Australian Journal of Basic and Applied Sciences, 5(12): 496-500, 2011 ISSN 1991-8178

Mutation Induction in Aspergillus terrus Using N-Methyl-N-Nitro-N-Nitrosoguanidine (NTG) and Gamma Rays
Mohamed H.Z. Mutwakil Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Kingdom of Saudi Arabia.
Abstract: Chemically and physically induced mutations of Aspergillus terrus strain isolated and identified from Saudi Arabia were studied by exposing the conidia of this fungus to N-methyl-N-nitroN-Nitrosoguanidine (NTG) at a concentration of 0.0075g/10ml and different doses of gamma rays at 20, 30, 40 and 50 KRad. The mutagens resulted in the induction of autotrophic and color mutants of conidia of A. terrus. The percentage of survival conidia and the percent of indicated mutants linearly decreased when exposing time to NTG increased. On the other hand, the survival percent of A. terrus linearly decreased when the dose of gamma rays increased in the second experiment. It was found that the majority of obtained mutants in two experiments were amino acid auxotrophs. Key words: Mutation induction, Aspergillus terrus, N-methyl-N-nitro-N-Nitrosoguanidine, Gamma rays. INTRODUCTION N-Methyl-N-nitro-N-nitrosoguanidine (NTG) has been widely used to induce mutations in bacteria. It has proved highly effective, so much so that it has been suggested to be the most potent chemical mutagen yet discovered (Adelberg, et al., 1965). They found that mutations to valine resistance and to autotrophy occurred at high frequency (up to 42-5 % auxotrophs) after exposure of E. coli to NTG under conditions such that about 5 % of the treated bacteria remained viable. NTG has also proved a very effective mutagen for yeast, though the results are less dramatic than with E. coli. (Megnet, 1965) with Schizosaccharomyces pombe found that, without selection, NTG-induced auxotrophs increased to a maximum frequency of about 8% at 20% survival. On the other hand Nordstrom (1967) obtained up to 50 % petite mutants among survivors of Saccharomyces cerevisiae after NTG treatment. Pal et al. (2005) isolated two cadmium resistant mutants (Cd1 and Cd2) of Aspergillus niger, among the six isolated by mutagenization with N-methyl N-nitro-N-nitrosoguanidine (MNNG). Selection and mapping of mutations affecting cysteine synthesis in A. nidulans was carried out. A new locus, cysE, is described, the mutants of which are deficient in in vivo conversion of O-acetylserine to cysteine, a step mediated by cysteine synthase. Three loci (cysB, C and E) were thus found to control this step in vivo, apparently without affecting the enzyme activity in vitro. By scoring for propargyl glycine sensitivity of cys mutants, chromosomal map positions were obtained for all five cysteine loci (A, B, C, D and E) (Cybis et al., 1988). The mutagenic effect of (N.T.G) on A. fumigates and A. nidulans were studied by Levadoux et al., (1981), Das et al., (1983), Baeshen and Sabir (1987) who found that NTG has proved a very effective mutagen and produce a great number of autotrophic mutants in these fungus. A. niger C was treated with UV and gamma irradiation (60Co), nitrogen mustard and colchicines. Mutagenic treatments resulted in substrains with either increased or decreased citric acid production, while some were unchanged in this respect. Low-yielding strains predominated in all the experiments. Analysis of the superior sub-strains showed gamma irradiation to be most effective in producing the greatest number of superior mutants (Das and Nandi, 1972). Gamma-ray exposure was used as a mutagenic agent for mutant induction from a strain of Humicola lutea 72 producing acid proteases. The second stage of irradiation resulted in a double active mutant (H. lutea 120-8). Further treatment gave rise to a triple active mutant (H. lutea 120-5) with a maximal proteolytic activity of 1,800~g tyrosine liberated from 2% casein/m1 culture filtrate/min at pH 3,0 and 40 ~ C. A change in the composition of proteolytic enzymes caused by gamma-ray exposure was established, using chromatography on DEAE-cellulose (Grigorov et al., 1983). Fadel and El-Batal (2000) studied the effect of gamma rays on A. niger wild type strain they obtained active enhanced isolates producing high level of amylolytic enzymes. Haq (2004) investigated that citric acid production by some selected mutant strains of A. niger from cane molasses in 250 ml Erlenmeyer flasks. A conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitrosoguanidine (MNNG). Out of 3,2-deoxy-d-glucose resistant variants, GCMC-7 was selected as the best mutant.
