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Isolation of Nitrosoguanidine-Induced Mutants of Streptococcus diacetilactis with Enhanced Ability to Produce Acetoin and Diacetyl
TECHNICAL
NOTES
Isolation of Nitrosoguanidine-lnduced Mutants of Streptococcus diacetilactis with Enhanced Ability to Produce Acetoin and Diacetyl Abstract
Streptococcus diacetilactis was treated with N-methyl-N'-nitro-N-nitrosoguanidine and plated on lactose-citrate-creatinine agar. Cultures started from slightly pink colonies produced lactic acid much more slowly than the original culture and produced much larger amounts of acetoin and diacetyl. Introduction N-methyl-N'-nitro -N-nitrosoguanidine (nitrosoguanidine), used widely by bacterial geneticists since an initial report by Mandell and Greenberg (6), appears to be the most potent chemical mutagen yet discovered. Adelberg et al. (1) found at least one mutation per treated Escherichia coli cell under conditions that permitted 50% survival, and Loprieno and Clarke (5) found nitrosoguanidine produced a higher ratio of mutagenesis to lethality than did other known mutagens, such as ultraviolet light or nitrous acid. The mutagenic action of nitrosoguanidine has been studied with other microorganisms, including Salmonella typhimurium (3), Schizosaccharomyces pombe (5), and Bacillus subtilis (9). Burrow et al. (2) used it in the isolation of diacetyl negative mutants of Streptococcus diacetilactis. This study was initiated to develop a procedure for isolating from S. diacetilactis mutants that produce larger amounts o£ acetoin and diacetyl than the parent culture.
phosphate buffer ( p i t 6.5, containing 0.01 MgSO4) and resuspended in 9 ml of the buffer. A 0.1-ml sample was removed for plating, and I ml of a nitrosoguanidine solution (1 mg/ml in 0.1 ~ sodium acetate buffer, p i t 4.6, sterilized by filtration) was added to the remainder. The mixture was mechanically shaken for 30 minutes in the dark at room temperature and filtered. The cells were washed with 5 ml of lactosecitrate broth, suspended in 15 ml of the broth, and 0.1 ml was removed for plating. The remainder was grown overnight at 22 C and replated. The lactose-citrate broth was that reported by Harvey and Collins (4) except that citrate was 2%. F o r selecting mutants with enhanced ability to produce acetoia and diacetyl, a plating medium was prepared by adding additionally 1.5% agar and 0.5% creatinine.ttC1. 2 The media were autoclaved at 121 C for 13 minutes. Acetoin and diacetyl were determined as the sum of both by the method of Westerfeld (8). Bacterial growth in lactose-citrate broth was followed by periodically measuring OD at 600 nm with a Beckman spectrophotometer~ Model DB, with 1-cm cuvettes, and p H was determined with a Radiometer p H meter, Model 22 (Copenhagen, Denmark).
