Vol. 127, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 817-828 Copyright © 1976 American Society for Microbiology
Bacillus pumilus Plasmid pPL10: Properties and Insertion into Bacillus subtilis 168 by Transformation PAUL S. LOVETT,* ELIZABETH J. DUVALL AND KATHLEEN M. KEGGINS Department ofBiological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228
Received for publication 6 March 1976
Bacillus pumilus ATCC 12140 harbors 10 or more copies per chromosome of each of two small plasmids. Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent. Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled deoxyribonucleic acid. Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; molecular weight, -4.4 x 106; -20 copies per chromosome; p = 1.698). Resulting Kill+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10. Transfer of pPL10 from strain L10 to B. pumilus strain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype. Strain NRS 576 naturally harbors about two copies per chromosome of a 28-million-dalton plasmid, pPL576. In Kill+ transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of about 11 molecules of pPL10 to 1 molecule of pPL576. Therefore, pPL576 and pPL10 are compatible plasmids. B. subtilis 168 is naturally resistant to pPL10-determined killing activity. Plasmid pPL10 was therefore inserted into B. subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient. The plasmid is stably maintained at an estimated 10 copies per chromosome in the spore- recipient and in spore+ transformants. pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoRl. The recent detection of a plasmid system in Bacillus pumilus NRS 576 provided the first demonstration that stably maintained extrachromosomal inheritance occurred in a member of the genetically related species B. pumilus, B. licheniformis, and B. subtilis (13). Subsequent examination of nearly 40 strains of B. subtilis and B. pumilus showed that about 10% contained covalently closed circular (CCC) duplex deoxyribonucleic acid (DNA) molecules of homogeneous size and buoyant density and of limited copy number, but of unknown genetic function (14, 15, 17). The absence of known selectable genetic functions for these plasmids initially precluded their manipulation by the standard genetic procedures of transduction and transformation. In an effort to detect plasmid-determined host functions, a detailed examination of the properties of each of the plasmid-containing Bacillus strains was initiated. In this report, evidence is presented demonstrating that a plasmid, pPL10, naturally harbored by B. pumilus ATCC 12140, determines the production of (and immunity or resistance to) a killing or bacteriocin-like activity.
Within the gram-positive bacterial group, B. subtilis 168 possesses the most extensively studied genetic system (29). This strain has been shown to lack detectable extrachromosomal CCC DNA molecules (15). The present study demonstrates that plasmid pPL10 can be transformed into B. subtilis 168, where it is stably maintained under nonselective growth conditions. The properties of pPL10 are such that with further development this plasmid should serve as a means for partial diploid construction in B. subtilis by taking advantage of current technology (4, 6). MATERIALS AND METHODS Bacteria, bacteriophages, and growth conditions. Strains of B. pumilus and B. subtilis used in this investigation are listed in Table 1. Bacteriophage SPP1 was obtained from R. Yasbin. All growth media have been reported (13-15). Incubation was at 37°C; liquid cultures were grown with rotary shaking. Plasmid-negative variants of ATCC 12140. Cultures of wild-type ATCC 12140, Li or L9, were mutagenized with nitrosoguanidine, and the washed survivors were grown overnight in Penassay broth 817
818
LOVETT, DUVALL, AND KEGGINS
TABLE 1. Strains of B. pumilus and B. subtilis Strain
Relevant properties
B. pumilus ATCC 12140
Li
LlSl L10 L9 L9S1 L9S2 L9S3 B. pumilus NRS 576
Cat-i
W20 B. subtilis 168 BR151 BR151 (4105)
Reference/ source
11
pMB1+ pMB2+ Lac+ Lac+ pPL10+ LacpMB1+ pMB2+ LysLac+ LysLysLyspPL576+ ade-100 ade-100
10 10
lys-3 trpC2 metBlO R. Yasbin lys-3 trpC2 metBlO R. Yasbin
4105
JH86 phe-I trpC2 spoB J. A. Hoch B. subtilis ATCC pPL2+ 11 7003 a pPL or pMB refers to the presence (+) of a specific plasmid. Abbreviations: Lac, lactose; lys, lysine; ade, adenine; trp, tryptophan; met, methionine; phe, phenylalanine; spo, sporulation mutation.