Corresponding Author: Mohamed H.Z. Mutwakil, Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Kingdom of Saudi Arabia. E-mail: mmutwakil@kau.edu.sa

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Singh et al. (2001) produced A. niger ORS-4.410, mutant of A. niger ORS-4 by repeated irradiation with UV rays. Treatment with chemical mutagens also resulted into mutant strains. The mutants differed from the parent strain morphologically and in gloconic acid production. The relationship between UV treatment dosage, conidial survival and frequency of mutation showed the maximum frequency of positive mutants (25%) obtained along with a conidial survival of 59% after second stage of UV radiation. Kattab and Bazaraa (2005) screened various strains of A. niger for extracellular glucose oxidase activity. The most effective producer strain FS-3 (15.9 U mL-1), was mutagenized using UV irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy D-glucose and 17 of these exhibited higher glucose activities (from 114.5 to 332.1%) than the original S-3 strain. Following determination of antifungal resistance of the highest producing mutants, four were selected and used in protoplast fusions in three different interspecific crosses. All fusants showed higher activities (from 285.5-394.2%) than the original strain. Moreover, of the 30 fusants isolated 19 showed higher glucose oxidase activity than their corresponding higher producing parental strain. Wong, et al. (2006) developed a statistical method named MPA (Mutagenesis Assistant program) to equip protein engineers with a tool to develop promising directed evolution strategies by comparing 19 mutagenesis methods. In this study the purpose of mutagenesis to select the colonies of A. terrus which produce autotrophic and colored mutants. Mutation procedures can be optimized in term of the type of mutagen and dose. Mutagens specificity can be taken into account and mutagenesis itself can be enhanced or directed in order to obtain the maximum frequency of desirable mutant type among the isolates to be screened. MATERIALS AND METHODS Microorganism: The test organism in this work, A. terrus Thom, was isolated from soil taken from Makkah- Jeddah road and identified at Commonwealth Mycological Institute at No. 249122 (El-Sharkawy et al., 1981). Media: Wild type and mutant strains of A. terrus were grown at Prune-Extract agar complete medium (Tallboys, 1960). Difco Czapek-Dox agar and supplemented (when supplemented with auxotrophs) medium, (Difco Laboratories, Detroit, Michigan, USA). Spore Counting: Counting of Spores 0.1ml of sore suspension was poured onto heamocytometer under the cover slip. By using light microscope, spores were counted (Kolmer et al., 1959). Chemical Mutagensis: N-methyl-N-nitrosoguanidine (MNNG) (Musilkova et al., 1978) was used to induce autotrophic mutants in A. terrus at concentration of 0.0075g/10ml spore suspension (Sabir et al., 1987). Mutagensis using MNNG: Stock solution 0.0075g of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was dissolved in 5 ml buffer saline. Different time intervals were selected for chemical mutagenesis. 5mL of MNNG stock solution and 5ml of Prune-Extract medium containing spore of A. terrus (1x107 spores ml-1) were added in flask, kept in water bath at 24oC. After interval time of 30 and 70 min. samples was centrifuged to collect cells and to remove mutagen from spore suspension. Serial dilution was done and about 80 to 100 spore spread at Prune-Extract agar medium. Mutagenesis Using Gamma Radiation: Wild type A. terrus spore suspension counted and added to 9 ml buffer saline in different 5 test tubes. 1ml taken from each tube as a control. Five different doses of gamma radiation were selected, which were 20, 30, 40, 50 and 60 KRad. samples taken at 0, 15, 30, 45, 60, 70 min. after exposure. A diluted sample spreads at PruneExtract agar medium (Gromada and Fjedurek, 1997). Selection of Mutant: Autotrophic mutants selection were isolated according to Fincham et al. (1979) and Pontecorvo (1949). Statistical Analysis: Data of survival percentage and frequency of auxotrophic mutants were subjected to Linear Regression

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calculations for the detection of linear relationship between concentration of mutagen and or time of exposure of gamma radiation to each concentration used and auxotrophic mutants frequency using Microsoft Excel 2002 for Ms-Windows (Sharma et al., 1970). RESULTS AND DISCUSSION Chemical and physical mutagens induce mutational, non-randomly distributed within a sequence and occur frequently in some sites than expected, originating mutagen-specific patterns of mutations. In recent procedures such as rational screening and genetic engineering have begun to make a significant contribution to this activity but mutagenesis and selection so called random screening is still a cost effective procedure and for reliable short term strain development is frequently the method of choice (Rowland, 1984).

