Vol. 124, No. 1 Printed in U.S.A.
JOURNAL OF BACrERIOLOGY, Oct. 1975, p. 48-54 Copyright 0 1975 American Society for Microbiology
Mutation of Bacillus subtilis Causing Hyperproduction of a-Amylase and Protease, and Its Synergistic Effect Y. YONEDA* AND B. MARUO Division of Enzymology, Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Received for publication 9 July 1975
Mutants that had a genetic lesion increasing the production of a-amylase and were isolated from a transformable strain of Bacillus subtilis Marburg by N-methyl-N'-nitro-N-nitrosoguanidine treatment. These mutants produced two to three times more a-amylase and five to 16 times more protease than their parent and were tentatively referred to as AP mutants. As this mutation seems to have occurred at a single gene of the bacterial chromosome and was not located near the a-amylase structural gene, the gene was designated as "pap." When pap- and amyR2 (an a-amylase regulator gene) or pap - and ProH coexisted in the same cell, synergistic effects of the two genetic characters were observed on the a-amylase and protease production, respectively. Upon introduction of the pap mutation, the following phenotypic changes were observed in addition to changes in a-amylase and protease productivity. (i) Mutants lost the character of competence for the transformation. (ii) When cells were cultured at 30 C for 30 h, mutant cells became filament owing to the formation of chains of cells. (iii) Autolysis of cells was decreased in the mutants. When pap was transferred to the wild strain by deoxyribonucleic acid-mediated transformation, the transformants showed all these phenotypic alterations simultaneously. protease simultaneously
in our laboratory by deoxyribonucleic acid (DNA)mediated transformation or mutagen treatment. Media. Bouillon-yeast extract (BY) medium contained the following components per liter of distilled water: bouillon, 5 g; yeast extract, 2 g; polypeptone, 10 g; and NaCl, 2 g. The pH was adjusted to 7.2 by NaOH. Assay of enzyme activity. Assay of a-amylase activity has been described previously (18). Hydrolysis of 100 Ag of soluble starch in 1 min at 40 C was defined as 1 U of enzyme activity. Determination of protease activity was described previously (16). One unit of enzyme activity was defined as the amount of enzyme that increased absorbancy at 275 nm by 0.001 per min at 40 C. To select mutants or transformants with high a-amylase or protease production, a-amylase or protease productivity was determined by measuring the halo size around a colony grown on a BY agar plate containing 1% soluble starch or 1% casein, respectively. Isolation of mutants with a-amylase hyperproductivity. B. subtilis 6160-1 and 6160-2 were grown in BY medium to 100 to 150 Klett units at 37 C, centrifuged, and washed with 0.05 M tris(hydroxymethyl)aminomethane-malate buffer. After cells were resuspended in tris(hydroxymethyl)aminomethane malate buffer, sterile N-methyl-N'-nitro-N-nitrosoguanidine was added to a final concentration of 100 jig/ml as described by Adelberg et al. (1). Treated cells were collected on a membrane filter, washed, and suspended in BY medium to give the same volume of original cell suspension. Glycerol was added to the cell
