Vol. 124, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, OCt. 1975, p. 484-490 Copyright 0 1975 American Society for Microbiology
Plasmid Deoxyribonucleic Acid in Bacillus subtilis and Bacillus pumilus PAUL S. LOVETT* AND MICHAEL G. BRAMUCCI Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228 Received for publication 7 March 1975
Two of eighteen strains of Bacillus subtilis examined contained covalently closed circular duplex deoxyribonucleic acid (DNA) of homogeneous size and buoyant density. Strain ATCC 15841 contained about 16 copies per chromosome of plasmid pPL1, a circular DNA element having a molecular weight of about 4.7 x 106 and a buoyant density of 1.700. Strain ATCC 7003 contained about one to two copies per chromosome of plasmid pPL2. pPL2 had a molecular weight of about 46 x 10' and a buoyant density of 1.696. Strain ATCC 7003 appeared to be closely related to B. subtilis 168 by genetic, physiological, and biochemical criteria. Strain ATCC 15841 appeared to be much less closely related. B. pumilus ATCC 12140 contained two size classes of covalently closed circular duplex DNA. The plasmids pMB1 and pMB2 had molecular weights of about 6.8 x 10 and 5.3 x 106, respectively, and were present in several copies per chromosome.
Two strains of Bacillus pumilus have been shown to contain extrachromosomal, covalently closed circular (CCC) duplex deoxyribonucleic acid (DNA) molecules of homogeneous size and buoyant density (13, 14, 17). These elements are present in cells at a relatively fixed copy number and appear biochemically comparable to the genetically characterized plasmids in Staphylococcus aureus and the enterics (4, 7, 20). The plasmid pPL576 carried by B. pumilus strain NRS 576 has been most extensively studied because its presence correlates with a phenotypic property of the host. Strain NRS 576 is oligosporogenic. Spontaneously occurring variants of this strain that form spores at an elevated frequency (the W mutants) lack the plasmid (13. 14). The presence of pPL576 clearly correlates with the reduced ability of the host strain to undergo sporulation. All available evidence indicates that pPL576 is an autonomously replicating DNA molecule and not, for example, a collection of similar-size DNA circles resulting from cyclization of random chromosome fragments. pPL576 has a chracteristic buoyant density (p = 1.698) distinct from that of the host chromosome (p = 1.701 [14]). The plasmid shares little homology with the host chromosome (14). Lastly, pPL576 contains a nonrandom (unique) base sequence as judged by its cleavage into three distinct linear fragments by EcoRl endonuclease (15). Physical studies suggest that pPL576 is distinct from a plasmid carried by B. pumilus strain ATCC 7065 (designated pPL7065 [14, 17]). The two elements differ in size, buovant
density, and copy number. No biological function has been attributed to pPL7065, hence it is classified as a cryptic plasmid. Before the isolation of pPL576 and pPL7065, the existence of the plasmids in sporeforming bacteria was suggested only by the detection of heterogeneous minicircles that comprise up to 30% of the DNA isolated from a strain of B. megaterium (3). Subsequent studies have shown that the majority of these minicircles are probably derived from the host chromosome and are therefore unlikely candidates for autonomous plasmids (9). However, the existence of plasmids as a minor fraction of the total minicircles is possible (2). The paucity of examples of Bacillus plasmids prompted us to screen several strains of