Vol. 129, No. 2 Printed in U.S.A.
OF BACTRIUOLOGY, Feb. 1977, p. 973-977 Copyright 0 1977 American Society for Microbiology
JOURNAL
Isolation of Acetyl Esterase Mutants of Bacillus subtilis 1681 THOMAS B. HIGERD Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina 29401
Received for publication 4 August 1976
Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified. By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity. All of the back-crossed esterase mutants were able to sporulate at wildtype frequency and produce exoprotease(s) and antibiotic(s). No difference in motility could be attributed to the esterase mutation. PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker. Many investigators have suggested that proteolytic and/or esterolytic enzymes may be involved directly in the process of bacterial sporulation (8, 13, 14, 18). At present, the biological function of the proteases cannot be defined, and the precise role for any individual protease cannot be specified. However, the isolation and characterization of mutants devoid of particular proteolytic or esterolytic activities will be useful for investigations in this area, particularly in regard to the apparent requirement that proteases be present for sporulation to proceed. Michel and Millet (15) have isolated a mutant of Bacillus subtilis that lacks the extracellular, zinc-containing neutral protease activity but sporulates normally. Thus, the activity of the neutral protease does not appear to be required for sporulation. In contrast, the alkaline serine protease, which also has esterolytic activity, appears to be required for sporogenesis, in that the temperature-sensitive serine protease mutant described by Leighton et al. (11) will not sporulate at the nonpermissive temperature. It is of interest, therefore, to isolate mutants devoid of esterolytic activity and to determine whether sporulation can proceed in the absence of an individual esterase activity. Two acetyl esterases have been identified in extracts of sporulating cells of B. subtilis (3, 6): esterase A, which has a molecular weight of about 160,000 and is present in logarithmically growing cells, and esterase B, which has a molecular weight of about 51,000 and appears after logarithmic growth ceases. In early-blocked asporogenous mutants, esterase B activity is greatly diminished (6).
This paper describes (i) a method that facilitates the isolation of mutants defective in esterolytic activity, and (ii) preliminary characterizations of mutants lacking detectable esterase A or esterase B activity. MATERIALS AND METHODS Bacterial strains. Bacterial strains used are listed in Table 1. Cultures were routinely transferred onto tryptose blood agar base (TBAB) agar (Difco). Detection of mutants. Mutant strains of B. subtilis 168 (trpC2) spores were isolated after treatment of the wild type with ethyl methane sulfonate as described by Ito and Spizizen (9). Mutagenized spores were plated on Spizizen minimal agar (19) supplemented with glucose (0.5%), tryptophan (8 ,ug/ml), and hydrolyzed casein (0.1%). After 48 h of growth at 37°C, the colonies were overlaid with 10 ml of melted soft agar (0.8%) in 0.5 M potassium phosphate buffer (pH 7.5) containing 5 mg 3-naphthyl acetate (6) and 5 mg of fast blue BB (Sigma). After another 4 h of incubation, colonies with diminished haloes were selected and streaked for isolation onto TBAB plates. For identification of esterase mutants, cells grown in the medium of Greenleaf and Losick (5) were harvested 3 h after logarithmic growth had ended. The cells were washed in 0.05 M tris(hy-
droxymethyl)aminomethane
(Tris)-hydrochloride
buffer (pH 7.5), resuspended in one-tenth their original volume in the same buffer, and frozen overnight. Cells of the thawed suspension were disrupted with a Branson model 185D sonic oscillator. After centrifugation at 40,000 x g for 30 min at 4°C, the cell-free extract was subjected to electrophoresis, and the gel was stained for esterase activity. Electrophoresis and esterase staining. A disc gel electrophoresis apparatus (ISCO model 419) with glass tubes (6 mm ID by 76 mm) was used. The height of the acrylamide gel column was 72 mm. T-he gel was prepared with a 0.14 M Tris-hydrochloride buffer (pH 7.5) containing 15% acrylamide, 0.4% N,N'-methylenebisacrylamide, and 0.12% (vol/vol) N,N,N',N'-tetramethylethylenediamine