To date, little is known about systems regulating the production of extracellular enzymes (11, 16, 19, 23, 26). Highly amylolytic bacilli, for instance, Bacillus subtilis var. amylosacchariticus (6) and Bacillus amyloliquefaciens (6, 17), produce a large amount of extracellular enzymes in addition to a-amylase. It is likely that there would be common mechanisms for regulating the production of these extracellular enzymes in addition to those for each enzyme. We have been studying the production of extracellular a-amylase (a-1,4-glucan 4-glucanohydrolase; EC 3.2.1.1) and protease in B. subtilis and reported the isolation of mutants referred to as AP mutants which showed hyperproduction of a-amylase and protease simultaneously from N-methyl-N'-nitrosoguanidine-treated B. subtilis Marburg strain (22). In this communication, we report the results of further characterization of these mutants. MATERIALS AND METHODS Bacterial strains. The bacterial strains used are listed in Table 1. Strains 6160 and Mu8u5u5 were donated by Y. Ikeda and H. Yoshikawa, respectively. B. subtilis var. amylosacchariticus was a gift from J. Fukumoto of Osaka City University. Bacillus natto IAM 1212 is a stock culture at the Institute of Applied Microbiology, The University of Tokyo. Other strains are derivatives of strain 6160 and they were obtained 48
a-AMYLASE AND PROTEASE HYPERPRODUCTION
VOL. 124, 1975
49
TABLE 1. Bacterial strains used B. subtilis Marburgb 6160 6160-1 6160-2
Origin
Genotypea
Strain
amyEm, amyRI, ProL purB6, trpB3, metB5, purB6, trpB3, metB5, str, amyEm, amyRI, ProL aro-116, trpB3, metB5, str, amyEm, amyR), ProL
Mu8u5u5 Mio1o NA64 NP58 YN9 YN21 YN118 YY88
thr, leuA8, metB5, aro-116, hisB2, metB5, str, purB6, metB5, purB6, trpB3, metB5, str, purB6, trpB3, metB5, str, purB6, trpB3, metB5, str, aro-116, trpB3, metB5, str, purB6, metB5, str,
amvEm, amyRI, ProL amyE1010, amyRI, ProL amyEm, amyR2, ProL amyEm, amyRI, ProH amyEm, amyRI, ProL, pap-9 amyEm, amyR21, ProL amyEm, amyRI, ProL, pap-118 amyEm, amyR2, ProL, pap-9
YY11O
purB6, metB5, str,
amyEm, amyR2, ProL, pap-118
YY154
trpB3, metB5, str,
amyEm, amyRI, ProH, pap-118
YY225
trpB3, metB5, str,
amyEm, amyR21, ProL, pap-118
Prototroph,
amyEn, amyR2, ProH
B. natto IAM 1212
Y. Ikeda Ultraviolet treatment of 6160 From strain 6160-1 by transformation from strain M03 (20) N. Sueoka K. Yamaguchi (20) K. Yamaguchi (18) H. Uehara (16) NTGC treatment of 6160-1 NTG treatment of 6160-1 NTG treatment of 6160-2 From strain NA64 by transformation from strain YN9 From strain NA64 by transformation from strain YN 118 From strain NP58 by transformation from strain YN118 From strain YN21 by transformation from strain YN 118 Stock culture
aThe genetic symbols of amyE and amyR mean structural and regulator genes of a-amylase (19). ProH and ProL mean the phenotypic expression of the gene corresponding to the high and low production of neutral protease (16). "These strains are derivatives of B. subtilis 168. c
NTG, N-methyl-N'-nitro-N-nitrosoguanidine.
suspension (final 20%), which was stored in liquid nitrogen. To isolate mutants, frozen cells were melted quickly and grown at 37 C for 3 h in BY medium. The culture was diluted properly and plated on a BY-agar medium containing 1% soluble starch. KI-I, solution (0.01 M) was sprayed on the colonies grown on the plate after an overnight incubation at 37 C. Colonies with a large unstained halo around them were selected, and the levels of a-amylase produced in the culture medium during 24 or 30 h of culture were assayed. Procedure of transformation. DNA used in the transformation was extracted from donor strains according to the method of Saito and Miura (13). The procedures employed in transformation experiments were the same as those described by H. Yoshikawa (24). The concentration of transforming DNA employed was about 1 Asg/ml. Assay of cross-reacting material production. Preparation of antisera against a-amylase and assay of cross-reacting activity were the same as those described by K. Yamane et al. (21).