the related species B. subtilis and B. pumilus for the presence of CCC duplex DNA. The isolation and properties of cryptic plasmids carried by two strains of B. subtilis and a strain of B. pumilus are the subject of the present report. MATERIALS AND METHODS Bacteria and growth conditions. B. subtilis strains examined for plasmids and their sources are shown in Table 1. B. subtilis strains ATCC 15841 and ATCC 7003 were the source of plasmids pPL1 and pPL2, respectively. Strains of B. subtilis 168 used for genetic transformations are shown in Table 2. B. pumilus ATCC 12140 was from R. Gordon. All growth media have been reported (13). Incubation was at 37 C; liquid cultures were grown with rotary shaking. Plasmid isolation. Cells were labeled with ['H Ithymidine (New England Nuclear Corp.) and lysed, and the lysates were centrifuged for 40 to 46 h in 484
PLASMIDS IN BACILLUS
VOL. 124, 1975
TABLE 1. Strains of Bacillus subtilis tested for CCC duplex DNAa of CCC bPresence Strain5 Strai' duplex DNAC
168 W23 ATCC 9799 ATCC 15562 ATCC 8188 ATCC 15561 ATCC 8472 ATCC 15245 ATCC 7003 ATCC 4925 ATCC 7972 ATCC 7480 ATCC 7059 ATCC 19659 ATCC 15575 ATCC 15841 ATCC 15818 ATCC 23059
0 0 0 0 0 0 0 0 + 0 0 0 0 0 0 + 0 0
485
E. coli DNA. Calculations of buoyant density were according to the equations of Schildkraut et al. (21). Transformation. Transforming DNA was prepared by a phenol method (18). Competent cells, prepared according to the method of Bott and Wilson (1), was shaken for 30 min at 37 C with saturating levels (5 gg/ml) of DNA. Transformation was terminated by addition of deoxyribonuclease (10 Ag/ml). Recipients were plated onto Spizizen minimal medium (22) supplemented with 0.05% acid-hydrolyzed casein when selection was for Trp+ transformants. Viable counts were determined by plating cells onto tryptose blood agar base (Difco). Results, were recorded after 25 to 30 h of incubation. Controls for DNA sterility and marker reversion were run. Mutant isolation. Mutants were isolated after treatment of cells with N-methyl-N'-nitrosoguanidine as previously described (19). Electron microscopy. DNA was mounted and stained with uranyl acetate as previously reported
(13).
Agarose-gel electrophoresis of DNA. The procedure followed was that described in detail by Helling et al. (8). Characterization of strains ATCC 7003 and a Twenty-milliliter cultures were grown for 18 to 20 h (stationary phase) in Spizizen minimal medium (22) ATCC 15841. Procedures for DNA hybridization by procedure and physiological containing 0.05% acid-hydrolyzed casein, 250 Ag of the membrane filter of the strains was performed as characterization deoxyadenosine per ml, and 0.05 mCi of [3H]thymidine. DNA extraction and demonstration of CCC previously described in detail (18). duplex DNA by dye-buoyant density centrifugation RESULTS was as previously described (14). " Strains 168 and W23 were from Frank Young and Lysates of 18 strains of B. subtilis were Kenneth Bott, respectively. Strain ATCC 9799 was examined for the presence of CCC duplex DNA purchased from the American Type Culture Collec- in dye-buoyant density gradients. DNA from 16 tion. All other strains were donated by Ruth Gordon. of the strains, including the highly transformac0, No detectable satellite peak in CsCl-ethidium ble strain 168 (21), contained no detectable bromide gradients (less than 0.2% of total cell DNA was in supercoiled form); +, strain contained detecta- levels of supercoiled DNA (less than 0.2%7; Table 1). Two of the strains, ATCC 15841 and ble levels of supercoiled DNA.
