Vol. 140, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 699-706
0021-9193/79/11-0699/08$02.00/0
Converting Bacteriophage for Sporulation and Crystal Formation in Bacillus thuringiensis FREDERICK J. PERLAK, CATHY L. MENI)ELSOHN, ANI) CURI'IS B. THORNE* Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003.
Received for publication 19 June 1979
Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted an oligosporogenic (sporulation frequency, 10-'), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the host used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10-6 to 10-5. Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis. The parasporal crystal produced by Bacillus thuringiensis is of special interest because of its toxicity to larval forms of certain insects (9). Previous studies have implicated a relationship between spores and crystals (6, 20). The spore coat has been shown to contain the same glycoprotein subunit found in the crystal (L. A. Bulla, Jr., K. J. Kramer, and L. I. Davidson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, 162, p. 165) and to exhibit a toxicity similar to that of the crystal (18). It is not unusual for strains to become acrystalliferous (Cry-) simultaneously with the loss of the ability to form spores (Spo-). However, Spo+ Cry- and Spo- Cry' mutants have been found (19), and phenotypic separation of spore and crystal formation by treating cells with chloramphenicol or exposing them to high temperatures has been reported (17). Meenakshi and Jayaraman (17) suggest that crystal protein synthesis and assembly can proceed to completion in the absence of the expression of any spore functions. A number of bacteriophages capable of converting spore-negative mutants of Bacillus subtillis or Bacillus pumilus to spore positive have been reported (2, 3, 11). In this report we describe a bacteriophage, TP-13, capable of converting an oligosporogenic (Osp), acrystalliferous mutant of B. thuringiensis to Spo+ Cry'. The phage also mediates generalized transduction in sensitive strains.
MATERIALS AND METHODS Organisms. T'he strains of bacteria used and their sources are given in T'able 1. Auxotrophic mutants of B. thuringiensis were isolated by the procedure of Iyer (10) after exposure of spores to UV light. Bacteriophage. Bacteriophages SP-15 (23) and CP-51 (26) have been described. TP-13 was isolated from soil as described in Results. TP-10 was also isolated in our laboratory from soil and will be described later (manuscript in preparation). Media and cultural conditions. The minimal media, Min 3 and Min 3C, as well as L-broth, PA broth and agar, and peptone diluent, were prepared as previously described (24). TBAB soft agar contained 11 g of tryptose blood agar base (I)ifco) and 100 g of D)ifco tryptone per liter. Motile cells were grown by inoculating L-broth with growth from a swarming colony on soft PA agar (0.35'S agar) and incubating on a rotary shaker at 37°C for 4 to 7 h.
Propagation and assay of phage. I'reviously described methods for propagation and assay of SIP-15 (16) and CP'-51 (24) were used. TP-13 was propagated on motile cells in T'BAB soft agar overlays on PlA agar plates (1.5('; agar). Plates were incubated for 18 h at 37°C. I'hage from each plate was harvested in 5 ml of PA broth, filtered through Millipore HA membranes (Millipore Corp., Bedford, Mass.), and stored at 4°C. TP-13 was assayed with motile cells of 4042A UM8-13 as the indicator in TBAB soft agar overlays on PA agar. Plaques were visible after 8 to 12 h at 30°C. Initially, soft PA agar overlays were used, but plaques were barely visible and could not be counted until colonies of converted cells grew within them. 699
700
PERLAK, MENDELSOHN, AND THORNE
J. BACTERIOL.
TABLE 1. List of strains and mutants Strain
Subspecies of B. thuringiensis
Characteristics
Origin/reference
Wild type Wild type Wild type Wild type Colonial variant of 4042 AdeAde- Osp CryWild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type cys-1 his-1 leu-2 trp-13 trp-17
lys-11
A. Yousten NRRLa NRRL NRRL NRRL UVb of 4042A UM8 at 430C NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL UV of 4060 UV of 4060 UV of 4060 UV of 4060 UV of 4060 UV of 4060
Wild type Wild type
NRRL ATCCC
B. lichenifornis 9945A
Wild type
ATCC
B. subtilis W23 168
Wild type trpC2
M. S. Fox M. S. Fox
B. thuringiensis 1715 4040 4041 4042 4042A 4042A UM8 4042A UM8-13 4043 4044 4045 4046 4047 4048 4049 4050 4055 4056 4057 4058 4059 4060 4060 UM25 4060 UM33 4060 UM52 4060 UM408 4060 UM473 4060 UM806 B.