RESULTS Selection and properties of AP mutants. The procedure for the isolation of mutants by N- methyl - N' - nitro - N- nitrosoguanidine treatment was as described in Materials and Methods. Fifteen strains with hyperproduction of a-amylase were isolated from 115,000 colonies of mutagenized B. subtilis Marburg strains 6160-1 and 6160-2. Nine strains produced high levels of a-amylase only and did not show any change in the productivity of other extracellular enzymes, i.e., protease and ribonuclease. An-
other six strains produced increased levels of protease simultaneously. The former were referred to as A mutants and the latter as AP mutants. The properties of A mutants will be reported elsewhere. In this paper, properties of representative AP mutants (YN9 and YN118) will be described. The parental strain produced 6.5 U of a-amylase and 3.9 U of protease per mg of cells into culture medium at 37 C for 24 h. AP mutants produced two to three times more a-amylase and four to 16 times more protease than their parent (Table 2). Figure 1 shows the time course of the production of a-amylase and protease at 30 C in the parental strain (6160) and AP mutant (YN9). It was shown that a-amylase and protease were produced into the culture medium when the cell growth entered the stationary phase, and that the rates of enzyme production were stimulated in strain YN9. The enzymatic properties of partially purified a-amylase produced by strains YN9 and 6160 were compared. No differences were observed in thermal stabilities, mobilities on polyacrylamide gel electrophoresis, and final products from soluble starch after complete digestion. Both a-amylases cross-reacted with rabbit antiserum against a-amylase produced by B. subtilis var. amylosacchariticus. Immunological reactivity against this serum showed that molecular activities of a-amylase produced by strains YN9 and 6160 were not different (unpublished data). From these results it is suggested that the
50
YONEDA AND MARUO
J. BACTERIOL.
structure of a-amylase of strain YN9 is not different from that of its parent. It has been known that B. subtilis produces two types of extracellular protease, i.e., alkaline protease (serine protease) and neutral protease (metal protease). Two proteases can be separated by diethylaminoethyl-Sephadex A-50 column chromatography (16). AP mutants produced four to 16 times more protease than their parent as a total activity. The proteases produced by the AP mutants (YN9, YN118) were separated into alkaline and neutral protease, and their activities were assayed. The results showed that, although production of
both proteases was elevated by the mutation, the stimulation in the production of alkaline protease was greater. This pattern was also observed in the transformants (YY88, YY110) when this mutation was transferred to another strain by DNA-mediated transformation (Table 3). The elevation of a-amylase and protease production was not due to the increase in total protein synthesis, because cell growth and the production of other enzymes (ribonuclease, alkaline phosphatase, a-,f3-glucosidase, and flgalactosidase) were not altered (3). It was obvious that the mutation stimulated mainly the production of a-amylase and protease. TABLE 2. Production of a-amylase and protease in Genetic analyses of the AP mutants. It is the parental and mutant strainsa possible that the increased production of a-amylase and protease was caused b-y the mutaEnzyme activity (U/mg of cells) Strain tions, one affecting a-amylase production and a-Amylase Protease the other protease production. Thus, we investigated whether this mutant was derived by a Parent single point mutation or not. DNA from strain 6160 6.5 3.9 YN9 was given to normal transformable strains AP mutant (6160, Mu8u5u5) and streptomycin-resistant YN 6 18.5 28.8 transformants were selected on BY agar plates YN 9 20.6 57.9 YN 19 19.7 43.6 containing 1% casein and 100 ug of streptomycin YN 22 13.8 20.7 per ml. Transformants with high protease proYN118 16.1 62.3 duction were selected by observing the halo size YN213 14.4 17.1 around the colonies. All the transformants A mutant (67/67) selected for high protease production YN 21 23.5 2.1 also showed high a-amylase production. The aCells were cultured in BY medium for 24 h at fact that the genetic character responsible for 37 C. the high production of two enzymes did not Activity Klitt kctvity wt" ("AlVn) 0 @ o 0 r Fa A." Ft a b Sao Soo 500 'V F~0 I3