CsCl-ethidium bromide gradients in the manner previously described in detail (14). Plasmid DNA was not further purified after isolation from CsCl-ethidium bromide gradients and exhaustive dialysis against TES buffer (14). Velocity and equilibrium centrifugation. Neutral and alkaline 5 to 20% sucrose gradients were prepared, run, and processed as previously described (13). Bacteriophage T7 DNA labeled with ["4C]thymidine was reference. T7 DNA was assigned an S value (sedimentation velocity) of 32 in neutral gradients and 37 in alkaline gradients (23). Equilibrium centrifugation of plasmid DNA in CsCl gradients was performed in a Beckman model E analytical ultracentrifuge as before (14). Reference was DNA from bacteriophage PBP1 (12) or Escherichia coli K-12. DNA from PBP1 propagated on B. pumilus strain NRRL B-3275 had a buoyant density of 1.690 relative to E. coli DNA (p = 1.710 [16]). The PBP1 DNA used in the present study was purified from phage propagated on strain ATCC 7065. This phage DNA had a buoyant density of 1.691 relative to
ATCC 7003, did contain supercoiled DNA (Fig. 1). The CCC duplex DNA comprised 4 (+0.4) % of the total DNA extracted from cells of both strains grown to log or stationary phase. pPLI: plasmid in B. subtilis ATCC 15841. The supercoiled DNA extracted from strain ATCC 15841 sedimented as a single component on neutral and alkaline sucrose gradients (Fig. 2). pPL1 had a sedimentation velocity of 25.5 (±1)S on neutral gradients and 45 (±1)S on alkaline gradients. The value of 25.5S on neutral gradients corresponds to a CCC duplex DNA molecule with a molecular weight of 4.7 x TABLE 2. Mutant derivatives of B. subtilis 168 used in transformations B. subtilis strain
168
BR151 SB5
Genotvpe
Source
Wild-type lys-3 trpC2 metBlO hisA1 ura-1 trpC2
F. Young R. Yasbin T. Higerd
486
LOVET1 AND BRAMUCCI
J. BACTRIOL.
10. (4). Accepting that the chromosome of B. subtilis has a molecular weight of about 2 x 10' (9, 10), we estimated that pPL1 was present in up to 16 copies per chromosome. pPL1 DNA banded as a single component in CsCI gradients (p = 1.700; Fig. 3). pPL2: plasmid in B. subtiis ATCC 7003. The supercoiled DNA extracted from strain :2 ATCC 7003 generally sedimented as two components on neutral and alkaline sucrose gradients. On neutral gradients (Fig. 4) the major peak had an S value of 75.3 (±2), corresponding to a CCC duplex molecule of 46.3 x 101 daltons (4). The minor peak had an S value of 48.0 (±1), corresponding to the open circular form of a plasmid with a molecular weight of 46.2 x 106 (4). On alkaline gradients (not shown), a major sedimented at greater than 120S and a peak 10 15 minor component sedimented at about 48S. FRACTIONS The gradient data demonstrate that strain FIG. 1. CsCI-ethidium bromide gradient centrifu- ATCC 7003 contained a single-size plasmid gation of DNA extracted from B. subtilis strains 168, species with a molecular weight of about 46 x ATCC 15841
ATCC 7003
11 K
0u
Al
ATCC 15841, and ATCC 7003. DNA labeled with ['H]thymidine was extracted (13) from 20-mi stationary-phase cultures of the strains. Lysates were cleared (12,000 x g, 30 min), and the supernatant fractions were mixed with CsCI and ethidium bromide and centrifuged at 36,000 rpm in a Ti50 rotor at 15 C for 40 h, as previously described (13). Gradients (5 ml) were collected in 15 to 18 fractions. Ten-microliter portions of each fraction were dried onto filter paper disks and counted.
10.
pPL2 CCC duplex DNA constituted approximately 4% of the DNA extracted from ATCC 7003. Therefore pPL2 was probably present in a limited number (one or two) of copies per chromosome. pPL2 was homogeneous with respect to buoyant density (p = 1.696; Fig. 3). Relatedness between B. subtilis 168 and
FRACTIONS
FIG. 2. Centrifugation of plasmid pPLJ and T7 DNA through 5 to 20%o neutral and alkaline sucrose gradients. pPLJ DNA was labeled with ['H]thymidine (solid circles). T7DNA was labeled with ["4C]thymidine (open circles). The neutral gradient was centrifuged in an SW50.1 rotor at 40,000 rpm for 4.5 h at 5 C. The alkaline gradient was centrifuged under similar conditions but for 3.5 h. Fractions were collected through holes pierced in the tube bottoms, precipitated with cold 5% trichloroacetic acid, and counted.
