Vol. 123, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, July 1975, p. 377-379 Copyright 0 1975 American Society for Microbiology
Evidence for a Nonrandom Base Sequence in a Bacillus pumilus Plasmid: EcoRl Endonuclease Digestion of pPL576 PAUL S. LOVETT* AND MICHAEL G. BRAMUCCI Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228 Received for publication 25 April 1975
EcoRl endonuclease digested the Bacillus pumilus plasmid pPL576 (molecular weight 28 x 106) into three distinct size classes of linear fragments. The molecular weights of the fragments are 13.0 x 106, 9.5 x 106, and 6.5 x 106 by sucrose gradient analysis. By electron microscope analysis the three fragments account for about 99% of the intact plasmid. These results indicate that pPL576 molecules contain a nonrandom base sequence, and are consistent with the interpretation that pPL576 is autonomous and not the result of cyclization of random chromosome fragments. -
Extrachromosomal circular duplex deoxyribonucleic acid (DNA) molecules of uniform size have been isolated from Bacillus pumilus strain NRS 576 (7). Although the specific genetic function(s) of this element is unknown, its presence correlates with the reduced ability of the host strain to form spores (7). pPL576 has been described as a plasmid primarily on the basis of its biochemical similarity to genetically characterized plasmids in enterics and Staphylococcus aureus (1, 5, 10). pPL576 is homogeneous in size and buoyant density and appears present in cells at a relatively fixed copy number (8). The use of the term plasmid to describe pPL576 implies that the element is autonomous and not the result of random fragmentation of the host chromosome into discrete linear pieces with subsequent cyclization. Such fragmentation could result, for example, from spontaneous induction of inducible phages carried by the host (6). Indeed, a large percentage of the small, heterogeneous circular DNA molecules isolated from B. megaterium strain 216 appears chromosomal in origin (6). pPL576 shares little homology with the chromosome of the host (8). This is consistent with the interpretation that the element is autonomous, and further predicts that all pPL576 molecules possess an identical base sequence. Certain endonucleases such as EcoRl cleave duplex DNA at sites within a length of specific base sequence (4). Cleavage of a population of plasmid molecules of identical base sequence should yield a discrete number of fragments whose additive molecular weight is equivalent
to that of the intact circle. Cleavage of a population of circular molecules of homogeneous size but random base sequence would result in numerous random size classes of linear fragments. In the present communication, we demonstrate that EcoRl endonuclease digestion of pPL576 cleaves the circular element into a limited number (three) of linear fragments whose additive size is equivalent to that of intact pPL576. These results indicate that pPL576 molecules possess a similar (presumably identical) base sequence. Accordingly, pPL576 appears not to result from cyclization of
random chromosome fragments. B. pumilus strain NRS 576 was the source of the plasmid pPL576 (7, 8). Plasmid DNA was isolated by dye-buoyant density gradient centrifugation (8). Neutral 5 to 20% sucrose gradients were prepared and run as before (7). DNA was prepared for electron microscopy by the aqueous technique of Davis et al. (2) or as previously described by using formamide (7). All other procedures were previously described
(8).
EcoRl restriction endonuclease was purchased from Miles Laboratories. Digestion of pPL576 was performed by incubation of 1 Ad of enzyme with 2 Ag (or less) of plasmid in 0.1 ml of 0.1 M tris(hydroxymethyl)aminomethane buffer, pH 7.2, 10-3 M MgCl2, and 0.05 M NaCl for 15 min at 37 C. Reactions were terminated by heating at 60 C for 5 min. These digestion conditions converted essentially all plasmid circles to three linear fragments as judged by analysis of the reaction mixture on sucrose 377