30C
20t
*/
300 200
10
/
100
50
20 10
1
10
/ 1-0-
oI 20
a*0
-
a
a
7 t 0 /io 0. *0~~~~~~~ 0~~ -0 -~~~~~
20
o0
0~~ *
8 10
JOURNAL OF BACrERIOLOGY, Oct. 1975, p. 48-54 Copyright 0 1975 American Society for Microbiology
Mutation of Bacillus subtilis Causing Hyperproduction of a-Amylase and Protease, and Its Synergistic Effect Y. YONEDA* AND B. MARUO Division of Enzymology, Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Received for publication 9 July 1975
Mutants that had a genetic lesion increasing the production of a-amylase and were isolated from a transformable strain of Bacillus subtilis Marburg by N-methyl-N'-nitro-N-nitrosoguanidine treatment. These mutants produced two to three times more a-amylase and five to 16 times more protease than their parent and were tentatively referred to as AP mutants. As this mutation seems to have occurred at a single gene of the bacterial chromosome and was not located near the a-amylase structural gene, the gene was designated as "pap." When pap- and amyR2 (an a-amylase regulator gene) or pap - and ProH coexisted in the same cell, synergistic effects of the two genetic characters were observed on the a-amylase and protease production, respectively. Upon introduction of the pap mutation, the following phenotypic changes were observed in addition to changes in a-amylase and protease productivity. (i) Mutants lost the character of competence for the transformation. (ii) When cells were cultured at 30 C for 30 h, mutant cells became filament owing to the formation of chains of cells. (iii) Autolysis of cells was decreased in the mutants. When pap was transferred to the wild strain by deoxyribonucleic acid-mediated transformation, the transformants showed all these phenotypic alterations simultaneously. protease simultaneously
in our laboratory by deoxyribonucleic acid (DNA)mediated transformation or mutagen treatment. Media. Bouillon-yeast extract (BY) medium contained the following components per liter of distilled water: bouillon, 5 g; yeast extract, 2 g; polypeptone, 10 g; and NaCl, 2 g. The pH was adjusted to 7.2 by NaOH. Assay of enzyme activity. Assay of a-amylase activity has been described previously (18). Hydrolysis of 100 Ag of soluble starch in 1 min at 40 C was defined as 1 U of enzyme activity. Determination of protease activity was described previously (16). One unit of enzyme activity was defined as the amount of enzyme that increased absorbancy at 275 nm by 0.001 per min at 40 C. To select mutants or transformants with high a-amylase or protease production, a-amylase or protease productivity was determined by measuring the halo size around a colony grown on a BY agar plate containing 1% soluble starch or 1% casein, respectively. Isolation of mutants with a-amylase hyperproductivity. B. subtilis 6160-1 and 6160-2 were grown in BY medium to 100 to 150 Klett units at 37 C, centrifuged, and washed with 0.05 M tris(hydroxymethyl)aminomethane-malate buffer. After cells were resuspended in tris(hydroxymethyl)aminomethane malate buffer, sterile N-methyl-N'-nitro-N-nitrosoguanidine was added to a final concentration of 100 jig/ml as described by Adelberg et al. (1). Treated cells were collected on a membrane filter, washed, and suspended in BY medium to give the same volume of original cell suspension. Glycerol was added to the cell
