Studies on the conidia of Aspergillus oryzae

5o5

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95987

STUDIES ON THE CONIDIA OF ASPERGILLUS ORYZAE.

VII. DEVELOPMENT OF PROTEIN SYNTHESIZING ACTIVITY DURING GERMINATION*

K O K I H O R I K O S H I AND Y O N O S U K E I K E D A

Department o[ Microbiology, The Institute o/ Physical and Chemical Research, Yamato-machi, Saitama Pre[. (Japan) (Received April i8th, 1968)

SUMMARY

A cell-free protein synthesizing system from Aspergillus oryzae has been established. Amino acid incorporation is dependent on ATP, ribosomes, supernatant, polyuridylic acid (poly U), and a critical concentration of magnesium. Amino acid incorporation in vivo into protein fractions during germination of conidia increases after a lag of 30-4 ° rain. The cell-free extract from dormant conidia has less capacity for protein synthesis; following germination protein synthesizing activity rises to 3-4 times that of the dormant system. Analysis of a "limiting factor" in the protein synthesizing system shows that a "limiting factor" is in the ribosome fraction which has lower phenylalanyl-transfer RNA (tRNA) binding activity than that of vegetative ribosomes.

INTRODUCTION

In dormant conidia of Aspergillus oryzae, only 8o-S ribosomal particles are observed. About 60 rain after the breakage of dormancy, polysomes are detected in conidia, and a rapid increase in protein synthesis is initiated~; soluble trehalase and bound fl-glucosidase increase after a lag of about 60 rain, and bound trehalase in about IOO rain (refs. 3, 4). However, D-mannitol dehydrogenase and soluble fl-glucosidase activities per conidium remain unchanged3,4. There is little information on cell-free protein synthesis in A. oryzae, although some properties of transfer RNA (tRNA) of A. oryzae have been reported by TANAKA et al. 5. Recently a cell-free amino acid incorporating system was prepared from conidia and vegetative cells of A. oryzae. This paper deals with (a) the characterization of the cell-flee protein synthesizing system, (b) the activation of the protein synthesizing machinery during germination, and (c) the nature of the defect in the protein synthesizing activity in dormant conidia. * Paper VI is ref. I. Abbreviations: poly U, polyuridylic acid; t R N A , t r a n s f e r R N A ; TMM buffer, o.oi M Tris-HC1 (pH 7.4) containing o.oi M MgCl,, 0.oo6 M mercaptoethanol, 0.o6 M KC1 and 0. 5 M spermidine.

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MATERIALS AND METHODS

Organism and cultivation The preparation of conidia, the medium used, and the growth conditions were reported previously 2.

Preparation o/cell-/ree extracts The cells were washed with o.oi M Tris-HC1 buffer (pH 7.4) containing o.oi M MgC12, 0.006 M mercaptoethanol, 0.06 M KC1, and 0.5 M spermidine (TMM buffer). The cell pellet was frozen and ground with 2-3 times its weight of quartz powder in a pre-chilled mortar (--20°). The enzymes were extracted with TMM buffer equivalent to 3 times the wet weight of the cells. The S-3o fraction was obtained by centrifuging the crude extract twice at 30 ooo x g for 20 rain. The upper 2/3 of the supernatant was saved and dialyzed against TMM buffer for 3 h. From this S-3o fraction, ribosomes were sedimented by centrifugation at lO5 ooo x g for 2 h, washed once with TMM buffer and finally resuspended in TMM buffer. The lO5 ooo × g supernatant was centrifuged at lO5 ooo × g for 3 h, and the upper 2/3 was saved as S-Ioo. To the ribosome fraction and the S-Ioo fraction, 1/5 vol. of glycerol was added and the preparations were kept at --IO °. Aminoacyl-tRNA synthetases were obtained by passing the cell-free extract through a DEAE-cellulose column as described b y YAMANE AND SUEOKA6. Glycerol was added to give a final concentration of 50 % and stored at --20 °.

Preparation o/tRNA tRN~A was prepared from the vegetative cells (IO h cultured) by the method of HOLLEY

7.

Reaction system/or protein synthesis The reaction mixture contained in a total volume of o.25 ml: 2.5 #moles Tris (pH 7.4); 0.25/,mole ATP; 1.25/,mole phosphoenolpyruvate; 5 #g pyruvate kinase (EC 2.7.1.4o); o.oi/*mole GTP; IO/*moles NH4C1; 1.5/,mole mercaptoethanol; 2.5 /*moles MgCI~; o.125/*mole spermidine; 200/*g tRNA; 50/*g polyuridylic acid, o.I/,C (IO m/*mole) E14qphenylalanine; and indicated amounts of S-3o, or S-Ioo and ribosomes. The reaction mixture was incubated at 35 ° for 20 rain. The reaction was stopped by the addition of an equal volume of io % trichloroacetic acid; the precipitate formed was heated at 9°0 for 15 rain, and kept at o ° for 60 rain. The resulting precipitates were filtered onto glass fiber filters and counted for radioactivity b y a Beckman scintillation spectrometer DPM IOO.