378
J. BACTERIOL.
NOTES
gradients and by electron microscopy. Increasing the enzyme level fivefold and the time of digestion to 3 h had no detectable effect on the extent of fragmentation. The experiments reported in the present study were performed with EcoRl from Miles. In earlier work, comparable results were obtained by using an enzyme preparation isolated from Escherichia coli RY13 and purified through phosphocellulose chromatography as described by Greene et al. (3). pPL576 digested with EcoRl sediments as three peaks in sucrose gradients (Fig. 1). Results comparable to those shown in Fig. 1 were obtained by increasing the enzyme level fivefold, and by increasing the digestion time up to 3 h. Hence, the conditions described in Fig. 1 appear to result in a limit digest. The sedimentation velocities of the three fragments (Fig. 1) are 25.5 (±0.5), 22.9 (+0.5), and 20.1 (±0.5) S (average of data from seven digests). These correspond to linear duplexes of 13.0 x 106, 9.5 x 106 and 6.5 x 106 daltons (11). The additive molecular weight of the three components is 29 x 106. This value is similar to the molecular weight reported for the intact plasmid determined by electron microscopy (28 x 106; [8]). Examination of an EcoRl limit digest of pPL576 by electron microscopy showed only linear molecules. Three size classes were present in about equal numbers. pPL576 open circles were added to a heat-inactivated EcoRl limit digest of the plasmid (0.05 ,g/ml). Grids were mounted in formamide and stained with uranyl acetate (7). Seventy-one linears were selected on the basis of photographic qualities and were compared in length to four intact plasmid circles. The majority of the linears fell into three groups representing 0.45, 0.32, and 0.22 (each ±0.01) the length of pPL576 (Fig. 2). The combined length of the fragments accounts for about 99% of the intact plasmid, suggesting that pPL576 has probably only three EcoRl-sensitive sites. To demonstrate that the EcoRl-generated fragments contained cohesive ends (9), a limit digest of pPL576 (0.05 ug/ml) was heated at 60 C for 5 min and placed at 1 to 2 C for 2 days. Electron microscopic examination (grids prepared at 4 C by aqueous method [2]) showed that 30 to 50% of the molecules were in circular form, and representatives of each size class were among the circles. Exposure of such a preparation to 37 C for 5 min eliminated the circles as judged by a qualitative screening of grids prepared at room temperature. The reported melting temperature of the hydrogen bonding between EcoRl-generated ends is about 6 C (9).
CONTROL
600
-
10
1
20
29
1
10
20
30 34
FRACTIONS 4 00-
~200.
1
20
40
52
FRACTIONS FIG. 1. Sedimentation of intact and EcoRIdigested pPL576 through 5 to 20%1o neutral sucrose gradients. [3H]thymidine-labeled plasmid (1.5 Mg) was incubated with 1 Ml of EcoRI endonuclease for 15 min at 37 C and then at 60 C for 5 min as described in the text. A control preparation incubated in parallel contained all reaction ingredients except the endonuclease. Upper panels: sedimentation of 0.05 ml of each of the two mixtures through sucrose gradients. Centrifugation was in the SW50.1 rotor at 15 C and 40,000 rpm for 4.5 h. Fractions were collected and precipitated with 5% trichloroacetic acid. Lower panel: A portion of the EcoRI-digested plasmid (10 MI) was mixed with 14C-labeled T7 DNA (0) and sedimented as above. T7 DNA was assigned a sedimentation velocity of 32 S (11).
These results indicate that the majority of pPL576 molecules are uniformly sensitive to EcoRl, and suggest that all plasmid molecules have a similar (presumably identical) base sequence. These data in conjunction with the limited homology between plasmid and chro-
VOL. 123, 1975
NOTES
the National Science Foundation, by Public Health Service grant AI-10331 from the National Institute of Allergy and Infectious Diseases, and by grant 74/18 from the American Cancer Society, Maryland Division, Inc.
20
r
E
or
15
z
10 20-
U
379
:-r
0.2
0.4
0.6
FRACTIONAL LENGTH OF PL 576
FIG. 2. Histogram of the lengths of EcoRIgenerated linear fragments of pPL576. pPL576 was digested with EcoRI, and the enzyme was heat inactivated (see text). Intact open circles of pPL576 were added, and the mixture was prepared for electron microscopy in the presence of formamide (7). Seventy-one linears were photographed and compared in length to four intact open circles. mosome (8) indicate that pPL576 does not result from cyclization of random chromosomal fragments, and suggest the element is autonomous.