PLASMIDS IN BACILLUS
VOL. 124, 1975
l 1
l
g o
JOURNAL OF BACTERIOLOGY, OCt. 1975, p. 484-490 Copyright 0 1975 American Society for Microbiology
Plasmid Deoxyribonucleic Acid in Bacillus subtilis and Bacillus pumilus PAUL S. LOVETT* AND MICHAEL G. BRAMUCCI Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228 Received for publication 7 March 1975
Two of eighteen strains of Bacillus subtilis examined contained covalently closed circular duplex deoxyribonucleic acid (DNA) of homogeneous size and buoyant density. Strain ATCC 15841 contained about 16 copies per chromosome of plasmid pPL1, a circular DNA element having a molecular weight of about 4.7 x 106 and a buoyant density of 1.700. Strain ATCC 7003 contained about one to two copies per chromosome of plasmid pPL2. pPL2 had a molecular weight of about 46 x 10' and a buoyant density of 1.696. Strain ATCC 7003 appeared to be closely related to B. subtilis 168 by genetic, physiological, and biochemical criteria. Strain ATCC 15841 appeared to be much less closely related. B. pumilus ATCC 12140 contained two size classes of covalently closed circular duplex DNA. The plasmids pMB1 and pMB2 had molecular weights of about 6.8 x 10 and 5.3 x 106, respectively, and were present in several copies per chromosome.
Two strains of Bacillus pumilus have been shown to contain extrachromosomal, covalently closed circular (CCC) duplex deoxyribonucleic acid (DNA) molecules of homogeneous size and buoyant density (13, 14, 17). These elements are present in cells at a relatively fixed copy number and appear biochemically comparable to the genetically characterized plasmids in Staphylococcus aureus and the enterics (4, 7, 20). The plasmid pPL576 carried by B. pumilus strain NRS 576 has been most extensively studied because its presence correlates with a phenotypic property of the host. Strain NRS 576 is oligosporogenic. Spontaneously occurring variants of this strain that form spores at an elevated frequency (the W mutants) lack the plasmid (13. 14). The presence of pPL576 clearly correlates with the reduced ability of the host strain to undergo sporulation. All available evidence indicates that pPL576 is an autonomously replicating DNA molecule and not, for example, a collection of similar-size DNA circles resulting from cyclization of random chromosome fragments. pPL576 has a chracteristic buoyant density (p = 1.698) distinct from that of the host chromosome (p = 1.701 [14]). The plasmid shares little homology with the host chromosome (14). Lastly, pPL576 contains a nonrandom (unique) base sequence as judged by its cleavage into three distinct linear fragments by EcoRl endonuclease (15). Physical studies suggest that pPL576 is distinct from a plasmid carried by B. pumilus strain ATCC 7065 (designated pPL7065 [14, 17]). The two elements differ in size, buovant
density, and copy number. No biological function has been attributed to pPL7065, hence it is classified as a cryptic plasmid. Before the isolation of pPL576 and pPL7065, the existence of the plasmids in sporeforming bacteria was suggested only by the detection of heterogeneous minicircles that comprise up to 30% of the DNA isolated from a strain of B. megaterium (3). Subsequent studies have shown that the majority of these minicircles are probably derived from the host chromosome and are therefore unlikely candidates for autonomous plasmids (9). However, the existence of plasmids as a minor fraction of the total minicircles is possible (2). The paucity of examples of Bacillus plasmids prompted us to screen several strains