Binding o] phenylalanyl-tRNA to ribosomes Ribosome-bound phenylalanyl-tRNA was assayed by the general procedure of NIRENBERG AND LEDERs. Each 5o-/.1 reaction mixture contained o.I M "Iris buffer (pH 7.2); 0.02 M magnesium acetate; 0.05 M KC1; 5o#g poly U; 5 #g (51oo counts] Biochim. Biophys. ,4cta, 166 (1968) 5o5-511

CELL-FREE PROTEIN SYNTHESIS OF

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rain) [14C]phenylalanyl-tRNA; and indicated amount of ribosomes. After 15 min at 25 ° incubation, ribosomal-bound phenylalanyl-tRNA which was retained by cellulose nitrate filters was determined.

Analysis Protein was determined by the method of LOWRY et al2 using bovine albumin as a standard. RNA was determined from ultraviolet absorption (260 m#).

Materials The reagents were obtained from the following source: [l*C3phenylalanine (I mC = 2 #moles) and 14C-labeled amino acid mixture (I #C = 5 #g), from Radiochemical centre; ATP, GTP, phosphoenolpyruvate, pyruvate kinase, and ribonuclease (EC 2.7.7.I6 ), Calbiochem, U.S.A.; poly U, Miles Chemical Co., U.S.A.

RESULTS

Incorporation o/ phenylalanine into protein directed by poly U in an in vitro system The protein-synthesizing activity was quite unstable; the preparation became inactive after 24 h at 5 °. Addition of glycerol (20 %) to the preparation was very effective in stabilizing the activity: S-3o, ribosomes, and S-Ioo did not lose their activities for at least I week at --IO °. The protein synthesizing activity was not affected by the presence of IO % glycerol. As shown in Table I, amino acid incorporation is dependent on ATP, S-3o, ribosomes, S-Ioo, and poly U. Omission of GTP

TABLE I REQUIREMENTS FOR THE INCORPORATION OF ?4C]PHENYLALANINE INTO PROTEIN BY THE CELLFREE S Y S T E M E x p t . I. T h e complete s y s t e m w h i c h c o n t a i n e d 800/zg S-3 o p r o t e i n is described in METHODS. E x p t . 2. The complete s y s t e m contained 45o/zg of S - i o o protein, a n d 15o/~g r i b o s o m a l protein.

Expt. No.

Conditions

Phenylalanine incorporated (pmoles)

Complete -- A T P --GTP --tRNA --poly U --S-3 o + r i b o n u c l e a s e (IO/*g)

432 i io 305 228 6 2 3

Complete ATP --S-Ioo --ribosome --poly U --tRNA

38o 5° 7° 8 5 I IO

-

-

Biochim. Biophys. Acta, I66 (1968) 5o5-51I

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K. HORIKOSHI, Y. IKEDA

resulted in about 3o % inhibition. The system was very sensitive to ribonuclease. Fig. I illustrates the time course of phenylalanine incorporation directed b y poly U. The incorporation was affected considerably b y p H and Mg z+ concentration. As shown in Fig. 2, the optimal Mg ~+ concentration for the system was about io mM and the optimal p H was in the range of 7.0-7.4.

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Fig. I. T i m e c o u r s e of p h e n y l a l a n i n e i n c o r p o r a t i o n i n t o protein. T h e r e a c t i o n m i x t u r e (0.25 ml) c o n t a i n e d 81o fig of S-3 o p r o t e i n a n d w a s i n c u b a t e d a t 35 ° for t h e desired t i m e . Fig. 2. T h e effect of Mg c o n c e n t r a t i o n on t h e i n c o r p o r a t i o n of p h e n y l a l a n i n e into protein. Cond i t i o n s are d e s c r i b e d in t h e t e x t , e x c e p t of Mg 2+ c o n c e n t r a t i o n , w h i c h are i n d i c a t e d in t h e figure. P r o t e i n of S-3 o a d d e d to t h e r e a c t i o n m i x t u r e w a s 81o jug.

