Vol. 131, No. 3
JOURNAL OF BACTZRIOLOGY, Sept. 1977, p. 1029-1032 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Selective Plasmid Transduction in Bacillus pumilus MICHAEL G. BRAMUCCI' AND PAUL S. LOVETT* Department ofBiological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228
Received for publication 1 April 1977
The inducible temperate bacteriophage 475 and a clear-plaque-forming variant, 475C1, mediated transduction of a 4.4 x 10"-dalton multicopy Bacillus pumilus plasmid, pPL10, at frequencies of 10-5 to 10-6 transductants per plaqueforming unit. 4)75- and 475C1-mediated transduction of several chromosome markers tested did not occur at a detectable frequency. 475-mediated plasmid transducing activity resides in particles that are similar to infectious particles in sedimentation velocity and buoyant density.
Several temperate bacteriophages have been isolated for members of the related species Bacillus subtilis andB. pumilus (1, 3, 4, 14). None of these phages has been shown to perform generalized transduction, and only the B. subtilis temperate phages 4105 and SP,8 have been reported to perform specialized transduction (12, 14). Previous attempts to detect transduction mediated by B. subtilis orB. pumilus temperate phages have been limited to studies of the transfer of host chromosome genes. Studies of plasmid transduction in Bacillus have only recently become possible as a result of the identification of a selectable host function that is plasmid determined in Bacillus (8). Plasmid pPL10 (-4.4 x 106 daltons; p= 1.698 g/cm3; 10 to 20 copies per chromosome) determines the production of, and immunity or resistance to, killing activity in strains of B. pumilus (8). pPL10 has been transferred among strains of B. pumilus by each of three generalized transducing phages (PBS1, PBP1, and PMB1) (2), and the plasmid has been transformed into B. subtilis 168 by an indirect selection technique (8). However, transduction studies of the plasmid in B. subtilis have not been possible because B. subtilis is naturally resistant to the pPL10-determined killing activity, and strains of B. subtilis harboring pPL10 only weakly express the Kill+ phenotype (8). We examined the ability of the inducible B. pumilus temperate phage 475 to mediate transduction of plasmid pPL10. 475 efficiently performs plasmid transduction, but it is incapable of mediating transduction of any of several chromosome markers. The ability of 475 to mediate pPL10 transduction was initially tested by inserting the plasI Present address: Uniformed Services University of the Health Sciences, Department of Microbiology, Bethesda, MD 20014.
mid into a mutant derivative of NRRL B-3275, strain 231-1-8 (Table 1), by transduction with PBP1 grown on strain L10 (Table 1; 5, 7, 8). Each of three Kill+ transductants retained the nutritional requirements of 231-1-8 (Ser Leu Lys), and each contained 4.4(±0.3)% ofthe total cell deoxyribonucleic acid (DNA) as covalently closed circular duplex (CCC) molecules (detected as previously described [8]) that sedimented at 25(± 1)S in neutral sucrose gradients relative to ['4C]thymidine-(New England Corp., Boston, Mass.) labeled T7 DNA (32S; 13). Mitomycin C induction (1) of strain 231-1-8 and the three plasmid-containing derivatives yielded 4)75 lysates with an infectious titer of about 108 plaque-forming units (PFU) per ml. Lysates induced from the plasmid-containing strains generated Kill+ transductants of strains W20 and Cat-1 at a frequency of 1 transductant per 10" PFU, whereas phages induced from strain 231-1-8 not containing pPL10 did not generate Kill+ transductants of either recipient. None of the lysates was capable of generating adenine-positive transductants of strains W20 or Cat-1. In other experiments, we were unable to demonstrate 4)75 transduction of chromosome markers try-2, gly-1, cys-1, lys-2, his-i, ade-1, leu-3, met-3, thr-1, and argA2 (9-11) in derivatives of the NRRL B-3275 strain and chromosome markers ade-100, trplOO, lys-100, cys-100, and ser-100 (1, 6) in derivatives of the NRS576 strain. Antiserum (1) neutralizing >99.9% of the PFU in a 475 lysate (10k PFU/ml) in 15 min at 37°C also neutralized all detectable transducing activity for the Kill+ marker, whereas antiserum prepared against a phage unrelated to 4)75 (phage PBP1; neutralization constant, 10; 5) had no neutralizing activity against 4)75 PFU or the Kill+ transducing activity. Moreover, incubation of a 475-transducing lysate at 650C