(Difco) (2). As an alternate to nitrosoguanidine, cells were grown overnight in Penassay broth containing 50 to 75 ug of acridine orange per ml or 10-5 M ethidium bromide (EB). In each case appropriate dilutions were spread onto tryptose blood agar base (TBAB; Difco) plates to yield about 300 colonies/ plate. After overnight incubation, a variable number of colonies sensitive to a killing activity elaborated by neighboring colonies were detected. Sensitive (S) variants isolated from a given strain such as Li were designated LlSl, etc. All of 24 S variants tested to date lacked detectable levels of CCC DNA. The Kill+ phenotype of a given clone was determined by cross-streaking (on nutrient agar) with a sensitive variant. In these tests, both members were derived from the same parental line. The killing activity of ATCC 12140 was assayed with plasmidnegative S variants of ATCC 12140. Kill+ transductants of NRS 576 were assayed by cross-streaking with wild-type NRS 576. Transduction. Transductior. by bacteriophage PBP1 was as previously described (12, 16). Kill+ transductants of sensitive (S) recipients were detected by mixing washed transduction mixtures (0.1 ml; -5 x 107 cells) with about 108 additional recipient cells (in 0.1 ml) in 2 ml of melted semisolid agar as in a phage plaque assay (2). The mixture was overlaid onto TBAB plates and incubated at 370C for 18 to 24 h. Kill+ transductants appeared as clear "plaques" containing a central colony. The frequency of Kill+ transductants was on the order of 1 transductant per 109 plaque-forming units (PFU). PBS1-mediated transduction was as previously described (3, 18). Transformation. B. subtilis was grown to competence as described by Bott and Wilson (1). Transformaton with phenol-purified DNA was essentially as previously described (15). The sporulation pheno-
J. BACTERIOL. type of JH86 transformants was judged by colony morphology (9). Plasmid isolation. Cells were labeled with [3H]or ['4C]thymidine (New England Nuclear Corp.), and DNA was extracted by the lysozyme-SarkosylPronase procedure and subjected to dye-buoyant density gradient centrifugation (15). Fractions comprising the plasmid peak were pooled, EB was removed by isoamyl alcohol extraction, and the DNA was dialyzed against TES buffer (13). ColElRSF1010 composite plasmid was isolated as described by Tanaka and Weisblum (27). Determination of the presence of CCC duplex DNA in a clone was performed by dye-buoyant density centrifugation of isotopically labeled DNA extracted from 20 ml of late log-phase cells. Absence of CCC DNA means that less than 0.2% of the total cell DNA was in the supercoiled configuration. Presence of CCC DNA in a clone means that greater than 2% of the total cell DNA was supercoiled. Percentage of contribution of plasmid to total cell DNA was estimated by weighing the areas under the peaks in CsCl-EB gradients prepared from noncleared lysates (14). Values specifically noted represent the average of two or three determinations. Gradient centrifugation. Neutral 5 to 20% sucrose gradients were prepared, centrifuged, and processed as previously reported (13). Bacteriophage T7 DNA, labeled with ['4C]thymidine, was reference. T7 DNA was assigned an S value (sedimentation velocity) of 32S (26). Equilibrium centrifugation of plasmid pPL10 DNA and Escherichia coli K-12 DNA (p = 1.710) in CsCl gradients using a model E analytical ultracentrifuge was as previously reported (15, 25). Endonuclease fragmentation of pPL10. The endonucleases Hind III and EcoRl were purchased from Miles Laboratories. Digested plasmid pPL10 (14C labeled) was mixed with EcoRl-digested composite plasmid (27) and subjected to agarose gel electrophoresis (8). Gels were cut in 2-mm slices. Each slice was dissolved in 0.5 ml of concentrated ammonium hydroxide and counted in Hydromix (Yorktown Research). Each of two lots ofEcoRl and Hind III was tested for their ability to fragment a fixed concentration of pPL1O. The experiments reported here were performed with twice the activity required for complete plasmid digestion. Assay of the Kill+ phenotype of pPL10 containing B. subtilis spoB. Cells of a given strain of B. pumilus that harbor pPL10 exhibit a killing activity against cells of the same strain that lack the plasmid. This killing activity was readily demonstrated by cross-streaking or by stabbing Kill+ clones into lawns of sensitive cells. By contrast, the killing activity of B. subtilis 168 spoB cells that contain pPL10 was difficult to detect by any procedure we have tested. The routine method used in this study to assay Kill+ B. subtilis spoB clones involved overnight growth of heavy streaks of B. subtilis on TBAB. The plates were then overlaid with 2 ml of semisolid agar seeded with about 108 W20 or L9S1 cells. In some cases the plates containing the streaks of B. subtilis were exposed to chloroform vapors prior to overlaying with semisolid agar. In either