To date, little is known about systems regulating the production of extracellular enzymes (11, 16, 19, 23, 26). Highly amylolytic bacilli, for instance, Bacillus subtilis var. amylosacchariticus (6) and Bacillus amyloliquefaciens (6, 17), produce a large amount of extracellular enzymes in addition to a-amylase. It is likely that there would be common mechanisms for regulating the production of these extracellular enzymes in addition to those for each enzyme. We have been studying the production of extracellular a-amylase (a-1,4-glucan 4-glucanohydrolase; EC 3.2.1.1) and protease in B. subtilis and reported the isolation of mutants referred to as AP mutants which showed hyperproduction of a-amylase and protease simultaneously from N-methyl-N'-nitrosoguanidine-treated B. subtilis Marburg strain (22). In this communication, we report the results of further characterization of these mutants. MATERIALS AND METHODS Bacterial strains. The bacterial strains used are listed in Table 1. Strains 6160 and Mu8u5u5 were donated by Y. Ikeda and H. Yoshikawa, respectively. B. subtilis var. amylosacchariticus was a gift from J. Fukumoto of Osaka City University. Bacillus natto IAM 1212 is a stock culture at the Institute of Applied Microbiology, The University of Tokyo. Other strains are derivatives of strain 6160 and they were obtained 48
a-AMYLASE AND PROTEASE HYPERPRODUCTION
VOL. 124, 1975
49
TABLE 1. Bacterial strains used B. subtilis Marburgb 6160 6160-1 6160-2
Origin
Genotypea
Strain
amyEm, amyRI, ProL purB6, trpB3, metB5, purB6, trpB3, metB5, str, amyEm, amyRI, ProL aro-116, trpB3, metB5, str, amyEm, amyR), ProL
Mu8u5u5 Mio1o NA64 NP58 YN9 YN21 YN118 YY88
thr, leuA8, metB5, aro-116, hisB2, metB5, str, purB6, metB5, purB6, trpB3, metB5, str, purB6, trpB3, metB5, str, purB6, trpB3, metB5, str, aro-116, trpB3, metB5, str, purB6, metB5, str,
amvEm, amyRI, ProL amyE1010, amyRI, ProL amyEm, amyR2, ProL amyEm, amyRI, ProH amyEm, amyRI, ProL, pap-9 amyEm, amyR21, ProL amyEm, amyRI, ProL, pap-118 amyEm, amyR2, ProL, pap-9
YY11O
purB6, metB5, str,
amyEm, amyR2, ProL, pap-118
YY154
trpB3, metB5, str,
amyEm, amyRI, ProH, pap-118
YY225
trpB3, metB5, str,
amyEm, amyR21, ProL, pap-118
Prototroph,
amyEn, amyR2, ProH
B. natto IAM 1212
Y. Ikeda Ultraviolet treatment of 6160 From strain 6160-1 by transformation from strain M03 (20) N. Sueoka K. Yamaguchi (20) K. Yamaguchi (18) H. Uehara (16) NTGC treatment of 6160-1 NTG treatment of 6160-1 NTG treatment of 6160-2 From strain NA64 by transformation from strain YN9 From strain NA64 by transformation from strain YN 118 From strain NP58 by transformation from strain YN118 From strain YN21 by transformation from strain YN 118 Stock culture
aThe genetic symbols of amyE and amyR mean structural and regulator genes of a-amylase (19). ProH and ProL mean the phenotypic expression of the gene corresponding to the high and low production of neutral protease (16). "These strains are derivatives of B. subtilis 168. c
NTG, N-methyl-N'-nitro-N-nitrosoguanidine.
suspension (final 20%), which was stored in liquid nitrogen. To isolate mutants, frozen cells were melted quickly and grown at 37 C for 3 h in BY medium. The culture was diluted properly and plated on a BY-agar medium containing 1% soluble starch. KI-I, solution (0.01 M) was sprayed on the colonies grown on the plate after an overnight incubation at 37 C. Colonies with a large unstained halo around them were selected, and the levels of a-amylase produced in the culture medium during 24 or 30 h of culture were assayed. Procedure of transformation. DNA used in the transformation was extracted from donor strains according to the method of Saito and Miura (13). The procedures employed in transformation experiments were the same as those described by H. Yoshikawa (24). The concentration of transforming DNA employed was about 1 Asg/ml. Assay of cross-reacting material production. Preparation of antisera against a-amylase and assay of cross-reacting activity were the same as those described by K. Yamane et al. (21).