1029
1030
J. BACTZRIOL.
NOTES TABLE 1. Strains of B. pumilus
Strain ATCC 12140 L10 NRRL B-3275
231-1-8 NRS 576 Cat-1 W20 W40
Relevant properties
Wild type; pMB2+ Lac; pPL10+
pMB1+
Origin/reference 8
8
1, 10
Wild type; lysogenic for 4)75 serC3 leu-3 Iys-2 4)75
9
Wild type; pPL576+ ade-100 pPL576+ ade-100 ade-100 cys-100 ser-
6 6 6 NGb of W20
100
GM45c
ade-100 ilv-100 Iys-100 Spo
1
ATCC Wild type 7061 a pPL or pMB refers to the presence (+) of a specific plasmid. Abbreviations: Lac, lactose; ser, serine; leu, leucine; lys, lysine; ade, adenine; cys, cysteine; ilv, isoleucine-valine; Spo, asporogenic. b Mutagenesis by nitrosoguanidine. c Strain sensitive to plaque formation by 4)75.
caused simultaneous inactivation ofthe plaqueforming and Kill+ transducing activities. These data suggest that the Kill+ transducing activity was mediated by 4)75. Derivatives of strain NRRL B-3275 that lack qb75 have not been isolated. Therefore, to establish a series of nearly isogenic strains that differ only in the presence or absence of 4)75 and pPL10, the mutant GM45 was used. GM45 is an asporogenic, plasmid-negative (lacks pPL576) derivative of strain NRS576 that is sensitive to 4)75 (plaque forming) and can be stably lysogenized by 4)75 (1). The recipient for transductions was W40, which is insensitive to 475 (plaque forming) but absorbs the phage (1). A series of derivatives of GM45 (Table 2) was induced with mitomycin C, and the resulting lysates were assayed for PFU and for Kill+ and Cys+ transducing activities. The data demonstrate that Kill+ transductants were obtained only when the induced lysate was prepared from a cell line that was both a 4)75 lysogen and harbored pPL10 (Table 2). No Cys+ transductants of W40 were detected. 4)75 lysates for gradient analyses were obtained by using a clear-plaque-forming variant of 4)75 (475C1) grown on strain GM45(pPL10). 4)75C1 generated Kill+ transductants at a frequency of 1 transductant per 105 PFU, deter-
mined by using W40 as a transduction recipient and GM45 as an indicator for plaque assays. Equilibrium centrifugation of a portion of the lysate in CsCl demonstrated that the buoyant densities of the PFU and Kill+ transducing activity were indistinguishable within the limits of the technique. Similarly, centrifugation of a portion of the lysate through a 5 to 20% neutral sucrose gradient demonstrated that the PFU and the transducing activity cosedimented (Fig. 1). These results indicate that the PFU and the transducing particles are very similar in size and DNA/protein ratio. Four Kill+ transductants of strain W20 generated by using 475C1 propagated on GM45(pPL10), three Kill+ transductants of strain W40 generated by-using a 475 containing mitomycin C-induced lysate of strain GM45(475 pPL10), and three Kill+ transductants of W20 generated by using a 475 containing mitomycin C-induced lysate of 231-1-8 (Table 2) were grown for plasmid isolation (8). In each case, the Kill+ transductants contained 3.2(±0.5)% of their total cell DNA as CCC molecules that sedimented at 25(±1)S in neutral sucrose gradients. Cat-1 is an adenine-requiring mutant of B. pumilus NRS576. Cat-1 harbors about two copies per chromosome of the 28 x 106-dalton plasmid pPL576 (6). We have previously shown that pPL576 and pPL10 can stably coexist in the same cell and, thus, are compatible plasmids (8). A Kill+ transductant of Cat-1 generated by using 475 induced from GM45(4)75 pPL10) was grown for plasmid isolation. Approximately 7% TABLz 2. Transducing activity in mitomycin Cinduced Iysates of derivatives of GM45a Source of induced
late
TranUductanm/ml
~ ~ ~Kil +
Cyst
JOURNAL OF BACTZRIOLOGY, Sept. 1977, p. 1029-1032 Copyright 0 1977 American Society for Microbiology
Printed in U.S.A.