Linda Davis provided excellent technical assistance. H. W. Boyer is thanked for generously providing E. coli RY13 and the method for EcoRl purification. This investigation was supported by grant GB-39299 from
LITERATURE CITED 1. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405. 2. Davis, R., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, p. 413-428. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 21. Academic Press, Inc., New York. 3. Greene, P. J., M. C. Betlach, H. M. Goodman, and H. W. Boyer. 1974. The EcoRl restriction endonuclease, p. 87-105. In R. B. Wickner (ed.), Methods in molecular biology. Marcel Dekker, Inc., New York. 4. Hedgpeth, J., H. M. Goodman, and H. W. Boyer. 1972. DNA nucleotide sequence restricted by the Rl endonuclease. Proc. Natl. Acad. Sci. U.S.A. 69:3448-3452. 5. Helinski, D. R., and D. B. Clewell. 1971. Circular DNA. Annu. Rev. Biochem. 40:899-942. 6. Henneberry, R. C., and B. C. Carlton. 1973. Characterization of the polydisperse closed circular deoxyribonucleic acid molecules of Bacillus megaterium. J. Bacteriol. 114:625-631. 7. Lovett, P. S. 1973. Plasmid in Bacillus pumilus and the enhanced sporulation of plasmid-negative variants. J. Bacteriol. 115:291-298. 8. Lovett, P. S., and M. G. Bramucci. 1974. Biochemical studies of two Bacillus pumilus plasmids. J. Bacteriol. 120:488-494. 9. Mertz, J. E., and R. W. Davis. 1972. Cleavage of DNA by Rl restriction endonuclease generates cohesive ends. Proc. Natl. Acad. Sci. U.S.A. 69:3370-3374. 10. Novick, R. P. 1969. Extrachromosomal inheritance in bacteria. Bacteriol. Rev. 33:210-263. 11. Studier, F. W. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390.
JOURNAL OF BACTERIOLOGY, July 1975, p. 377-379 Copyright 0 1975 American Society for Microbiology
Evidence for a Nonrandom Base Sequence in a Bacillus pumilus Plasmid: EcoRl Endonuclease Digestion of pPL576 PAUL S. LOVETT* AND MICHAEL G. BRAMUCCI Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228 Received for publication 25 April 1975
EcoRl endonuclease digested the Bacillus pumilus plasmid pPL576 (molecular weight 28 x 106) into three distinct size classes of linear fragments. The molecular weights of the fragments are 13.0 x 106, 9.5 x 106, and 6.5 x 106 by sucrose gradient analysis. By electron microscope analysis the three fragments account for about 99% of the intact plasmid. These results indicate that pPL576 molecules contain a nonrandom base sequence, and are consistent with the interpretation that pPL576 is autonomous and not the result of cyclization of random chromosome fragments. -
Extrachromosomal circular duplex deoxyribonucleic acid (DNA) molecules of uniform size have been isolated from Bacillus pumilus strain NRS 576 (7). Although the specific genetic function(s) of this element is unknown, its presence correlates with the reduced ability of the host strain to form spores (7). pPL576 has been described as a plasmid primarily on the basis of its biochemical similarity to genetically characterized plasmids in enterics and Staphylococcus aureus (1, 5, 10). pPL576 is homogeneous in size and buoyant density and appears present in cells at a relatively fixed copy number (8). The use of the term plasmid to describe pPL576 implies that the element is autonomous and not the result of random fragmentation of the host chromosome into discrete linear pieces with subsequent cyclization. Such fragmentation could result, for example, from spontaneous induction of inducible phages carried by the host (6). Indeed, a large percentage of the small, heterogeneous circular DNA molecules isolated from B. megaterium strain 216 appears chromosomal in origin (6). pPL576 shares little homology with the chromosome of the host (8). This is consistent with the interpretation that the element is autonomous, and further predicts that all pPL576 molecules possess an identical base sequence. Certain endonucleases such as EcoRl cleave duplex DNA at sites within a length of specific base sequence (4). Cleavage of a population of plasmid molecules of identical base sequence should yield a discrete number of fragments whose additive molecular weight is equivalent
to that of the intact circle. Cleavage of a population of circular molecules of homogeneous size but random base sequence would result in numerous random size classes of linear fragments. In the present communication, we demonstrate that EcoRl endonuclease digestion of pPL576 cleaves the circular element into a limited number (three) of linear fragments whose additive size is equivalent to that of intact pPL576. These results indicate that pPL576 molecules possess a similar (presumably identical) base sequence. Accordingly, pPL576 appears not to result from cyclization of
random chromosome fragments. B. pumilus strain NRS 576 was the source of the plasmid pPL576 (7, 8). Plasmid DNA was isolated by dye-buoyant density gradient centrifugation (8). Neutral 5 to 20% sucrose gradients were prepared and run as before (7). DNA was prepared for electron microscopy by the aqueous technique of Davis et al. (2) or as previously described by using formamide (7). All other procedures were previously described
(8).