BACILLUS PLASMID pPL10
VOL. 127, 1976
the overlaid plates were incubated for an additional 24 h. Under ideal conditions, streaks of B. subtilis spoB (pPL10) exhibited a weak zone of killing of the W20 cells. This killing was not detected with B. subtilis spoB cells that lacked pPL10. Using a spoB mutant that stably maintains pPL10, we have found that we can detect killing activity in only about one-third of the tests. This is not due to loss of the plasmid, but rather it seems to be a technical problem in detecting the activity. We have not encountered this difficulty when the plasmid is carried by B. pumilus strains. The difficulty in detecting the Kill+ phenotype in pPL10-containing spoB mutants of B. subtilis indicates that the frequency we determined for plasmid uptake by B. subtilis during transformation is probably too low. Efforts to improve this assay are in progress. case,
RESULTS
Plasmid-negative variants of B pumilus ATCC 12140. B. pumilus ATCC 12140 and two mutant derivatives, Li (Lac+) and L9 (Lac+ Lys-), each contain two species of CCC DNA molecules having sedimentation velocities (S values) of about 28 and 25S (15). These plasmids were designated as pMB1 and PMB2, respectively, and both were present in cells at many copies (10 or more) per chromosome (15). Variants of each of the three ATCC 12140 plasmid-containing parents were isolated that were sensitive to a killing activity produced by the parents (see Materials and Methods). Each of over 50 sensitive (S) variants of Li and L9 retained the characteristic nutritional property of the parent, and each of 24 S variants tested lacked detectable levels of CCC duplex DNA (7 60 20 40 77 SLICE NUMBER
FIG. 9. Agarose gel electrophoresis of EcoRl-digested pPL1O. Plasmid pPLIO from strain LIO was digested with EcoRI and subjected to electrophoresis as in Fig. 8, but for only 8 h.
826
LOVETT. DUVALL, AND KEGGINS
J. BACTERIOL.
600
0
20
40 SLICE NUMBER
60
72
FIG. 10. Agarose gel electrophoresis ofHind III-digested pPL1O and EcoRl-digested pPL1O. pPLIO labeled with [14C]thymidine was digested to completion with Hind III. pPL10 labeled with [3H]thymidine was digested to completion with EcoRI . Each were heat inactivated at 65°C for 10 min, mixed, and subjected to electrophoresis as in Fig. 8.
been shown to determine a positive host function, a killing activity. The host range of the plasmid-determined killing activity, assayed by cross-streaking, includes S variants of ATCC 12140 and B. pumilus NRS 576. Several other strains of Bacillus, including B. pumilus NRRL B-3275 (18) and B. subtilis 168, appear to be resistant to the killing activity. As shown in the present study, B. subtilis 168 can maintain pPL10 when the plasmid is introduced by transformation. Similarly, B. pumilus NRRL B-3275 can maintain pPL10 when the plasmid is introduced by transduction, using an indirect selection procedure (M. Bramucci and P. S. Lovett, manuscript in preparation). Therefore, apparent insensitivity of a strain to the plasmiddetermined killing activity does not indicate that the strain is unable to maintain pPL10. Although pPL10 was originally detected in B. pumilus, the plasmid appears to be stably inherited in B. subtilis 168. We have not yet encountered loss of pPL10 from B9 and B10 during over 100 successive single-colony transfers on rich media. Additionally, the plasmid is
present in each of the following types of derivatives of B9: spore+ and Phe+ transformants and transductants, 4105 lysogens, and derivates that contain the dna-i or divlV-BI mutations (Lovett, unpublished data; 22, 28). In all cases the plasmid was detected as CCC duplex molecules sedimenting at 25(± 1)S. Although the plasmid is maintained in B. subtilis 168, the plasmid copy number we determined in B9 and B10 (-10 per chromosome) is lower than the value of 20 copies per chromosome that we found when the plasmid was present inB. pumilus strains L10 and W20. At present, we do not know if the apparent reduced copy number of pPL10 in B. subtilis is real or an artifact possibly resulting from elevated levels of nucleases in B. subtilis relative to B. pumilus strains. Apparent similarities exist between the Bacillus pPL10 plasmid system and the plasmiddetermined bacteriocin systems detected in other species of bacteria (20, 21). For instance, since pPL10 determines the production of a killing activity, the plasmid must also confer on the host a form of resistance or immunity to the
BACILLUS PLASMID pPL10
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827
This investigation was supported by Public Health Service grant AI-10331 from the National Institute of Allergy and Infectious Diseases and by a grant from the American Cancer Society, Maryland Division, Inc.