of the related species B. subtilis and B. pumilus for the presence of CCC duplex DNA. The isolation and properties of cryptic plasmids carried by two strains of B. subtilis and a strain of B. pumilus are the subject of the present report. MATERIALS AND METHODS Bacteria and growth conditions. B. subtilis strains examined for plasmids and their sources are shown in Table 1. B. subtilis strains ATCC 15841 and ATCC 7003 were the source of plasmids pPL1 and pPL2, respectively. Strains of B. subtilis 168 used for genetic transformations are shown in Table 2. B. pumilus ATCC 12140 was from R. Gordon. All growth media have been reported (13). Incubation was at 37 C; liquid cultures were grown with rotary shaking. Plasmid isolation. Cells were labeled with ['H Ithymidine (New England Nuclear Corp.) and lysed, and the lysates were centrifuged for 40 to 46 h in 484
PLASMIDS IN BACILLUS
VOL. 124, 1975
TABLE 1. Strains of Bacillus subtilis tested for CCC duplex DNAa of CCC bPresence Strain5 Strai' duplex DNAC
168 W23 ATCC 9799 ATCC 15562 ATCC 8188 ATCC 15561 ATCC 8472 ATCC 15245 ATCC 7003 ATCC 4925 ATCC 7972 ATCC 7480 ATCC 7059 ATCC 19659 ATCC 15575 ATCC 15841 ATCC 15818 ATCC 23059
0 0 0 0 0 0 0 0 + 0 0 0 0 0 0 + 0 0
485
E. coli DNA. Calculations of buoyant density were according to the equations of Schildkraut et al. (21). Transformation. Transforming DNA was prepared by a phenol method (18). Competent cells, prepared according to the method of Bott and Wilson (1), was shaken for 30 min at 37 C with saturating levels (5 gg/ml) of DNA. Transformation was terminated by addition of deoxyribonuclease (10 Ag/ml). Recipients were plated onto Spizizen minimal medium (22) supplemented with 0.05% acid-hydrolyzed casein when selection was for Trp+ transformants. Viable counts were determined by plating cells onto tryptose blood agar base (Difco). Results, were recorded after 25 to 30 h of incubation. Controls for DNA sterility and marker reversion were run. Mutant isolation. Mutants were isolated after treatment of cells with N-methyl-N'-nitrosoguanidine as previously described (19). Electron microscopy. DNA was mounted and stained with uranyl acetate as previously reported
(13).
Agarose-gel electrophoresis of DNA. The procedure followed was that described in detail by Helling et al. (8). Characterization of strains ATCC 7003 and a Twenty-milliliter cultures were grown for 18 to 20 h (stationary phase) in Spizizen minimal medium (22) ATCC 15841. Procedures for DNA hybridization by procedure and physiological containing 0.05% acid-hydrolyzed casein, 250 Ag of the membrane filter of the strains was performed as characterization deoxyadenosine per ml, and 0.05 mCi of [3H]thymidine. DNA extraction and demonstration of CCC previously described in detail (18). duplex DNA by dye-buoyant density centrifugation RESULTS was as previously described (14). " Strains 168 and W23 were from Frank Young and Lysates of 18 strains of B. subtilis were Kenneth Bott, respectively. Strain ATCC 9799 was examined for the presence of CCC duplex DNA purchased from the American Type Culture Collec- in dye-buoyant density gradients. DNA from 16 tion. All other strains were donated by Ruth Gordon. of the strains, including the highly transformac0, No detectable satellite peak in CsCl-ethidium ble strain 168 (21), contained no detectable bromide gradients (less than 0.2% of total cell DNA was in supercoiled form); +, strain contained detecta- levels of supercoiled DNA (less than 0.2%7; Table 1). Two of the strains, ATCC 15841 and ble levels of supercoiled DNA.