Development o~ protein-synthesizing activity in the in vitro system during germination To determine whether a functional protein-synthesizing machinery is carried over from dormant state, an estimate of the capacity of the in vitro incorporating activity of the extracts of conidia and cells during various stages of germination was undertaken. The germination medium (3-1) which had been inoculated with 1.5 g of conidia was placed in a 5-1 erlenmeyer flask and incubated at 32 ° on a rotary shaker. A 4oo-ml aliquot of the broth was taken after o, 20, 40, 60, 12o, 18o, and 36o min. Conidia were collected by centrifugation, washed with TMM buffer, and cellfree extracts were prepared b y the method described above. The enzymes were extracted with 1.5 ml of TMM buffer. The protein-synthesizing activity in o.I ml of the S-3o was determined b y the methods described in the METHODS section. As shown in Fig. 3, radioactivity in the protein fraction increases after a lag of about 30-40 rain, and specific activities (counts/rain per mg protein) of S-3o have constant values after 12o min of cultivation. The specific activity of germinated conidia was about 3 times higher than that of dormant conidia.

Incorporation o/amino acids into protein/raction in the in vivo system Four mg of conidia were suspended in 4 ml of the germination medium which contained 4 #C of amino acid mixture and incubated at 32 ° on a rotary shaker. An Biochim. Biophys. Acta, 166 (I968) 5o5-511

CELL-FREE PROTEIN SYNTHESIS OF

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Fig. 3- I n vitro analysis of the protein-synthesizing capacity during germination. Conidia were germinated as described in the text. E x t r a c t s were prepared from the cultures during various stages of germination and assayed for the ability to incorporate [14C]phenylalanine into protein in the presence of poly U. Fig. 4. Incorporation of 1*C-labeled amino acids into hot trichloroacetic acid-insoluble fractions during germination. An amino acid mixture containing 4 #C was added to 4 ml of germination medium in which 4 mg of conidia were inoculated. An o.5-ml aliquot of the b r o t h was withdrawn at indicated intervals and ra dioactivity in the hot trichloroacetic acid-insoluble fraction was measured.

o.5-ml aliquot of the broth was withdrawn from the culture after o, 15, 30, 45, 60, and 85 rain. To the sample, 0.5 ml of io % trichloroacetic acid was added and heated at IOO° for 15 rain. The resulting precipitate was collected onto a millipore filter, and counted for radioactivity. As shown in Fig. 4, radioactivity in hot trichloroacetic acid-insoluble fraction increases after a lag of about 30 min showing a good coincidence with the results of the in vitro study.

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Fig. 5. Binding of phenylalanyl-tRNA to ribosomes (subtracted values from non-specific binding in the absence of poly U). The reaction mixture (Tris-HC1 (pH 7.2), 5/zmoles; magnesium acetate, I #mole; KC1, 2.5/zmoles; poly U, 5o#g; [14C]phenylalanyl-tRNA, 51oo counts/rain; and indicated amounts of ribosomes, total 5o/zl) was incubated at 25 ° for 15 rain. Biochim. Biophys. Acta, x66 (1968) 5o5-51I

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K. HORIKOSHI, Y. IKEDA

Analysis o~ a "limiting/actor" in the cell-/ree amino acid incorporating system [rom dormant conidia To examine the location of a "limiting factor" in the cell-free system of dormant conidia, ribosomes and S-Ioo from the dormant and vegetative cells (6 h cultured) were prepared and phenylalanine incorporation into protein was tested in a series of heterologous combinations. The protein added per 0.25 ml of the reaction mixture was 39 °/~g of vegetative S-Ioo, 14o #g of vegetative ribosomes, 300/~g of conidia S-Ioo, and IiO #g of conidia ribosomes. The results which are shown in Table II indicate that a "limiting factor" in the conidia extract is not in the S-Ioo fraction, but in the ribosome fraction. T A B L E II ANALYSIS OF T H E " L I M I T I N G F A C T O R " IN T H E C E L L - F R E E AMINO ACID I N C O R P O R A T I N G SYSTEM

V e g e t a t i v e cell e x t r a c t s were p r e p a r e d f r o m 6-h c u l t u r e d cells, a n d conidia e x t r a c t s were p r e p a r e d f r o m d o r m a n t conidia. T h e p r o t e i n a d d e d p e r o.25 m l of t h e r e a c t i o n m i x t u r e s w a s 39o/~g of v e g a t a t i v e S-Ioo, 3oo/~g of c o n i d i a S-ioo, 1 4 o / , g of v e g e t a t i v e r i b o s o m e s , a n d i io/~g of conidia r i b o s o m e s , as indicated. O t h e r c o n d i t i o n s are t h e s a m e as in METHODS.