thuringiensis finitimus alesti sotto
dendrolimus kenyae galleriae entomocidus entomocidus-limassol aizawai morrisoni tolworthi kurstaki canadensis subtoxicus darmstadiensis toumanoffi thompsoni
cereus
569 6464
B. pumilus Wild type P. Lovett 12140 a NRRL, Agricultural Research Service, Northern Regional Laboratory, U.S. Department of Agriculture, Peoria, Ill. b Mutagenesis by UV light. ATCC, American Type Culture Collection, Rockville, Md. Purification of TP-13. Sixty milliliters of lysate containing 8 x 109 plaque-forming units (PFU)/ml was concentrated by centrifugation for 2 h at 30,000 rpm in a type 50 (Beckman) rotor at 4°C. Each phage pellet was overlaid with 0.5 ml of 1% peptone, held at 4°C for 16 h, and then gently resuspended by pipette. One milliliter of the concentrated phage suspension was mixed with 3.5 g of CsCl and TNM buffer (0.05 M Tris-hydrochloride, 0.05 M NaCl, 0.01 M MgSO4, pH 7.0) to a final volume of 5 ml (average buoyant density, 1.520 g/cm3) and centrifuged in an SW50.1 rotor at 4°C for 24 to 30 h at 35,000 rpm. The phage band, which was clearly visible, was collected dropwise through the tube bottom as a single fraction. The
purified phage was dialyzed against TNM buffer for 24 h at 5C. Phage antiserum. Eight milliliters of phage suspension, purified as described above and containing 101 PFU, was homogenized with 10 ml of Freund incomplete adjuvant (Difco). Each of two rabbits was injected with 4 ml intramuscularly and 4 ml subcutaneously. The rabbits were bled after 5 weeks, and serum was heated at 56°C for 30 min. Velocity constants (K values) were calculated by the method described by Adams (1). Host range determinations. Motile cells and a suspension of TP-13 were cross-streaked on PA agar. Sensitivity to the phage was indicated by a decrease
CONVERT'ING I'HAGE T''l-1'3
VOL. 140, 1979
in the turbidity of the bacterial growth in the phage area.
Transduction. For transduction of 4042A UM8-13 to Spo+ with CP-51, transduction mixtures (24) were spread on PA agar plates and incubated at 37°C for 72 h. Growth from each plate was harvested in 5 ml of water, heated at 650C for 30 min to kill vegetative cells, and plated on PA agar for enumeration. The presence
of
spores was
confirmed by phase-contrast
microscopy.
For transduction with TP-13, mixtures containing 0.8 to 0.9 ml of motile culture (approximately 5 x 108 cells) and 0.1 to 0.2 ml of TP-13 lysate (108 to 109 PFU) in 20-mm tubes were incubated on a rotary shaker at 370C for 15 min. Samples (0.1 ml) were
plated on appropriate selective media, and plates were incubated at 370C for 36 to 48 h. Electron microscopy. Samples of purified TP-13 were negatively stained (1% uranyl acetate, pH 3.9, or 0.4% ammonium molybdate, pH 7.0) on Formvarcoated, carbon-reinforced grids and examined in a Philips EM-200 electron microscope. Isolation and characterization of TP-13 DNA. Phage DNA for melting point and buoyant density determinations was prepared by a modification of the procedure described by Yelton and Thorne (26). After two deproteinizations with phenol saturated with SSC (pH 8.0) (SSC = 0.15 M NaCl plus 0.015 M sodium citrate), a final deproteinization with a chloroformisoamyl alcohol (24:1) mixture was performed to remove excess phenol (5). The DNA was then dialyzed against SSC at pH 7.0. The melting temperature of TP-13 DNA was determined by the method of Marmur and Doty (15) with a Gilford linear thermosensor. For buoyant density determinations the DNA was mixed with CsCl in SSC (pH 7.0) to achieve an average density of 1.74 g/cm3 and centrifuged for 44 h at 35,000 rpm in an SW50.1 rotor at 40C. Fractions were collected dropwise through the tube bottom for determination of refractive index and absorbance at 260 nm. B. subtilis W23 DNA prepared by the procedure of Marmur (13) and SP-15 DNA were used as standards in determination of melting temperature and buoyant density. RESULTS
Isolation of TP-13 and conversion of an Osp Cry- mutant to Spo+ Cry+. Attempts to isolate a spore-converting phage for B. thuringiensis were unsuccessful until a particular sporulation mutant, UM8-13, derived from 4042A UM8, was used as the indicator. The parent strain, UM8, is an adenine auxotroph capable of sporulation and crystal formation (Fig. 1A). Mutant UM8-13, isolated by the procedure of Yousten (28), is oligosporogenic and acrystalliferous (Fig. 1B). The few spores produced by UM8-13 (
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 699-706
0021-9193/79/11-0699/08$02.00/0
Converting Bacteriophage for Sporulation and Crystal Formation in Bacillus thuringiensis FREDERICK J. PERLAK, CATHY L. MENI)ELSOHN, ANI) CURI'IS B. THORNE* Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003.