RESULTS Selection and properties of AP mutants. The procedure for the isolation of mutants by N- methyl - N' - nitro - N- nitrosoguanidine treatment was as described in Materials and Methods. Fifteen strains with hyperproduction of a-amylase were isolated from 115,000 colonies of mutagenized B. subtilis Marburg strains 6160-1 and 6160-2. Nine strains produced high levels of a-amylase only and did not show any change in the productivity of other extracellular enzymes, i.e., protease and ribonuclease. An-
other six strains produced increased levels of protease simultaneously. The former were referred to as A mutants and the latter as AP mutants. The properties of A mutants will be reported elsewhere. In this paper, properties of representative AP mutants (YN9 and YN118) will be described. The parental strain produced 6.5 U of a-amylase and 3.9 U of protease per mg of cells into culture medium at 37 C for 24 h. AP mutants produced two to three times more a-amylase and four to 16 times more protease than their parent (Table 2). Figure 1 shows the time course of the production of a-amylase and protease at 30 C in the parental strain (6160) and AP mutant (YN9). It was shown that a-amylase and protease were produced into the culture medium when the cell growth entered the stationary phase, and that the rates of enzyme production were stimulated in strain YN9. The enzymatic properties of partially purified a-amylase produced by strains YN9 and 6160 were compared. No differences were observed in thermal stabilities, mobilities on polyacrylamide gel electrophoresis, and final products from soluble starch after complete digestion. Both a-amylases cross-reacted with rabbit antiserum against a-amylase produced by B. subtilis var. amylosacchariticus. Immunological reactivity against this serum showed that molecular activities of a-amylase produced by strains YN9 and 6160 were not different (unpublished data). From these results it is suggested that the
50
YONEDA AND MARUO
J. BACTERIOL.
structure of a-amylase of strain YN9 is not different from that of its parent. It has been known that B. subtilis produces two types of extracellular protease, i.e., alkaline protease (serine protease) and neutral protease (metal protease). Two proteases can be separated by diethylaminoethyl-Sephadex A-50 column chromatography (16). AP mutants produced four to 16 times more protease than their parent as a total activity. The proteases produced by the AP mutants (YN9, YN118) were separated into alkaline and neutral protease, and their activities were assayed. The results showed that, although production of
both proteases was elevated by the mutation, the stimulation in the production of alkaline protease was greater. This pattern was also observed in the transformants (YY88, YY110) when this mutation was transferred to another strain by DNA-mediated transformation (Table 3). The elevation of a-amylase and protease production was not due to the increase in total protein synthesis, because cell growth and the production of other enzymes (ribonuclease, alkaline phosphatase, a-,f3-glucosidase, and flgalactosidase) were not altered (3). It was obvious that the mutation stimulated mainly the production of a-amylase and protease. TABLE 2. Production of a-amylase and protease in Genetic analyses of the AP mutants. It is the parental and mutant strainsa possible that the increased production of a-amylase and protease was caused b-y the mutaEnzyme activity (U/mg of cells) Strain tions, one affecting a-amylase production and a-Amylase Protease the other protease production. Thus, we investigated whether this mutant was derived by a Parent single point mutation or not. DNA from strain 6160 6.5 3.9 YN9 was given to normal transformable strains AP mutant (6160, Mu8u5u5) and streptomycin-resistant YN 6 18.5 28.8 transformants were selected on BY agar plates YN 9 20.6 57.9 YN 19 19.7 43.6 containing 1% casein and 100 ug of streptomycin YN 22 13.8 20.7 per ml. Transformants with high protease proYN118 16.1 62.3 duction were selected by observing the halo size YN213 14.4 17.1 around the colonies. All the transformants A mutant (67/67) selected for high protease production YN 21 23.5 2.1 also showed high a-amylase production. The aCells were cultured in BY medium for 24 h at fact that the genetic character responsible for 37 C. the high production of two enzymes did not Activity Klitt kctvity wt" ("AlVn) 0 @ o 0 r Fa A." Ft a b Sao Soo 500 'V F~0 I3
30C
20t
*/
300 200
10
/
100
50
20 10
1
10
/ 1-0-
oI 20
a*0
-
a
a
7 t 0 /io 0. *0~~~~~~~ 0~~ -0 -~~~~~
20
o0
0~~ *
8 10