CsCl-ethidium bromide gradients in the manner previously described in detail (14). Plasmid DNA was not further purified after isolation from CsCl-ethidium bromide gradients and exhaustive dialysis against TES buffer (14). Velocity and equilibrium centrifugation. Neutral and alkaline 5 to 20% sucrose gradients were prepared, run, and processed as previously described (13). Bacteriophage T7 DNA labeled with ["4C]thymidine was reference. T7 DNA was assigned an S value (sedimentation velocity) of 32 in neutral gradients and 37 in alkaline gradients (23). Equilibrium centrifugation of plasmid DNA in CsCl gradients was performed in a Beckman model E analytical ultracentrifuge as before (14). Reference was DNA from bacteriophage PBP1 (12) or Escherichia coli K-12. DNA from PBP1 propagated on B. pumilus strain NRRL B-3275 had a buoyant density of 1.690 relative to E. coli DNA (p = 1.710 [16]). The PBP1 DNA used in the present study was purified from phage propagated on strain ATCC 7065. This phage DNA had a buoyant density of 1.691 relative to
ATCC 7003, did contain supercoiled DNA (Fig. 1). The CCC duplex DNA comprised 4 (+0.4) % of the total DNA extracted from cells of both strains grown to log or stationary phase. pPLI: plasmid in B. subtilis ATCC 15841. The supercoiled DNA extracted from strain ATCC 15841 sedimented as a single component on neutral and alkaline sucrose gradients (Fig. 2). pPL1 had a sedimentation velocity of 25.5 (±1)S on neutral gradients and 45 (±1)S on alkaline gradients. The value of 25.5S on neutral gradients corresponds to a CCC duplex DNA molecule with a molecular weight of 4.7 x TABLE 2. Mutant derivatives of B. subtilis 168 used in transformations B. subtilis strain
168
BR151 SB5
Genotvpe
Source
Wild-type lys-3 trpC2 metBlO hisA1 ura-1 trpC2
F. Young R. Yasbin T. Higerd
486
LOVET1 AND BRAMUCCI
J. BACTRIOL.
10. (4). Accepting that the chromosome of B. subtilis has a molecular weight of about 2 x 10' (9, 10), we estimated that pPL1 was present in up to 16 copies per chromosome. pPL1 DNA banded as a single component in CsCI gradients (p = 1.700; Fig. 3). pPL2: plasmid in B. subtiis ATCC 7003. The supercoiled DNA extracted from strain :2 ATCC 7003 generally sedimented as two components on neutral and alkaline sucrose gradients. On neutral gradients (Fig. 4) the major peak had an S value of 75.3 (±2), corresponding to a CCC duplex molecule of 46.3 x 101 daltons (4). The minor peak had an S value of 48.0 (±1), corresponding to the open circular form of a plasmid with a molecular weight of 46.2 x 106 (4). On alkaline gradients (not shown), a major sedimented at greater than 120S and a peak 10 15 minor component sedimented at about 48S. FRACTIONS The gradient data demonstrate that strain FIG. 1. CsCI-ethidium bromide gradient centrifu- ATCC 7003 contained a single-size plasmid gation of DNA extracted from B. subtilis strains 168, species with a molecular weight of about 46 x ATCC 15841
ATCC 7003
11 K
0u
Al
ATCC 15841, and ATCC 7003. DNA labeled with ['H]thymidine was extracted (13) from 20-mi stationary-phase cultures of the strains. Lysates were cleared (12,000 x g, 30 min), and the supernatant fractions were mixed with CsCI and ethidium bromide and centrifuged at 36,000 rpm in a Ti50 rotor at 15 C for 40 h, as previously described (13). Gradients (5 ml) were collected in 15 to 18 fractions. Ten-microliter portions of each fraction were dried onto filter paper disks and counted.
10.
pPL2 CCC duplex DNA constituted approximately 4% of the DNA extracted from ATCC 7003. Therefore pPL2 was probably present in a limited number (one or two) of copies per chromosome. pPL2 was homogeneous with respect to buoyant density (p = 1.696; Fig. 3). Relatedness between B. subtilis 168 and
FRACTIONS
FIG. 2. Centrifugation of plasmid pPLJ and T7 DNA through 5 to 20%o neutral and alkaline sucrose gradients. pPLJ DNA was labeled with ['H]thymidine (solid circles). T7DNA was labeled with ["4C]thymidine (open circles). The neutral gradient was centrifuged in an SW50.1 rotor at 40,000 rpm for 4.5 h at 5 C. The alkaline gradient was centrifuged under similar conditions but for 3.5 h. Fractions were collected through holes pierced in the tube bottoms, precipitated with cold 5% trichloroacetic acid, and counted.
PLASMIDS IN BACILLUS
VOL. 124, 1975
l 1
l
g o