Received for publication 19 June 1979
Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted an oligosporogenic (sporulation frequency, 10-'), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the host used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10-6 to 10-5. Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis. The parasporal crystal produced by Bacillus thuringiensis is of special interest because of its toxicity to larval forms of certain insects (9). Previous studies have implicated a relationship between spores and crystals (6, 20). The spore coat has been shown to contain the same glycoprotein subunit found in the crystal (L. A. Bulla, Jr., K. J. Kramer, and L. I. Davidson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, 162, p. 165) and to exhibit a toxicity similar to that of the crystal (18). It is not unusual for strains to become acrystalliferous (Cry-) simultaneously with the loss of the ability to form spores (Spo-). However, Spo+ Cry- and Spo- Cry' mutants have been found (19), and phenotypic separation of spore and crystal formation by treating cells with chloramphenicol or exposing them to high temperatures has been reported (17). Meenakshi and Jayaraman (17) suggest that crystal protein synthesis and assembly can proceed to completion in the absence of the expression of any spore functions. A number of bacteriophages capable of converting spore-negative mutants of Bacillus subtillis or Bacillus pumilus to spore positive have been reported (2, 3, 11). In this report we describe a bacteriophage, TP-13, capable of converting an oligosporogenic (Osp), acrystalliferous mutant of B. thuringiensis to Spo+ Cry'. The phage also mediates generalized transduction in sensitive strains.
MATERIALS AND METHODS Organisms. T'he strains of bacteria used and their sources are given in T'able 1. Auxotrophic mutants of B. thuringiensis were isolated by the procedure of Iyer (10) after exposure of spores to UV light. Bacteriophage. Bacteriophages SP-15 (23) and CP-51 (26) have been described. TP-13 was isolated from soil as described in Results. TP-10 was also isolated in our laboratory from soil and will be described later (manuscript in preparation). Media and cultural conditions. The minimal media, Min 3 and Min 3C, as well as L-broth, PA broth and agar, and peptone diluent, were prepared as previously described (24). TBAB soft agar contained 11 g of tryptose blood agar base (I)ifco) and 100 g of D)ifco tryptone per liter. Motile cells were grown by inoculating L-broth with growth from a swarming colony on soft PA agar (0.35'S agar) and incubating on a rotary shaker at 37°C for 4 to 7 h.
Propagation and assay of phage. I'reviously described methods for propagation and assay of SIP-15 (16) and CP'-51 (24) were used. TP-13 was propagated on motile cells in T'BAB soft agar overlays on PlA agar plates (1.5('; agar). Plates were incubated for 18 h at 37°C. I'hage from each plate was harvested in 5 ml of PA broth, filtered through Millipore HA membranes (Millipore Corp., Bedford, Mass.), and stored at 4°C. TP-13 was assayed with motile cells of 4042A UM8-13 as the indicator in TBAB soft agar overlays on PA agar. Plaques were visible after 8 to 12 h at 30°C. Initially, soft PA agar overlays were used, but plaques were barely visible and could not be counted until colonies of converted cells grew within them. 699
700
PERLAK, MENDELSOHN, AND THORNE
J. BACTERIOL.
TABLE 1. List of strains and mutants Strain
Subspecies of B. thuringiensis
Characteristics
Origin/reference
Wild type Wild type Wild type Wild type Colonial variant of 4042 AdeAde- Osp CryWild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type cys-1 his-1 leu-2 trp-13 trp-17
lys-11
A. Yousten NRRLa NRRL NRRL NRRL UVb of 4042A UM8 at 430C NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL NRRL UV of 4060 UV of 4060 UV of 4060 UV of 4060 UV of 4060 UV of 4060
Wild type Wild type
NRRL ATCCC
B. lichenifornis 9945A
Wild type
ATCC
B. subtilis W23 168
Wild type trpC2
M. S. Fox M. S. Fox
B. thuringiensis 1715 4040 4041 4042 4042A 4042A UM8 4042A UM8-13 4043 4044 4045 4046 4047 4048 4049 4050 4055 4056 4057 4058 4059 4060 4060 UM25 4060 UM33 4060 UM52 4060 UM408 4060 UM473 4060 UM806 B.