1 Publication no. 71 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston.
973
974
HIGERD
TABLE 1. Bacillus strains used Strain Origin J. Spizizen B. subtilis 168 (trpC2) B. subtilis BD92 (hisA trpC2 cysB F. Young pro) J. Hoch B. subtilis GSY1026 (trpC2 metB) J. Hoch B. subtilis SB5 (trpC2 hisA ura) J. Spiizen B. subtilis SpoA12 ATCCa B. amyloliquefaciens ATCC 23842 ATCC B. brevis ATCC 8246 J. Spizizen B. cereus ATCC 14579 ATCC B. circulans ATCC 4513 ATCC B. coagulans ATCC 7050 J. Spizizen B. licheniformis ATCC 14580 ATCC B. macerans ATCC 8244 J. Spizizen B. megaterium ATCC 14581 P. Lovett B. pumilis ATCC 7061 a ATCC, American Type Culture Collection, Rockville, Md. catalyzed with an equal volume of 0.07% ammonium persulfate. Both upper and lower reservoirs of the apparatus were filled with 0.01 M Tris-hydrochloride buffer (pH 7.5). The lower electrode served as the anode. A 50-,ul sample of the test solution containing approximately 75 ,ug of protein in 10% sucrose was layered onto the above gel. Electrophoresis was performed at 4°C with a loading amperage of 2 mA per tube for 10 min and a constant running current of 5 mA per tube for 65 min. After electrophoresis, the gel was removed from the glass tubes and stained for esterase activity. The staining procedure was the same as described previously (6), except that 50 mg of fast blue BB and,0. 1 M phosphate buffer (pH 7.5) were used. Esterase and protein assays. Esterase activity was assayed spectrophotometrically with p-nitrophenyl acetate as the substrate (17). This reaction was carried out at room temperature and in the presence of 0.5 M phosphate buffer (pH 7.5). Protein determination was performed according to the method of Koch and Putnam (10), using bovine serum albumin as the standard. Specific activity refers to the number of units of enzyme activity (1 unit hydrolyzed 1 mmol ofp-nitrophenyl acetate per min) per milligram of protein. Spores were prepared from 72-h cultures growing on the medium of Greenleaf and Losick (5) and washed exhaustively in distilled water. The washed spores were heated to 80°C for 10 min and added to fresh medium, upon which germination and growth ensued. At appropriate times, 2.5-ml samples were removed and diluted twofold in 0.05 M potassium phosphate buffer (pH 7.5), and the absorbance was determined at 660 nm. Samples of 100 ml were removed at various times, and the cells were washed in 0.1 M Tris-hydrochloride buffer (pH 7.5) and frozen in 10 ml of buffer overnight. The washed cells were sonicated and assayed for esterase activity and protein content after centrifugation at 40,000 x g for 15 min. Sporulation and antibiotic test. The ability of the mutants to sporulate and to produce antibiotic was determined by the method of Ito and Spizizen (8).
J. BACTZRIOL.
Extracellular protease determination. Agar plates containing Spizizen minimal salts (19) with 0.5% glucose and yeast extract were supplemented with 0.35% casein (Difco) or hemoglobin (Fisher). After 36 h of growth of 37°C, the presence or absence of zones of clearing around isolated colonies was recorded. Transformation and transduction. Back-crossing of all the original esterase mutants of B. subtilis 168 (trpC2) into GSY1026 was performed by the procedure of Anagnostopoulos and Spizizen (1), using saturating levels of deoxyribonucleic acid. For obtaining PBS1 transducing lysates, the method of Hoch (7) was used.