Experlmental Procedure
Streptococcus diacetilact~ 18-16 was, propagated at 22 C in sterile litmus milk routinely and in lactose-citrate broth immediately before treatment. The procedure for treating with Nmethyl-N'-nitro-N-nitrosoguanidineI was a modification of that reported by Adelberg et a]. (1). A culture of S. diacetilactis grown for two days in citrate broth was diluted to an optical density COD) of 0.1 at 450 nm with lactose-citrate broth, and 10 ml were filtered through a Millipore filter (pore size, 0.45 ~). The cells were washed with 0.2 ~ potassium 1 K & K Laboratories, Hollywood, California. 2 Calbiochemical Co., Los Angeles, California. 282
Results and Discussion
Plate counts indicated that about 80% of S. diacetilactis survived treatment with nitrosoguauidine. This result is approximately the same as 81% reported by Adelberg et al. (1) for a similar exposure of E. coli to nitrosoguanidine. There were several failures in attempts to identify colonies of S. diacetilactis with enhanced ability to produce acetoin and diacetyl. These included: addition of 1.0% or 0.1% hydroxylamine plus 0.03% NiClz.6H20 or addition of 0.02% urea to the plating medium; spraying colonies with an aqueous solution of 1.0% hydroxylamine, 2.0% sodium acetate, and 0.03% NiCl2.6tt20 (7); and spraying colonies with an aqueous solution of 0.5% creatine followed by 5% a-naphthol in 2.5 N a O H (8). Success was achieved by the addition of 0.5% creatinine or 0.5% creatine to the lactose-citrate agar. W i t h creatinine or creatine added to the
TECHNICAL
lactose-citrate agar those colonies with enhanced ability to produce acetoin and diacetyl were slightly pink in contrast to white parental colonies. Isolates from white colonies produced amounts of acetoin and diacetyl similar to those produced by the parent culture, but these amounts were insufficient to give detectably pink colonies on the plating medium. Additionally, the slightly pink colonies were smaller and flatter, and some had a ring near the center. Bacteria from white and pink colonies were cocci in chains , but those from pink colonies were somewhat larger. We studied three isolates from pink colonies and two from white colonies. Results with the isolates from pink colonies were similar in all tests and different from results with isolates from white colonies. They grew and produced lactic acid much more slowly in lactose-citrate broth or litmus milk and required 10 to 12 days to coagulate litmus milk. The isolates from white colonies grew more rapidly in broth or litmus milk, coagulated litmus milk in less than 48 hours, and apparently were the parental type. I n lactose-citrate broth they produced maximal acetoin plus diaeetyl (2.4 ~g/ml) in 33 hours (OD ---- 5.0). Isolates from the slightly pink colonies produced more acetoin plus diaeetyl, 4.2 ~g/ml in 33 hours (0D ---1.7) and 15.2 ~g/ml in 10 days (0D ~-- 6.0). At 10 days the p i t in these cultures had decreased only to 5.2 to 5.3. The diacetyl negative mutants isolated by Burrow et al. (2) following treatment with nitrosoguanidine produced acid more rapidly than the parent culture. The mutants we isolated produced acid more slowly than the parent culture and, judged by the rates of production of acid and acetoin plus diacetyl, reverted to the parental type upon being propagated serially at 10-day intervals for two months in litmus milk. The procedure we developed is
283
NOTES
proposed for attempts to isolate stable fastgrowing mutants with enhanced ability to produce diacetyl and acetoin. LINDA
F. CHUANG and E. B. COLLINS,
Department of Food Science and Technology, University of California, Davis 95616 References
(1) Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine in J~scherichia coli K12. Biochem. Biophys. Res. Comm., 18: 788. (2) Burrow, C. D., W. ]~. Sandine, P. R. Elliker, and C. Speckman. 1970. Characterization of diacetyl negative mutants of Streptococcus diaveti~aetis. J. Dairy Sci., 53:121. (3) Eisenstark, A., R. Eisenstark, and R. Yah Sickle. 1965. Mutation of Salmonella $yphimurium by nitrosoguanidine. Mutation Res., 2:1. (4) Harvey, R. J., and E. B. Collins. 1961. Role of citritase in acetoin formation by Streptococcus diacetilactis and Leueonostoc eiSrovorum. J. Bacteriol., 82:954. (5) Loprieno, N., and C. H. Clarke. 1965. Investigations on reversions to methionine independence induced by mutagens in Schizosacvharomyves pombe. Mutation Res., 2: 312. (6) Mandell, J. D., and J. Greenberg. 1960. A new chemical mutagen for bacteria, 1methyl-3-nitro-l-nitrosoguanldine. Biochem. Biophys. Res. Comm., 3: 575. (7) Stotz, E., and J. Raborg. 1943. A colorimetric determination of acetoin and diacetyl. J. Biol. Chem., 150: 25. (8) Westerfetd, W. W. 1945. A colorimetric determination of blood acetoin. J. Biol. Chem., 161: 495. (9) Yoshida, T., and S. Yuki. 1968. Action of N-methy]-N'-nitro-N-nitrosoguanidinein Bacillus s~ebtilis. Japanese J. Genetics, 43: 173.