thuringiensis finitimus alesti sotto
dendrolimus kenyae galleriae entomocidus entomocidus-limassol aizawai morrisoni tolworthi kurstaki canadensis subtoxicus darmstadiensis toumanoffi thompsoni
cereus
569 6464
B. pumilus Wild type P. Lovett 12140 a NRRL, Agricultural Research Service, Northern Regional Laboratory, U.S. Department of Agriculture, Peoria, Ill. b Mutagenesis by UV light. ATCC, American Type Culture Collection, Rockville, Md. Purification of TP-13. Sixty milliliters of lysate containing 8 x 109 plaque-forming units (PFU)/ml was concentrated by centrifugation for 2 h at 30,000 rpm in a type 50 (Beckman) rotor at 4°C. Each phage pellet was overlaid with 0.5 ml of 1% peptone, held at 4°C for 16 h, and then gently resuspended by pipette. One milliliter of the concentrated phage suspension was mixed with 3.5 g of CsCl and TNM buffer (0.05 M Tris-hydrochloride, 0.05 M NaCl, 0.01 M MgSO4, pH 7.0) to a final volume of 5 ml (average buoyant density, 1.520 g/cm3) and centrifuged in an SW50.1 rotor at 4°C for 24 to 30 h at 35,000 rpm. The phage band, which was clearly visible, was collected dropwise through the tube bottom as a single fraction. The
purified phage was dialyzed against TNM buffer for 24 h at 5C. Phage antiserum. Eight milliliters of phage suspension, purified as described above and containing 101 PFU, was homogenized with 10 ml of Freund incomplete adjuvant (Difco). Each of two rabbits was injected with 4 ml intramuscularly and 4 ml subcutaneously. The rabbits were bled after 5 weeks, and serum was heated at 56°C for 30 min. Velocity constants (K values) were calculated by the method described by Adams (1). Host range determinations. Motile cells and a suspension of TP-13 were cross-streaked on PA agar. Sensitivity to the phage was indicated by a decrease
CONVERT'ING I'HAGE T''l-1'3
VOL. 140, 1979
in the turbidity of the bacterial growth in the phage area.
Transduction. For transduction of 4042A UM8-13 to Spo+ with CP-51, transduction mixtures (24) were spread on PA agar plates and incubated at 37°C for 72 h. Growth from each plate was harvested in 5 ml of water, heated at 650C for 30 min to kill vegetative cells, and plated on PA agar for enumeration. The presence
of
spores was
confirmed by phase-contrast
microscopy.
For transduction with TP-13, mixtures containing 0.8 to 0.9 ml of motile culture (approximately 5 x 108 cells) and 0.1 to 0.2 ml of TP-13 lysate (108 to 109 PFU) in 20-mm tubes were incubated on a rotary shaker at 370C for 15 min. Samples (0.1 ml) were
plated on appropriate selective media, and plates were incubated at 370C for 36 to 48 h. Electron microscopy. Samples of purified TP-13 were negatively stained (1% uranyl acetate, pH 3.9, or 0.4% ammonium molybdate, pH 7.0) on Formvarcoated, carbon-reinforced grids and examined in a Philips EM-200 electron microscope. Isolation and characterization of TP-13 DNA. Phage DNA for melting point and buoyant density determinations was prepared by a modification of the procedure described by Yelton and Thorne (26). After two deproteinizations with phenol saturated with SSC (pH 8.0) (SSC = 0.15 M NaCl plus 0.015 M sodium citrate), a final deproteinization with a chloroformisoamyl alcohol (24:1) mixture was performed to remove excess phenol (5). The DNA was then dialyzed against SSC at pH 7.0. The melting temperature of TP-13 DNA was determined by the method of Marmur and Doty (15) with a Gilford linear thermosensor. For buoyant density determinations the DNA was mixed with CsCl in SSC (pH 7.0) to achieve an average density of 1.74 g/cm3 and centrifuged for 44 h at 35,000 rpm in an SW50.1 rotor at 40C. Fractions were collected dropwise through the tube bottom for determination of refractive index and absorbance at 260 nm. B. subtilis W23 DNA prepared by the procedure of Marmur (13) and SP-15 DNA were used as standards in determination of melting temperature and buoyant density. RESULTS
Isolation of TP-13 and conversion of an Osp Cry- mutant to Spo+ Cry+. Attempts to isolate a spore-converting phage for B. thuringiensis were unsuccessful until a particular sporulation mutant, UM8-13, derived from 4042A UM8, was used as the indicator. The parent strain, UM8, is an adenine auxotroph capable of sporulation and crystal formation (Fig. 1A). Mutant UM8-13, isolated by the procedure of Yousten (28), is oligosporogenic and acrystalliferous (Fig. 1B). The few spores produced by UM8-13 (