C-)
50 30 SLICE NUMBER FIG. 11. Agarose gel electrophoresis of EcoRI-digested pPL10 isolated from B9 and B. pumilus L10. Approximately 0.1 pg of ['4C]thymidine-labeled pPL10 isolated from B. pumilus L10 was combined with 0.1 pg of [3H]thymidine-labeled pPL10 isolated from B9. The mixture was digested with EcoRI and subjected to electrophoresis as in Fig. 8.
killing activity. In this respect pPL10 appears analogous to the colicinogenic factors (20). However, the chemical nature of the pPL10determined killing activity and the host range of the activity remained to be determined. Therefore, a more extensive comparison between pPL10 and the well-studied bacteriocin systems is not warranted at this time. The lack of a general method for performing genetic complementation in B. subtilis 168, coupled with recent developments in in vitro construction of plasmids (4, 6), suggests a potential use for pPL10 in the generation of diploid strains. The stability of pPL10 in B. subtilis and the small size of the plasmid suggest that, with further modification, pPL10 may be useful for such experiments. ACKNOWLEDGMENTS J. A. Hoch, N. Mendelson, N. Sueoka, and R. Yasbin generously provided mutant derivatives ofB. subtilis 168.
LITERATURE CITED 1. Bott, K. F., and G. A. Wilson. 1967. Development of competence in the Bacillus subtilis transformation system. J. Bacteriol. 94:562-570. 2. Bramucci, M. G., and P. S. Lovett. 197. Temperate bacteriophage infectious for asporogenic variants of Bacillus pumilus. J. Virol. 14:1281-1287. 3. Bramucci, M. G., and P. S. Lovett. 1976. Low frequency PBS1-mediated plasmid transduction in Bacillus pumilus. J. Bacteriol. 127:829-31. 4. Clarke, L., and J. Carbon. 1975. Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome. Proc. Natl. Acad. Sci. U.S.A. 72:4361-4365. 5. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 36:361-405. 6. Cohen, S. N., A. C. Y. Chang, H. W. Boyer, and R. B. Helling. 1973. Construction of biologically functional plasmids in vitro. Proc. Natl. Acad. Sci. U.S.A. 72:3240-3244. 7. Hedgpeth, J., H. M. Goodman, and H. W. Boyer. 1972. DNA nucleotide sequence restricted by Rl endonuclease. Proc. Natl. Acad. Sci. U.S.A. 69:3448-3452. 8. Helling, R. B., H. B. Goodman, and H. W. Boyer. 1974. Analysis of endonuclease EcoRl fragments of DNA from lambdoid bacteriophages and other viruses by agarose gel electrophoresis. J. Virol. 14:1235-1244. 9. Hoch, J. A., and J. L. Mathews. 1973. Chromosomal location of pleiotropic negative sporulation mutations in Bacillus subtilis. Genetics 73:215-228. 10. Kavenoff, R. 1972. Characterization of the Bacillus subtilis genome by sedimentation. J. Mol. Biol. 72:801806. 11. Klotz, L. C., and B. H. Zimm. 1972. Size of DNA determined by viscoelastic measurements: results on bacteriophages, Bacillus subtilis and Escherichia coli. J. Mol. Biol. 72:779-800. 12. Lovett, P. S. 1972. PBP1: a flagella-specific bacteriophage mediating transduction in Bacillus pumilus. Virology 47:743-752. 13. Lovett, P. S. 1973. Plasmid inBacillus pumilus and the enhanced sporulation of plasmid-negative variants. J. Bacteriol. 115:291-298. 14. Lovett, P. S., and M. G. Bramucci. 1974. Biochemical studies of two Bacillus pumilus plasmnids. J. Bacteriol. 120E.488-494. 15. Lovett, P. S., and M. G. Bramucci. 1975. Plasmid deoxyribonucleic acid in Bacillus subtilis and Bacillus pumilus. J. Bacteriol. 124:484-490. 16. Lovett, P. S., D. Bramucci, M. G. Bramucci, and B. D. Burdick. 1974. Some properties of the PBP1 transduction system in Bacillus pumilus. J. Virol. 13:81-84. 17. Lovett, P. S., and B. D. Burdick. 1973. Cryptic plasmid in Bacillus pumilus ATCC 7065. Biochem. Biophys. Res. Commun. 54:365-370. 18. Lovett, P. S., and F. E. Young. 1970. Genetic analysis in Bacillus pumilus by PBS1-mediated transduction. J. Bacteriol. 101:603-608. 19. Mertz, J. E., and R. W. Davis. 1972. Cleavage of DNA by RI restriction endonuclease generates cohesive ends. Proc. Natl. Acad. Sci. U.S.A. 69:3370-3374. 20. Nomura, M. 1967. Colicins and related bacteriocins. Annu. Rev. Microbiol. 21:257-284. 21. Novick, R. P. 1969. Extrachromosomal inheritance in bacteria. Bacteriol. Rev. 33:210-257. 22. Reeve, J. N., N. H. Mendelson, S. I. Coyne, L. L. Hallock, and R. M. Cole. 1973. Minicells of Bacillus