RESULTS To establish a method for scoring esterasenegative mutants, B. subtilis strains 168 (trpC2) and SR22 (spoA12 trpC2) were used as the test organisms; 168 contains both esterases A and B, and SR22 has diminished esterase B activity (6). Initially, the organisms were grown on minimal salts medium and on complex media (e.g., TBAB), but the zone size and intensity of the naphthyl-azo dye complex surrounding the two strains were indistinguishable. In addition, when the substrate overlay was not strongly buffered, broad haloes surrounded colonies of both SR22 and 168. The medium found to be most suitable for producing contrasting zone sizes was minimal medium supplemented with glucose and casein hydrolysate, as described in Materials and Methods. From the 3,500 colonies observed, five mutants were finally selected that exhibited reduced zone size. To diminish the possibility of multiple mutations leading to spurious phenotypic properties, the mutants were back-crossed into strains GSY1026 and SB5 by transformation and transduction, respectively, and compared with the parental strains. Four of the mutants (EB-1, -2, -3, and 4) showed no esterase B activity, and the fifth (EA-1) showed no esterase A activity (Fig. 1). All the mutants isolated produced antibiotic(s) active against SR22 or B. amyloliquefaciens H. In addition, all five mutants were found to be motile when examined microscopically or by swarming on soft-agar overlay plates. Extracellular proteolytic activity, presumably from the alkaline protease (16), as measured on nutrient agar plates containing casein or hemoglobin, was also detected in all five mutants. These mutants were able to sporulate at the same frequency (approximately 85%) as the parental strain. To determine the time course of esterase production in these mutants, spores of EA-1 and EB-1 were placed in fresh medium. The culture was monitored for growth, and cells were har-
ESTERASE MUTANTS OF B. SUBTILIS
VOL. 129, 1977
WILD TYPE
MUTANTS: EB-2
EB- 1
EB-3
EB-4
975
EA - 1
*1FIG. 1. Histochemical staining for esterase activity after polyacrylamide gel electrophoresis of crude extracts from the wild type and mutants of B. subtilis.
vested at various intervals and assayed for esterolytic activity and protein content. Esterase A activity first appeared during the early exponential phase of the growth curve of mutant EB-1 (Fig. 2). EA-1, however, produced detectable levels of esterase B activity near the end of the exponential phase. These results are compatible with those of an earlier study of the kinetics of appearance of the two esterase activities in the wild type and the electrophoretic esterase profiles of several early-blocked asporogenous mutants (6). The location of the esterase B (estB) mutation was determined by transducing auxotrophic markers with lysates of PBS1 made on the esterase mutants. Two-factor crosses carried out by transduction demonstrated that EB1, EB-3, and EB-4 were linked to the hisA locus. The average frequencies of cotransduction of the esterase B marker with hisA and eysB were 52 and 1%, respectively. No linkage was observed between the esterase A marker and hisA; attempts to map esterase A on the chromosome have been unsuccessful. Electrophoretic patterns of esterases from different microorganisms grown on the same complex medium have been compared previously as an aid in taxonomy (2, 4, 12). Extracts from approximately 20 auxotrophic mutants of
B. subtilis have been examined electrophoretically; to date, all the strains have demonstrated the esterase pattern of B. subtilis 168, although this pattern was not shared with other Bacillus species (Fig. 3). More strains must be examined, but it appears that it may be possible to identify species of the genus by comparative polyacrylamide gel electrophoresis of esterases from crude extracts.
DISCUSSION In an initial attempt to characterize the "nonspecific" esterase of procaryotes, mutants specifically lacking an identified esterase have been isolated for the first time. The mutants retain the ability to support phage growth, although Bernard Reilly (personal communication) has noted that the burst size of bacteriophage 029 is decreased 10- and 100-fold in EA-1 and EB-1, respectively, as compared with the parental strains. This effect appears to be specific for o29 in that normal burst sizes occur after infection with o25 or PBS1. In addition, their ability to produce antibiotic(s) and extracellular protease(s) is not affected appreciably. Neither esterase A nor esterase B activity appears to be required for sporulation, although esterase B activity is greatly diminished in the SpoA and SpoB groups of early-blocked asporo-