Selective Plasmid Transduction in Bacillus pumilus MICHAEL G. BRAMUCCI' AND PAUL S. LOVETT* Department ofBiological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228
Received for publication 1 April 1977
The inducible temperate bacteriophage 475 and a clear-plaque-forming variant, 475C1, mediated transduction of a 4.4 x 10"-dalton multicopy Bacillus pumilus plasmid, pPL10, at frequencies of 10-5 to 10-6 transductants per plaqueforming unit. 4)75- and 475C1-mediated transduction of several chromosome markers tested did not occur at a detectable frequency. 475-mediated plasmid transducing activity resides in particles that are similar to infectious particles in sedimentation velocity and buoyant density.
Several temperate bacteriophages have been isolated for members of the related species Bacillus subtilis andB. pumilus (1, 3, 4, 14). None of these phages has been shown to perform generalized transduction, and only the B. subtilis temperate phages 4105 and SP,8 have been reported to perform specialized transduction (12, 14). Previous attempts to detect transduction mediated by B. subtilis orB. pumilus temperate phages have been limited to studies of the transfer of host chromosome genes. Studies of plasmid transduction in Bacillus have only recently become possible as a result of the identification of a selectable host function that is plasmid determined in Bacillus (8). Plasmid pPL10 (-4.4 x 106 daltons; p= 1.698 g/cm3; 10 to 20 copies per chromosome) determines the production of, and immunity or resistance to, killing activity in strains of B. pumilus (8). pPL10 has been transferred among strains of B. pumilus by each of three generalized transducing phages (PBS1, PBP1, and PMB1) (2), and the plasmid has been transformed into B. subtilis 168 by an indirect selection technique (8). However, transduction studies of the plasmid in B. subtilis have not been possible because B. subtilis is naturally resistant to the pPL10-determined killing activity, and strains of B. subtilis harboring pPL10 only weakly express the Kill+ phenotype (8). We examined the ability of the inducible B. pumilus temperate phage 475 to mediate transduction of plasmid pPL10. 475 efficiently performs plasmid transduction, but it is incapable of mediating transduction of any of several chromosome markers. The ability of 475 to mediate pPL10 transduction was initially tested by inserting the plasI Present address: Uniformed Services University of the Health Sciences, Department of Microbiology, Bethesda, MD 20014.
mid into a mutant derivative of NRRL B-3275, strain 231-1-8 (Table 1), by transduction with PBP1 grown on strain L10 (Table 1; 5, 7, 8). Each of three Kill+ transductants retained the nutritional requirements of 231-1-8 (Ser Leu Lys), and each contained 4.4(±0.3)% ofthe total cell deoxyribonucleic acid (DNA) as covalently closed circular duplex (CCC) molecules (detected as previously described [8]) that sedimented at 25(± 1)S in neutral sucrose gradients relative to ['4C]thymidine-(New England Corp., Boston, Mass.) labeled T7 DNA (32S; 13). Mitomycin C induction (1) of strain 231-1-8 and the three plasmid-containing derivatives yielded 4)75 lysates with an infectious titer of about 108 plaque-forming units (PFU) per ml. Lysates induced from the plasmid-containing strains generated Kill+ transductants of strains W20 and Cat-1 at a frequency of 1 transductant per 10" PFU, whereas phages induced from strain 231-1-8 not containing pPL10 did not generate Kill+ transductants of either recipient. None of the lysates was capable of generating adenine-positive transductants of strains W20 or Cat-1. In other experiments, we were unable to demonstrate 4)75 transduction of chromosome markers try-2, gly-1, cys-1, lys-2, his-i, ade-1, leu-3, met-3, thr-1, and argA2 (9-11) in derivatives of the NRRL B-3275 strain and chromosome markers ade-100, trplOO, lys-100, cys-100, and ser-100 (1, 6) in derivatives of the NRS576 strain. Antiserum (1) neutralizing >99.9% of the PFU in a 475 lysate (10k PFU/ml) in 15 min at 37°C also neutralized all detectable transducing activity for the Kill+ marker, whereas antiserum prepared against a phage unrelated to 4)75 (phage PBP1; neutralization constant, 10; 5) had no neutralizing activity against 4)75 PFU or the Kill+ transducing activity. Moreover, incubation of a 475-transducing lysate at 650C
1029
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J. BACTZRIOL.