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subtilis J. Bacteriol. 114:860-873. 23. Roy, P. H., and H. 0. Smith. 1973. DNA methylases of Hemophilus influenzae Rd. II. Partial recognition site base sequence. J. Mol. Biol. 81:445-459. 24. Rutberg, L. 1969. Mapping of a temperate bacteriophage active on Bacillus subtilis. J. Virol. 3:38-44. 25. Schildkraut, C. K., J. Marmur, and P. Doty. 1962. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J. Mol. Biol. 4:430-443. 26. Studier, F. W. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390.
J. BACTERIOL. 27. Tanaka, T., and B. Weisblum. 1975. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bacteriol. 121:354-362. 28. White, K., and N. Sueoka. 1973. Temperature-sensitive DNA synthesis mutants ofBacillus subtilis. Genetics 73:185-214. 29. Young, F. E., and G. A. Wilson. 1972. Genetics ofBacillus subtilis and other gram-positive sporulating bacilli, p. 77-106. In H. 0 Halvorson, R. S. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C.
JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 817-828 Copyright © 1976 American Society for Microbiology
Bacillus pumilus Plasmid pPL10: Properties and Insertion into Bacillus subtilis 168 by Transformation PAUL S. LOVETT,* ELIZABETH J. DUVALL AND KATHLEEN M. KEGGINS Department ofBiological Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21228
Received for publication 6 March 1976
Bacillus pumilus ATCC 12140 harbors 10 or more copies per chromosome of each of two small plasmids. Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent. Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled deoxyribonucleic acid. Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; molecular weight, -4.4 x 106; -20 copies per chromosome; p = 1.698). Resulting Kill+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10. Transfer of pPL10 from strain L10 to B. pumilus strain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype. Strain NRS 576 naturally harbors about two copies per chromosome of a 28-million-dalton plasmid, pPL576. In Kill+ transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of about 11 molecules of pPL10 to 1 molecule of pPL576. Therefore, pPL576 and pPL10 are compatible plasmids. B. subtilis 168 is naturally resistant to pPL10-determined killing activity. Plasmid pPL10 was therefore inserted into B. subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient. The plasmid is stably maintained at an estimated 10 copies per chromosome in the spore- recipient and in spore+ transformants. pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoRl. The recent detection of a plasmid system in Bacillus pumilus NRS 576 provided the first demonstration that stably maintained extrachromosomal inheritance occurred in a member of the genetically related species B. pumilus, B. licheniformis, and B. subtilis (13). Subsequent examination of nearly 40 strains of B. subtilis and B. pumilus showed that about 10% contained covalently closed circular (CCC) duplex deoxyribonucleic acid (DNA) molecules of homogeneous size and buoyant density and of limited copy number, but of unknown genetic function (14, 15, 17). The absence of known selectable genetic functions for these plasmids initially precluded their manipulation by the standard genetic procedures of transduction and transformation. In an effort to detect plasmid-determined host functions, a detailed examination of the properties of each of the plasmid-containing Bacillus strains was initiated. In this report, evidence is presented demonstrating that a plasmid, pPL10, naturally harbored by B. pumilus ATCC 12140, determines the production of (and immunity or resistance to) a killing or bacteriocin-like activity.