Vegetative cell extracts

Conidia extracts

S-•oo

Ribosome

S-xoo

+

+ +

+ ~-

+

Ribosome

Phenylalanine incorporated (pmoles) 380 3o4

+

IIO

+

lO5

Binding o/ phenylalanyl-tRNA to ribosomes To reconfirm the results obtained above, the binding capacity of phenylalanyltRNA to ribosomal particles directed by poly U was assayed. Results are shown in Fig. 5. At lower ribosome concentrations, ribosomes from dormant conidia have about 4 ° °/o of the binding ability of vegetative cell ribosomes. At higher concentrations of ribosomes (over 24o#g), no difference could be observed under the tested condition.

DISCUSSION

A cell-free amino acid incorporating system from A. oryzae has been established and found remarkably similar to that of Bacillus subtilis 1°. In vivo experiments show that the incorporation of amino acids into hot trichloroacetic acid-insoluble fractions during germination increases after a lag of about 30-40 rain. In vitro protein synthesizing capacity increases after a lag of about 30-40 rain. From these results, it appears that the protein synthesizing machinery which is carried over from the dormant state is activated and/or increases during germination. The activity of the germinated conidia is three times higher than that of dormant conidia. It is, therefore, quite clear that dormant conidia have a "limiting factor" in the protein synthesizing machinery. A "limiting factor" could be in the following points: soluble flaction (tRNA, Biochim. Biophys. Acta, 166 (i968) 5 o 5 - 5 I I

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amino acyl-tRNA synthetases, transfer enzyme), and/or ribosomes. No differences between the tRNA and amino acyl-tRNA synthetases of conidia and germinated conidia have been observed. Although details will be discussed elsewhere, phenylalanyl-tRNA and phenylalanyl-tRNA synthetase are detected in the soluble fractions of dormant and germinated conidia. From the results in Table II, no "limiting factor" can be detected in the conidia S-Ioo because vegetative ribosomes plus conidia S-Ioo has the same activity as that of vegetative ribosomes plus vegetative S-Ioo, but the "limiting factor" appears to be in the conidia ribosome fraction. These results are confirmed by binding experiments, which show that ribosomes from germinated conidia have higher activity than that of dormant conidia ribosomes. From these findings, it can be concluded that ribosomes are activated during germination, and raise the capacity of the protein synthesizing system. Dormant spores of Bacillus cereus T have defects in the transfer enzyme and ribosomes, and heat treatment causes activation of the transfer enzyme in the soluble fraction, but not of ribosomes n. Dormant conidia of A. oryzae have reserve materials, D-mannitol 3, trehalose 1, polyphosphate TM and other various compounds 13. And conidia of A. oryzae are characterized by high level of endogenous respiration 14. Quantitative comparison of the heat sensitivity of dormant conidia and vegetative cells of Aspergillus niger reveals that the conidia show only 5 ° higher resistance than vegetative cells15. From the results described above, we conclude that conidia of A. oryzae are not in a true dormant state, but in a semi-dormant state.

ACKNOWLEDGEMENT The authors would like to express their thanks to Dr. R. H. DoI for his critical reading of the manuscript. REFERENCES K. HORIKOSHI AND Y. IKEDA, J. Bacteriol., 91 (1966) 1883. K. HORIKOSHI, Y. OHTAKA AND Y. IKEDA, J. Agr. Biol. Chem. Japan, 29 (1965) 724 . K. HORIKOSHI, S. hDA AND Y. IKEDA, J. Bacteriol., 89 (1965) 326. K. HORIKOSHI AND Y. IKEDA, Bioeh{m. Biophys. Acta, i o i (1965) 352. K. TANAKA, A. IV[OTOHASHI,K. MIURA AND T. YANAGITA, J. Gen. Appl. Microbiol., 12 (1966) 277, 6 T. YAMANE AND N. SUEOKA, Proc. Natl. Acad, Sci. U.S., 5 ° (1963) lO93. 7 R. W. HOLL~Y, Biochem, Biophys. Res. Commun., Io (1963) 186. 8 M. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. 9 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265. IO K. HORIKOSHI AND R. H. DoI, Arch. Biochem. Biophys., 122 (1967) 685. I I Y. KOBAYASHI AND H. O. HALVORSON, Arch. Biochem. Biophys., 123 (1968) 622. 12 A. NISHI, J. Bacteriol., 81 (1961) IO. 13 T. YANAGITA, in E. ZEUTHEN, Synchrony in cell division, Wiley, N e w York, 1964, p. 415. t 4 G. TERUI AND T. MOCHIZUKI, Technol. Rept. Osaka Univ., 5 (1955) 218. 15 T. YANAOITA AND S. YAMAGUCHI, Appl. Microbiol., 6 (1958) 375. I 2 3 4 5

Biochim. Biophys. Acta, 166 (1968) 5o5-511