EcoRl restriction endonuclease was purchased from Miles Laboratories. Digestion of pPL576 was performed by incubation of 1 Ad of enzyme with 2 Ag (or less) of plasmid in 0.1 ml of 0.1 M tris(hydroxymethyl)aminomethane buffer, pH 7.2, 10-3 M MgCl2, and 0.05 M NaCl for 15 min at 37 C. Reactions were terminated by heating at 60 C for 5 min. These digestion conditions converted essentially all plasmid circles to three linear fragments as judged by analysis of the reaction mixture on sucrose 377
378
J. BACTERIOL.
NOTES
gradients and by electron microscopy. Increasing the enzyme level fivefold and the time of digestion to 3 h had no detectable effect on the extent of fragmentation. The experiments reported in the present study were performed with EcoRl from Miles. In earlier work, comparable results were obtained by using an enzyme preparation isolated from Escherichia coli RY13 and purified through phosphocellulose chromatography as described by Greene et al. (3). pPL576 digested with EcoRl sediments as three peaks in sucrose gradients (Fig. 1). Results comparable to those shown in Fig. 1 were obtained by increasing the enzyme level fivefold, and by increasing the digestion time up to 3 h. Hence, the conditions described in Fig. 1 appear to result in a limit digest. The sedimentation velocities of the three fragments (Fig. 1) are 25.5 (±0.5), 22.9 (+0.5), and 20.1 (±0.5) S (average of data from seven digests). These correspond to linear duplexes of 13.0 x 106, 9.5 x 106 and 6.5 x 106 daltons (11). The additive molecular weight of the three components is 29 x 106. This value is similar to the molecular weight reported for the intact plasmid determined by electron microscopy (28 x 106; [8]). Examination of an EcoRl limit digest of pPL576 by electron microscopy showed only linear molecules. Three size classes were present in about equal numbers. pPL576 open circles were added to a heat-inactivated EcoRl limit digest of the plasmid (0.05 ,g/ml). Grids were mounted in formamide and stained with uranyl acetate (7). Seventy-one linears were selected on the basis of photographic qualities and were compared in length to four intact plasmid circles. The majority of the linears fell into three groups representing 0.45, 0.32, and 0.22 (each ±0.01) the length of pPL576 (Fig. 2). The combined length of the fragments accounts for about 99% of the intact plasmid, suggesting that pPL576 has probably only three EcoRl-sensitive sites. To demonstrate that the EcoRl-generated fragments contained cohesive ends (9), a limit digest of pPL576 (0.05 ug/ml) was heated at 60 C for 5 min and placed at 1 to 2 C for 2 days. Electron microscopic examination (grids prepared at 4 C by aqueous method [2]) showed that 30 to 50% of the molecules were in circular form, and representatives of each size class were among the circles. Exposure of such a preparation to 37 C for 5 min eliminated the circles as judged by a qualitative screening of grids prepared at room temperature. The reported melting temperature of the hydrogen bonding between EcoRl-generated ends is about 6 C (9).