Fig. 1: Growth of wild type Aspergillus terrus strain. Two different experiments were performed in this study to instigate the effect of N-methyl-N-nitro-NNitrosoguanidine (NMNG) and Gamma rays in the induction of autotrophic and colored mutants in the conidia of A.terrus.Conidial spores of wild type A. terrus treated with N-methyl-N-nitro-N-Nitrosoguanidine (N.T.G) at a concentration of 0.0075gm/ml spore suspension. The survival percentage and number of produced mutant at different time intervals (0.30 and 70 min.) were indicated in Table (1) and Fig. (1). Table (1) shows that the time treatments were 20 and 70 min. and the number of treated spores were 200 spores. The number of germinated spores decreased to become 144 at 20min. and 88 at 70min. comparing to 200 at 0 treated times. Data also showed that the number of un-germinated spores decreased at 20 min. and become to 46 and 112 at 70 min treatment time survival percentage also decreased to 27 and 44 respectively. The percentage of auxotrophic mutant 1% each and colored mutant 0.333 of each treated spores. Results also showed that the survival percentage and recovery of treated spores of A. terrus with (NTG) and the ability of spore germination percentage. Data showed that spore germination decreased as confirmed by linear regression calculation, when exposing time increased, and the survival percentage decreased with exposing time increased. The highest possible percentage of mutation ca. 44% at exposure time of 70min. data also indicated that 50% survival mutant produced when treated spores with (NTG) for 58min. the lethal dose of (NTG) was low at all exposing times. Table (1) and Fig. (1) represented that when treated water suspension conidial spores of A. terrus to (NTG) at a concentration of 0.0075/10ml at different time intervals it were 0. 30, and 70min., the survival percentage decreased when exposing mutagenic time increased and mutagenic percentage increased linearly when exposing time increased, and the lethal effect of mutagen very low but auxotrophic and colored mutant produced. These results indicated that the effect of (NTG) as a mutagen on DNA level, these results are in agreement with Ingle (1972), Tayle (1975), Musilkova et al. (1978) and Baeshen and Sabir (1987).
Table 1: The survival percentage and number of produced mutant at different time intervals (0.30 and 70 min.). Treatment/min. No. of No. of No. of un- Survival No. of Autotrophic treated germinated germinated (%) isolated mutants spores spores spores Colonies No. % 0 200 200 100 300 20 200 154 46 77 300 1.00 1 70 200 88 112 44 300 2.00 1 Colored mutants No. % 1 0.333 1 0.333

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To obtain an instant mutant, high dose of mutagen required. For this purpose, 0.0075g/10ml for 20min. dose rate, produced 46 un-germinated spore comparing with 0.0075g/10ml for70 min. dose rate that produced 112 un-germinated spore. this showed, exposure of spore suspension to (NNG) gave kill which was determined as the best mutant having the ability to hyper produce an autotrophic mutants, these data are in agreement with Musikova et al. (1978) they reported that (NNG) strongly influenced the variability of A. niger as the number of spores capable of growth, decreased with the duration of treatment while morphological/biochemical mutants considerably increased. It has been well documented that N-methyl-N-nitrosoguanidine (NNG) is one of the strong and multi-potential carcinogen that has been frequently reported inducing mutations (Zhu et al., 2000; Lino and Tersa, 1998). Mutation with Gamma Rays: Gamma rays, high energy beams, very penetrating substantial thickness of heavy metals as leads, steel or concrete to shield them, so