JOUR~AL OF DAIRY SCIEI~CE VOL. 54, ~O. 2
NOTES
Isolation of Nitrosoguanidine-lnduced Mutants of Streptococcus diacetilactis with Enhanced Ability to Produce Acetoin and Diacetyl Abstract
Streptococcus diacetilactis was treated with N-methyl-N'-nitro-N-nitrosoguanidine and plated on lactose-citrate-creatinine agar. Cultures started from slightly pink colonies produced lactic acid much more slowly than the original culture and produced much larger amounts of acetoin and diacetyl. Introduction N-methyl-N'-nitro -N-nitrosoguanidine (nitrosoguanidine), used widely by bacterial geneticists since an initial report by Mandell and Greenberg (6), appears to be the most potent chemical mutagen yet discovered. Adelberg et al. (1) found at least one mutation per treated Escherichia coli cell under conditions that permitted 50% survival, and Loprieno and Clarke (5) found nitrosoguanidine produced a higher ratio of mutagenesis to lethality than did other known mutagens, such as ultraviolet light or nitrous acid. The mutagenic action of nitrosoguanidine has been studied with other microorganisms, including Salmonella typhimurium (3), Schizosaccharomyces pombe (5), and Bacillus subtilis (9). Burrow et al. (2) used it in the isolation of diacetyl negative mutants of Streptococcus diacetilactis. This study was initiated to develop a procedure for isolating from S. diacetilactis mutants that produce larger amounts o£ acetoin and diacetyl than the parent culture.
phosphate buffer ( p i t 6.5, containing 0.01 MgSO4) and resuspended in 9 ml of the buffer. A 0.1-ml sample was removed for plating, and I ml of a nitrosoguanidine solution (1 mg/ml in 0.1 ~ sodium acetate buffer, p i t 4.6, sterilized by filtration) was added to the remainder. The mixture was mechanically shaken for 30 minutes in the dark at room temperature and filtered. The cells were washed with 5 ml of lactosecitrate broth, suspended in 15 ml of the broth, and 0.1 ml was removed for plating. The remainder was grown overnight at 22 C and replated. The lactose-citrate broth was that reported by Harvey and Collins (4) except that citrate was 2%. F o r selecting mutants with enhanced ability to produce acetoia and diacetyl, a plating medium was prepared by adding additionally 1.5% agar and 0.5% creatinine.ttC1. 2 The media were autoclaved at 121 C for 13 minutes. Acetoin and diacetyl were determined as the sum of both by the method of Westerfeld (8). Bacterial growth in lactose-citrate broth was followed by periodically measuring OD at 600 nm with a Beckman spectrophotometer~ Model DB, with 1-cm cuvettes, and p H was determined with a Radiometer p H meter, Model 22 (Copenhagen, Denmark).
Experlmental Procedure
Streptococcus diacetilact~ 18-16 was, propagated at 22 C in sterile litmus milk routinely and in lactose-citrate broth immediately before treatment. The procedure for treating with Nmethyl-N'-nitro-N-nitrosoguanidineI was a modification of that reported by Adelberg et a]. (1). A culture of S. diacetilactis grown for two days in citrate broth was diluted to an optical density COD) of 0.1 at 450 nm with lactose-citrate broth, and 10 ml were filtered through a Millipore filter (pore size, 0.45 ~). The cells were washed with 0.2 ~ potassium 1 K & K Laboratories, Hollywood, California. 2 Calbiochemical Co., Los Angeles, California. 282
Results and Discussion
Plate counts indicated that about 80% of S. diacetilactis survived treatment with nitrosoguauidine. This result is approximately the same as 81% reported by Adelberg et al. (1) for a similar exposure of E. coli to nitrosoguanidine. There were several failures in attempts to identify colonies of S. diacetilactis with enhanced ability to produce acetoin and diacetyl. These included: addition of 1.0% or 0.1% hydroxylamine plus 0.03% NiClz.6H20 or addition of 0.02% urea to the plating medium; spraying colonies with an aqueous solution of 1.0% hydroxylamine, 2.0% sodium acetate, and 0.03% NiCl2.6tt20 (7); and spraying colonies with an aqueous solution of 0.5% creatine followed by 5% a-naphthol in 2.5 N a O H (8). Success was achieved by the addition of 0.5% creatinine or 0.5% creatine to the lactose-citrate agar. W i t h creatinine or creatine added to the