CONTROL
600
-
10
1
20
29
1
10
20
30 34
FRACTIONS 4 00-
~200.
1
20
40
52
FRACTIONS FIG. 1. Sedimentation of intact and EcoRIdigested pPL576 through 5 to 20%1o neutral sucrose gradients. [3H]thymidine-labeled plasmid (1.5 Mg) was incubated with 1 Ml of EcoRI endonuclease for 15 min at 37 C and then at 60 C for 5 min as described in the text. A control preparation incubated in parallel contained all reaction ingredients except the endonuclease. Upper panels: sedimentation of 0.05 ml of each of the two mixtures through sucrose gradients. Centrifugation was in the SW50.1 rotor at 15 C and 40,000 rpm for 4.5 h. Fractions were collected and precipitated with 5% trichloroacetic acid. Lower panel: A portion of the EcoRI-digested plasmid (10 MI) was mixed with 14C-labeled T7 DNA (0) and sedimented as above. T7 DNA was assigned a sedimentation velocity of 32 S (11).
These results indicate that the majority of pPL576 molecules are uniformly sensitive to EcoRl, and suggest that all plasmid molecules have a similar (presumably identical) base sequence. These data in conjunction with the limited homology between plasmid and chro-
VOL. 123, 1975
NOTES
the National Science Foundation, by Public Health Service grant AI-10331 from the National Institute of Allergy and Infectious Diseases, and by grant 74/18 from the American Cancer Society, Maryland Division, Inc.
20
r
E
or
15
z
10 20-
U
379
:-r
0.2
0.4
0.6
FRACTIONAL LENGTH OF PL 576
FIG. 2. Histogram of the lengths of EcoRIgenerated linear fragments of pPL576. pPL576 was digested with EcoRI, and the enzyme was heat inactivated (see text). Intact open circles of pPL576 were added, and the mixture was prepared for electron microscopy in the presence of formamide (7). Seventy-one linears were photographed and compared in length to four intact open circles. mosome (8) indicate that pPL576 does not result from cyclization of random chromosomal fragments, and suggest the element is autonomous.
Linda Davis provided excellent technical assistance. H. W. Boyer is thanked for generously providing E. coli RY13 and the method for EcoRl purification. This investigation was supported by grant GB-39299 from
LITERATURE CITED 1. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405. 2. Davis, R., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, p. 413-428. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 21. Academic Press, Inc., New York. 3. Greene, P. J., M. C. Betlach, H. M. Goodman, and H. W. Boyer. 1974. The EcoRl restriction endonuclease, p. 87-105. In R. B. Wickner (ed.), Methods in molecular biology. Marcel Dekker, Inc., New York. 4. Hedgpeth, J., H. M. Goodman, and H. W. Boyer. 1972. DNA nucleotide sequence restricted by the Rl endonuclease. Proc. Natl. Acad. Sci. U.S.A. 69:3448-3452. 5. Helinski, D. R., and D. B. Clewell. 1971. Circular DNA. Annu. Rev. Biochem. 40:899-942. 6. Henneberry, R. C., and B. C. Carlton. 1973. Characterization of the polydisperse closed circular deoxyribonucleic acid molecules of Bacillus megaterium. J. Bacteriol. 114:625-631. 7. Lovett, P. S. 1973. Plasmid in Bacillus pumilus and the enhanced sporulation of plasmid-negative variants. J. Bacteriol. 115:291-298. 8. Lovett, P. S., and M. G. Bramucci. 1974. Biochemical studies of two Bacillus pumilus plasmids. J. Bacteriol. 120:488-494. 9. Mertz, J. E., and R. W. Davis. 1972. Cleavage of DNA by Rl restriction endonuclease generates cohesive ends. Proc. Natl. Acad. Sci. U.S.A. 69:3370-3374. 10. Novick, R. P. 1969. Extrachromosomal inheritance in bacteria. Bacteriol. Rev. 33:210-263. 11. Studier, F. W. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390.