976
J. BACTERIOL.
HIGERD
0
10
0
c4l
*0
01
D
D5
t
~
Time
(hoursi
Time
hours)
FIG. 2. Kinetics of growth and appearance of esterolytic activity. Symbols: 0, absorbance; U, specific activity. osj A
6
C
OF BACTRIUOLOGY, Feb. 1977, p. 973-977 Copyright 0 1977 American Society for Microbiology
JOURNAL
Isolation of Acetyl Esterase Mutants of Bacillus subtilis 1681 THOMAS B. HIGERD Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina 29401
Received for publication 4 August 1976
Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified. By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity. All of the back-crossed esterase mutants were able to sporulate at wildtype frequency and produce exoprotease(s) and antibiotic(s). No difference in motility could be attributed to the esterase mutation. PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker. Many investigators have suggested that proteolytic and/or esterolytic enzymes may be involved directly in the process of bacterial sporulation (8, 13, 14, 18). At present, the biological function of the proteases cannot be defined, and the precise role for any individual protease cannot be specified. However, the isolation and characterization of mutants devoid of particular proteolytic or esterolytic activities will be useful for investigations in this area, particularly in regard to the apparent requirement that proteases be present for sporulation to proceed. Michel and Millet (15) have isolated a mutant of Bacillus subtilis that lacks the extracellular, zinc-containing neutral protease activity but sporulates normally. Thus, the activity of the neutral protease does not appear to be required for sporulation. In contrast, the alkaline serine protease, which also has esterolytic activity, appears to be required for sporogenesis, in that the temperature-sensitive serine protease mutant described by Leighton et al. (11) will not sporulate at the nonpermissive temperature. It is of interest, therefore, to isolate mutants devoid of esterolytic activity and to determine whether sporulation can proceed in the absence of an individual esterase activity. Two acetyl esterases have been identified in extracts of sporulating cells of B. subtilis (3, 6): esterase A, which has a molecular weight of about 160,000 and is present in logarithmically growing cells, and esterase B, which has a molecular weight of about 51,000 and appears after logarithmic growth ceases. In early-blocked asporogenous mutants, esterase B activity is greatly diminished (6).
This paper describes (i) a method that facilitates the isolation of mutants defective in esterolytic activity, and (ii) preliminary characterizations of mutants lacking detectable esterase A or esterase B activity. MATERIALS AND METHODS Bacterial strains. Bacterial strains used are listed in Table 1. Cultures were routinely transferred onto tryptose blood agar base (TBAB) agar (Difco). Detection of mutants. Mutant strains of B. subtilis 168 (trpC2) spores were isolated after treatment of the wild type with ethyl methane sulfonate as described by Ito and Spizizen (9). Mutagenized spores were plated on Spizizen minimal agar (19) supplemented with glucose (0.5%), tryptophan (8 ,ug/ml), and hydrolyzed casein (0.1%). After 48 h of growth at 37°C, the colonies were overlaid with 10 ml of melted soft agar (0.8%) in 0.5 M potassium phosphate buffer (pH 7.5) containing 5 mg 3-naphthyl acetate (6) and 5 mg of fast blue BB (Sigma). After another 4 h of incubation, colonies with diminished haloes were selected and streaked for isolation onto TBAB plates. For identification of esterase mutants, cells grown in the medium of Greenleaf and Losick (5) were harvested 3 h after logarithmic growth had ended. The cells were washed in 0.05 M tris(hy-
droxymethyl)aminomethane
(Tris)-hydrochloride
buffer (pH 7.5), resuspended in one-tenth their original volume in the same buffer, and frozen overnight. Cells of the thawed suspension were disrupted with a Branson model 185D sonic oscillator. After centrifugation at 40,000 x g for 30 min at 4°C, the cell-free extract was subjected to electrophoresis, and the gel was stained for esterase activity. Electrophoresis and esterase staining. A disc gel electrophoresis apparatus (ISCO model 419) with glass tubes (6 mm ID by 76 mm) was used. The height of the acrylamide gel column was 72 mm. T-he gel was prepared with a 0.14 M Tris-hydrochloride buffer (pH 7.5) containing 15% acrylamide, 0.4% N,N'-methylenebisacrylamide, and 0.12% (vol/vol) N,N,N',N'-tetramethylethylenediamine