Within the gram-positive bacterial group, B. subtilis 168 possesses the most extensively studied genetic system (29). This strain has been shown to lack detectable extrachromosomal CCC DNA molecules (15). The present study demonstrates that plasmid pPL10 can be transformed into B. subtilis 168, where it is stably maintained under nonselective growth conditions. The properties of pPL10 are such that with further development this plasmid should serve as a means for partial diploid construction in B. subtilis by taking advantage of current technology (4, 6). MATERIALS AND METHODS Bacteria, bacteriophages, and growth conditions. Strains of B. pumilus and B. subtilis used in this investigation are listed in Table 1. Bacteriophage SPP1 was obtained from R. Yasbin. All growth media have been reported (13-15). Incubation was at 37°C; liquid cultures were grown with rotary shaking. Plasmid-negative variants of ATCC 12140. Cultures of wild-type ATCC 12140, Li or L9, were mutagenized with nitrosoguanidine, and the washed survivors were grown overnight in Penassay broth 817
818
LOVETT, DUVALL, AND KEGGINS
TABLE 1. Strains of B. pumilus and B. subtilis Strain
Relevant properties
B. pumilus ATCC 12140
Li
LlSl L10 L9 L9S1 L9S2 L9S3 B. pumilus NRS 576
Cat-i
W20 B. subtilis 168 BR151 BR151 (4105)
Reference/ source
11
pMB1+ pMB2+ Lac+ Lac+ pPL10+ LacpMB1+ pMB2+ LysLac+ LysLysLyspPL576+ ade-100 ade-100
10 10
lys-3 trpC2 metBlO R. Yasbin lys-3 trpC2 metBlO R. Yasbin
4105
JH86 phe-I trpC2 spoB J. A. Hoch B. subtilis ATCC pPL2+ 11 7003 a pPL or pMB refers to the presence (+) of a specific plasmid. Abbreviations: Lac, lactose; lys, lysine; ade, adenine; trp, tryptophan; met, methionine; phe, phenylalanine; spo, sporulation mutation.
(Difco) (2). As an alternate to nitrosoguanidine, cells were grown overnight in Penassay broth containing 50 to 75 ug of acridine orange per ml or 10-5 M ethidium bromide (EB). In each case appropriate dilutions were spread onto tryptose blood agar base (TBAB; Difco) plates to yield about 300 colonies/ plate. After overnight incubation, a variable number of colonies sensitive to a killing activity elaborated by neighboring colonies were detected. Sensitive (S) variants isolated from a given strain such as Li were designated LlSl, etc. All of 24 S variants tested to date lacked detectable levels of CCC DNA. The Kill+ phenotype of a given clone was determined by cross-streaking (on nutrient agar) with a sensitive variant. In these tests, both members were derived from the same parental line. The killing activity of ATCC 12140 was assayed with plasmidnegative S variants of ATCC 12140. Kill+ transductants of NRS 576 were assayed by cross-streaking with wild-type NRS 576. Transduction. Transductior. by bacteriophage PBP1 was as previously described (12, 16). Kill+ transductants of sensitive (S) recipients were detected by mixing washed transduction mixtures (0.1 ml; -5 x 107 cells) with about 108 additional recipient cells (in 0.1 ml) in 2 ml of melted semisolid agar as in a phage plaque assay (2). The mixture was overlaid onto TBAB plates and incubated at 370C for 18 to 24 h. Kill+ transductants appeared as clear "plaques" containing a central colony. The frequency of Kill+ transductants was on the order of 1 transductant per 109 plaque-forming units (PFU). PBS1-mediated transduction was as previously described (3, 18). Transformation. B. subtilis was grown to competence as described by Bott and Wilson (1). Transformaton with phenol-purified DNA was essentially as previously described (15). The sporulation pheno-