TECHNICAL
lactose-citrate agar those colonies with enhanced ability to produce acetoin and diacetyl were slightly pink in contrast to white parental colonies. Isolates from white colonies produced amounts of acetoin and diacetyl similar to those produced by the parent culture, but these amounts were insufficient to give detectably pink colonies on the plating medium. Additionally, the slightly pink colonies were smaller and flatter, and some had a ring near the center. Bacteria from white and pink colonies were cocci in chains , but those from pink colonies were somewhat larger. We studied three isolates from pink colonies and two from white colonies. Results with the isolates from pink colonies were similar in all tests and different from results with isolates from white colonies. They grew and produced lactic acid much more slowly in lactose-citrate broth or litmus milk and required 10 to 12 days to coagulate litmus milk. The isolates from white colonies grew more rapidly in broth or litmus milk, coagulated litmus milk in less than 48 hours, and apparently were the parental type. I n lactose-citrate broth they produced maximal acetoin plus diaeetyl (2.4 ~g/ml) in 33 hours (OD ---- 5.0). Isolates from the slightly pink colonies produced more acetoin plus diaeetyl, 4.2 ~g/ml in 33 hours (0D ---1.7) and 15.2 ~g/ml in 10 days (0D ~-- 6.0). At 10 days the p i t in these cultures had decreased only to 5.2 to 5.3. The diacetyl negative mutants isolated by Burrow et al. (2) following treatment with nitrosoguanidine produced acid more rapidly than the parent culture. The mutants we isolated produced acid more slowly than the parent culture and, judged by the rates of production of acid and acetoin plus diacetyl, reverted to the parental type upon being propagated serially at 10-day intervals for two months in litmus milk. The procedure we developed is
283
NOTES
proposed for attempts to isolate stable fastgrowing mutants with enhanced ability to produce diacetyl and acetoin. LINDA
F. CHUANG and E. B. COLLINS,
Department of Food Science and Technology, University of California, Davis 95616 References
(1) Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine in J~scherichia coli K12. Biochem. Biophys. Res. Comm., 18: 788. (2) Burrow, C. D., W. ]~. Sandine, P. R. Elliker, and C. Speckman. 1970. Characterization of diacetyl negative mutants of Streptococcus diaveti~aetis. J. Dairy Sci., 53:121. (3) Eisenstark, A., R. Eisenstark, and R. Yah Sickle. 1965. Mutation of Salmonella $yphimurium by nitrosoguanidine. Mutation Res., 2:1. (4) Harvey, R. J., and E. B. Collins. 1961. Role of citritase in acetoin formation by Streptococcus diacetilactis and Leueonostoc eiSrovorum. J. Bacteriol., 82:954. (5) Loprieno, N., and C. H. Clarke. 1965. Investigations on reversions to methionine independence induced by mutagens in Schizosacvharomyves pombe. Mutation Res., 2: 312. (6) Mandell, J. D., and J. Greenberg. 1960. A new chemical mutagen for bacteria, 1methyl-3-nitro-l-nitrosoguanldine. Biochem. Biophys. Res. Comm., 3: 575. (7) Stotz, E., and J. Raborg. 1943. A colorimetric determination of acetoin and diacetyl. J. Biol. Chem., 150: 25. (8) Westerfetd, W. W. 1945. A colorimetric determination of blood acetoin. J. Biol. Chem., 161: 495. (9) Yoshida, T., and S. Yuki. 1968. Action of N-methy]-N'-nitro-N-nitrosoguanidinein Bacillus s~ebtilis. Japanese J. Genetics, 43: 173.
JOUR~AL OF DAIRY SCIEI~CE VOL. 54, ~O. 2