1 Publication no. 71 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston.
973
974
HIGERD
TABLE 1. Bacillus strains used Strain Origin J. Spizizen B. subtilis 168 (trpC2) B. subtilis BD92 (hisA trpC2 cysB F. Young pro) J. Hoch B. subtilis GSY1026 (trpC2 metB) J. Hoch B. subtilis SB5 (trpC2 hisA ura) J. Spiizen B. subtilis SpoA12 ATCCa B. amyloliquefaciens ATCC 23842 ATCC B. brevis ATCC 8246 J. Spizizen B. cereus ATCC 14579 ATCC B. circulans ATCC 4513 ATCC B. coagulans ATCC 7050 J. Spizizen B. licheniformis ATCC 14580 ATCC B. macerans ATCC 8244 J. Spizizen B. megaterium ATCC 14581 P. Lovett B. pumilis ATCC 7061 a ATCC, American Type Culture Collection, Rockville, Md. catalyzed with an equal volume of 0.07% ammonium persulfate. Both upper and lower reservoirs of the apparatus were filled with 0.01 M Tris-hydrochloride buffer (pH 7.5). The lower electrode served as the anode. A 50-,ul sample of the test solution containing approximately 75 ,ug of protein in 10% sucrose was layered onto the above gel. Electrophoresis was performed at 4°C with a loading amperage of 2 mA per tube for 10 min and a constant running current of 5 mA per tube for 65 min. After electrophoresis, the gel was removed from the glass tubes and stained for esterase activity. The staining procedure was the same as described previously (6), except that 50 mg of fast blue BB and,0. 1 M phosphate buffer (pH 7.5) were used. Esterase and protein assays. Esterase activity was assayed spectrophotometrically with p-nitrophenyl acetate as the substrate (17). This reaction was carried out at room temperature and in the presence of 0.5 M phosphate buffer (pH 7.5). Protein determination was performed according to the method of Koch and Putnam (10), using bovine serum albumin as the standard. Specific activity refers to the number of units of enzyme activity (1 unit hydrolyzed 1 mmol ofp-nitrophenyl acetate per min) per milligram of protein. Spores were prepared from 72-h cultures growing on the medium of Greenleaf and Losick (5) and washed exhaustively in distilled water. The washed spores were heated to 80°C for 10 min and added to fresh medium, upon which germination and growth ensued. At appropriate times, 2.5-ml samples were removed and diluted twofold in 0.05 M potassium phosphate buffer (pH 7.5), and the absorbance was determined at 660 nm. Samples of 100 ml were removed at various times, and the cells were washed in 0.1 M Tris-hydrochloride buffer (pH 7.5) and frozen in 10 ml of buffer overnight. The washed cells were sonicated and assayed for esterase activity and protein content after centrifugation at 40,000 x g for 15 min. Sporulation and antibiotic test. The ability of the mutants to sporulate and to produce antibiotic was determined by the method of Ito and Spizizen (8).
J. BACTZRIOL.
Extracellular protease determination. Agar plates containing Spizizen minimal salts (19) with 0.5% glucose and yeast extract were supplemented with 0.35% casein (Difco) or hemoglobin (Fisher). After 36 h of growth of 37°C, the presence or absence of zones of clearing around isolated colonies was recorded. Transformation and transduction. Back-crossing of all the original esterase mutants of B. subtilis 168 (trpC2) into GSY1026 was performed by the procedure of Anagnostopoulos and Spizizen (1), using saturating levels of deoxyribonucleic acid. For obtaining PBS1 transducing lysates, the method of Hoch (7) was used.
