (1952) 34 53. 2 ASATO, Y., AND C. E. FOLSOME, in preparation, 1969. 3 BAZlY, M. J., Sexuality in a Blue-green alga: Genetic recombination in Anacystis nidulans, Nature, 218 (i969) 282-283 . 4 CARR, N. G., .aND J. PEARCE, Photoheterotrophism in blue-green algae, Biochcm..1., 99 (I960) 28 pp. 5 DAVIES, B. D., Isolation of biochemicallv deficient m u t a n t s of bacteria by penicillin, .l. ,4 m. Chem. Soc., 7o (1948) 4267 . 6 JACOB, F., S. BRENXER AND F. CUZIN, ()n the regulation of DNA replication in bacteria, ('old Spring Harbor Syrup. Quant. Biol., 28 (1963) 329-348. 7 KRATZ, W. A., AND J. MYERS, Nutrition and growth of several Blue-green algae, Am. ,]. 13otany, 42 (1955) 282-287. 8 I
9 KUMAR, 1t. D., Action of mutagenic chemicals on Anacvslis nidula~zs, 11 I. N-methvl-N'-nitro N-nitrosoguanidine, Arch. Mil¢robiol., 63 (1968) 95 to2. lO IA, S. L., G. P. R E D E I AND (~. S. (;On,VANS, A p h y l o g e n e t i c comparison of m u t a t i o n spectra, Biol. Gcn. (;e~zet., lOO (i967) 77-83. I I SINGll, H. N., Genetic control of sporulation in the Blue green alga, Anabaena d,lio/um Bha radwaja, P/anta, 75 (I967) 33 38. 12 SINGI[, l{. N., AND l{. SINHa, ( ; e u e t i c r e c o m b i n a t i o n in Blue-green alga, (7.vli~tdrospcrmun~

ma/us

l
Nalure,

207 (1005) 7 8 2 - 7 S 3 .

13 Slx(;H, 11. N., H. 1). t
I0 \:AN BAALI,;N, ('., Quantitative surface plating of coccoid tglue-green algae, .]. t)hyco/., i {1q65) ~9 22. 17 \:AN ICAALEN, ('., Mutation of the Blue green alga, Anacvstis nidula*~s, .S'ch'~*cc, 149 (1905) 7o. 18 XeVERBIN, | t .

ANI) (7. S. RUPERT, P r e s e n c e of p h o t o r e a c t i v a t i n g

e n z y m e in b l u e g r e e n a l g a l

cells, Photochem. Photobiol., 7 (I968) 225 230. 19 \Vu, J. H., R. A. LEWlN AND H. WERBIN, Photoreactivation of UV irradiated Blue-green algal virus LPP-~, l"irology, 31 (1957) 637-664 . Mutation Res., 8 (I969) 531-53 o