NOTES TABLE 1. Strains of B. pumilus
Strain ATCC 12140 L10 NRRL B-3275
231-1-8 NRS 576 Cat-1 W20 W40
Relevant properties
Wild type; pMB2+ Lac; pPL10+
pMB1+
Origin/reference 8
8
1, 10
Wild type; lysogenic for 4)75 serC3 leu-3 Iys-2 4)75
9
Wild type; pPL576+ ade-100 pPL576+ ade-100 ade-100 cys-100 ser-
6 6 6 NGb of W20
100
GM45c
ade-100 ilv-100 Iys-100 Spo
1
ATCC Wild type 7061 a pPL or pMB refers to the presence (+) of a specific plasmid. Abbreviations: Lac, lactose; ser, serine; leu, leucine; lys, lysine; ade, adenine; cys, cysteine; ilv, isoleucine-valine; Spo, asporogenic. b Mutagenesis by nitrosoguanidine. c Strain sensitive to plaque formation by 4)75.
caused simultaneous inactivation ofthe plaqueforming and Kill+ transducing activities. These data suggest that the Kill+ transducing activity was mediated by 4)75. Derivatives of strain NRRL B-3275 that lack qb75 have not been isolated. Therefore, to establish a series of nearly isogenic strains that differ only in the presence or absence of 4)75 and pPL10, the mutant GM45 was used. GM45 is an asporogenic, plasmid-negative (lacks pPL576) derivative of strain NRS576 that is sensitive to 4)75 (plaque forming) and can be stably lysogenized by 4)75 (1). The recipient for transductions was W40, which is insensitive to 475 (plaque forming) but absorbs the phage (1). A series of derivatives of GM45 (Table 2) was induced with mitomycin C, and the resulting lysates were assayed for PFU and for Kill+ and Cys+ transducing activities. The data demonstrate that Kill+ transductants were obtained only when the induced lysate was prepared from a cell line that was both a 4)75 lysogen and harbored pPL10 (Table 2). No Cys+ transductants of W40 were detected. 4)75 lysates for gradient analyses were obtained by using a clear-plaque-forming variant of 4)75 (475C1) grown on strain GM45(pPL10). 4)75C1 generated Kill+ transductants at a frequency of 1 transductant per 105 PFU, deter-
mined by using W40 as a transduction recipient and GM45 as an indicator for plaque assays. Equilibrium centrifugation of a portion of the lysate in CsCl demonstrated that the buoyant densities of the PFU and Kill+ transducing activity were indistinguishable within the limits of the technique. Similarly, centrifugation of a portion of the lysate through a 5 to 20% neutral sucrose gradient demonstrated that the PFU and the transducing activity cosedimented (Fig. 1). These results indicate that the PFU and the transducing particles are very similar in size and DNA/protein ratio. Four Kill+ transductants of strain W20 generated by using 475C1 propagated on GM45(pPL10), three Kill+ transductants of strain W40 generated by-using a 475 containing mitomycin C-induced lysate of strain GM45(475 pPL10), and three Kill+ transductants of W20 generated by using a 475 containing mitomycin C-induced lysate of 231-1-8 (Table 2) were grown for plasmid isolation (8). In each case, the Kill+ transductants contained 3.2(±0.5)% of their total cell DNA as CCC molecules that sedimented at 25(±1)S in neutral sucrose gradients. Cat-1 is an adenine-requiring mutant of B. pumilus NRS576. Cat-1 harbors about two copies per chromosome of the 28 x 106-dalton plasmid pPL576 (6). We have previously shown that pPL576 and pPL10 can stably coexist in the same cell and, thus, are compatible plasmids (8). A Kill+ transductant of Cat-1 generated by using 475 induced from GM45(4)75 pPL10) was grown for plasmid isolation. Approximately 7% TABLz 2. Transducing activity in mitomycin Cinduced Iysates of derivatives of GM45a Source of induced
late
TranUductanm/ml
~ ~ ~Kil +
Cyst