RESULTS To establish a method for scoring esterasenegative mutants, B. subtilis strains 168 (trpC2) and SR22 (spoA12 trpC2) were used as the test organisms; 168 contains both esterases A and B, and SR22 has diminished esterase B activity (6). Initially, the organisms were grown on minimal salts medium and on complex media (e.g., TBAB), but the zone size and intensity of the naphthyl-azo dye complex surrounding the two strains were indistinguishable. In addition, when the substrate overlay was not strongly buffered, broad haloes surrounded colonies of both SR22 and 168. The medium found to be most suitable for producing contrasting zone sizes was minimal medium supplemented with glucose and casein hydrolysate, as described in Materials and Methods. From the 3,500 colonies observed, five mutants were finally selected that exhibited reduced zone size. To diminish the possibility of multiple mutations leading to spurious phenotypic properties, the mutants were back-crossed into strains GSY1026 and SB5 by transformation and transduction, respectively, and compared with the parental strains. Four of the mutants (EB-1, -2, -3, and 4) showed no esterase B activity, and the fifth (EA-1) showed no esterase A activity (Fig. 1). All the mutants isolated produced antibiotic(s) active against SR22 or B. amyloliquefaciens H. In addition, all five mutants were found to be motile when examined microscopically or by swarming on soft-agar overlay plates. Extracellular proteolytic activity, presumably from the alkaline protease (16), as measured on nutrient agar plates containing casein or hemoglobin, was also detected in all five mutants. These mutants were able to sporulate at the same frequency (approximately 85%) as the parental strain. To determine the time course of esterase production in these mutants, spores of EA-1 and EB-1 were placed in fresh medium. The culture was monitored for growth, and cells were har-
ESTERASE MUTANTS OF B. SUBTILIS
VOL. 129, 1977
WILD TYPE
MUTANTS: EB-2
EB- 1
EB-3
EB-4
975
EA - 1
*1FIG. 1. Histochemical staining for esterase activity after polyacrylamide gel electrophoresis of crude extracts from the wild type and mutants of B. subtilis.
vested at various intervals and assayed for esterolytic activity and protein content. Esterase A activity first appeared during the early exponential phase of the growth curve of mutant EB-1 (Fig. 2). EA-1, however, produced detectable levels of esterase B activity near the end of the exponential phase. These results are compatible with those of an earlier study of the kinetics of appearance of the two esterase activities in the wild type and the electrophoretic esterase profiles of several early-blocked asporogenous mutants (6). The location of the esterase B (estB) mutation was determined by transducing auxotrophic markers with lysates of PBS1 made on the esterase mutants. Two-factor crosses carried out by transduction demonstrated that EB1, EB-3, and EB-4 were linked to the hisA locus. The average frequencies of cotransduction of the esterase B marker with hisA and eysB were 52 and 1%, respectively. No linkage was observed between the esterase A marker and hisA; attempts to map esterase A on the chromosome have been unsuccessful. Electrophoretic patterns of esterases from different microorganisms grown on the same complex medium have been compared previously as an aid in taxonomy (2, 4, 12). Extracts from approximately 20 auxotrophic mutants of
B. subtilis have been examined electrophoretically; to date, all the strains have demonstrated the esterase pattern of B. subtilis 168, although this pattern was not shared with other Bacillus species (Fig. 3). More strains must be examined, but it appears that it may be possible to identify species of the genus by comparative polyacrylamide gel electrophoresis of esterases from crude extracts.
DISCUSSION In an initial attempt to characterize the "nonspecific" esterase of procaryotes, mutants specifically lacking an identified esterase have been isolated for the first time. The mutants retain the ability to support phage growth, although Bernard Reilly (personal communication) has noted that the burst size of bacteriophage 029 is decreased 10- and 100-fold in EA-1 and EB-1, respectively, as compared with the parental strains. This effect appears to be specific for o29 in that normal burst sizes occur after infection with o25 or PBS1. In addition, their ability to produce antibiotic(s) and extracellular protease(s) is not affected appreciably. Neither esterase A nor esterase B activity appears to be required for sporulation, although esterase B activity is greatly diminished in the SpoA and SpoB groups of early-blocked asporo-
976
J. BACTERIOL.
HIGERD
0
10
0
c4l
*0
01
D
D5
t
~
Time
(hoursi
Time
hours)
FIG. 2. Kinetics of growth and appearance of esterolytic activity. Symbols: 0, absorbance; U, specific activity. osj A
6
C