that, gamma radiation was used as a mutagen. Various doses of -rays were used to induce mutation in A. terrus it was found that a dosage of (80) KRad dose rate was optimum for the mutation of A. terrus. produced 60% killing of the fungal spores (Table 2). Mutants of A. terrus were isolated as the methods of Smith and wood (1991) which carried out in semi-dark room. The dose of (80) KRad. produced a 60% killing of spore suspension. The high dose of irradiation decreased the frequency of positive mutations and the number of viable colonies as described by Petruccioli et al, (1995). Kattab and Bazaraa (2005) reported that 11 out of 200 mutants colonies of A. niger were isolated after exposing to UV treatment. The data also revealed that the number of resistance colonies increased by increasing the exposing time. Results are in agreement with Park et al. (2000). REFERENCES Adelberg, E.A., M. Mandel and C.C. Chen, 1965. Optimal conditions for mutagenesis by N-methyl-NnitroNnitrosoguanidine in Escherichia coli K12 Biochem. Biophys. Res. Commun., pp: 788-795. Baeshen, N.A. and J.M. Sabir, 1987. Some genetic studies on Aspergillus terrus Thom., Researches Sci. K.A.U., pp: 215-224 Cybis, J., R. Natorff, I. Lewandowska, W. Prazmo and A. Paszewski, 1988. Mutation affecting cysteine synthesis in Aspergillus nidulans: characterization and chromosome mapping. Genet. Res. Camb., 51: 85-88. Das, A. and P. Nandi, 1972. Specific effects of mutagens on Aspergillus niger producing citric acid. Folia Microbiol., 17: 248-250. Das, T.K. and K. Sen, 1983. Induced mutation developing delta-9 desaturase defective unsaturated FattyAcid requiring mutant of Aspergillus nidulance., Indian J. Exp. Biol., 6: 339-342. El-Sharkawy, H.M., A. Malibari, M.K.Z. Elshaieb and K. Tawfik, 1981. Some edapho-ecological factor controlling the distribution of soil fungi in the western region of Saudi Arabia., Bull. Fac., K.A.U. Jeddah, 5: 103-115. Fadel, M. and A.I. El-Batal, 2000. studies on activation of amylolytic enzymes production by gamma irradiated Aspergillus niger using some surfactants and natural oils under solid state fermentation. Pakistan Journal of Biological Seience, 3(10): 1762-1768. Fincham, J.R.S., R. Day and A. Radford, 1979. Fungal Genetics., University of California press. Berkley and Los Angles. Grigorov, I., P. Aleksieva, A. Djerova, P. Sheremetska and B.Tchorbanov, 1983. Selection of Gamma-Ray Mutants from a Strain of Humicola lutea 72, Producing Acid Proteases. Eur. J. Appl. Microbiol. Biotechnol., 17: 355-357. Gromada, A. and J. Fideurek, 1997. Selective isolation of Aspergillus niger mutants with enhance glucose oxidase production. J. Appl. Microbiol., 82: 648-652. Haq, I., S. Ali, M.A. Qadeer and J. Iqbal, 2004. Citric acid production by selected mutants of Aspergillus niger from cane molasses. Bioresourse echnology, 93(2): 125-130. Ingle, M.R., 1972. Factors affecting the formation and stability of Verticillum diploid. Ph..D. thesis, University of Dundee. Jan, C., N. Renata, L. Irmina, P. Wieslawa and P. Andrzej, 1988. Mutations affecting cysteine synthesis in Aspergillus nidulans: characterization and chromosome mapping. Genetical Research, 51: 85-88. Kalmer, J.A., E.H Spaulding and H.W. Robinson, 1959. Approved Laboratory techniques 5th ed. Appleton. New York., pp: 54-60. Kattab, A.A. and W.A. Bazaraa, 2005. Screening mutagenesis and protoplast fusion of Aspergillus niger for the enhancement extracellular glucose oxidase production. J. Ind. Microbiol. Biotechnol., 32: 289-294. Levadoux, W.L., K.F. Gregory and A. Tailor, 1981. Sequential cold sensitive mutations in Asprigillus Fumigatus analysis by the paraseqtual cycle. J. Bacteriol., 27(3): 295-203.

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