J. BACTERIOL. type of JH86 transformants was judged by colony morphology (9). Plasmid isolation. Cells were labeled with [3H]or ['4C]thymidine (New England Nuclear Corp.), and DNA was extracted by the lysozyme-SarkosylPronase procedure and subjected to dye-buoyant density gradient centrifugation (15). Fractions comprising the plasmid peak were pooled, EB was removed by isoamyl alcohol extraction, and the DNA was dialyzed against TES buffer (13). ColElRSF1010 composite plasmid was isolated as described by Tanaka and Weisblum (27). Determination of the presence of CCC duplex DNA in a clone was performed by dye-buoyant density centrifugation of isotopically labeled DNA extracted from 20 ml of late log-phase cells. Absence of CCC DNA means that less than 0.2% of the total cell DNA was in the supercoiled configuration. Presence of CCC DNA in a clone means that greater than 2% of the total cell DNA was supercoiled. Percentage of contribution of plasmid to total cell DNA was estimated by weighing the areas under the peaks in CsCl-EB gradients prepared from noncleared lysates (14). Values specifically noted represent the average of two or three determinations. Gradient centrifugation. Neutral 5 to 20% sucrose gradients were prepared, centrifuged, and processed as previously reported (13). Bacteriophage T7 DNA, labeled with ['4C]thymidine, was reference. T7 DNA was assigned an S value (sedimentation velocity) of 32S (26). Equilibrium centrifugation of plasmid pPL10 DNA and Escherichia coli K-12 DNA (p = 1.710) in CsCl gradients using a model E analytical ultracentrifuge was as previously reported (15, 25). Endonuclease fragmentation of pPL10. The endonucleases Hind III and EcoRl were purchased from Miles Laboratories. Digested plasmid pPL10 (14C labeled) was mixed with EcoRl-digested composite plasmid (27) and subjected to agarose gel electrophoresis (8). Gels were cut in 2-mm slices. Each slice was dissolved in 0.5 ml of concentrated ammonium hydroxide and counted in Hydromix (Yorktown Research). Each of two lots ofEcoRl and Hind III was tested for their ability to fragment a fixed concentration of pPL1O. The experiments reported here were performed with twice the activity required for complete plasmid digestion. Assay of the Kill+ phenotype of pPL10 containing B. subtilis spoB. Cells of a given strain of B. pumilus that harbor pPL10 exhibit a killing activity against cells of the same strain that lack the plasmid. This killing activity was readily demonstrated by cross-streaking or by stabbing Kill+ clones into lawns of sensitive cells. By contrast, the killing activity of B. subtilis 168 spoB cells that contain pPL10 was difficult to detect by any procedure we have tested. The routine method used in this study to assay Kill+ B. subtilis spoB clones involved overnight growth of heavy streaks of B. subtilis on TBAB. The plates were then overlaid with 2 ml of semisolid agar seeded with about 108 W20 or L9S1 cells. In some cases the plates containing the streaks of B. subtilis were exposed to chloroform vapors prior to overlaying with semisolid agar. In either
BACILLUS PLASMID pPL10
VOL. 127, 1976
the overlaid plates were incubated for an additional 24 h. Under ideal conditions, streaks of B. subtilis spoB (pPL10) exhibited a weak zone of killing of the W20 cells. This killing was not detected with B. subtilis spoB cells that lacked pPL10. Using a spoB mutant that stably maintains pPL10, we have found that we can detect killing activity in only about one-third of the tests. This is not due to loss of the plasmid, but rather it seems to be a technical problem in detecting the activity. We have not encountered this difficulty when the plasmid is carried by B. pumilus strains. The difficulty in detecting the Kill+ phenotype in pPL10-containing spoB mutants of B. subtilis indicates that the frequency we determined for plasmid uptake by B. subtilis during transformation is probably too low. Efforts to improve this assay are in progress. case,
RESULTS
Plasmid-negative variants of B pumilus ATCC 12140. B. pumilus ATCC 12140 and two mutant derivatives, Li (Lac+) and L9 (Lac+ Lys-), each contain two species of CCC DNA molecules having sedimentation velocities (S values) of about 28 and 25S (15). These plasmids were designated as pMB1 and PMB2, respectively, and both were present in cells at many copies (10 or more) per chromosome (15). Variants of each of the three ATCC 12140 plasmid-containing parents were isolated that were sensitive to a killing activity produced by the parents (see Materials and Methods). Each of over 50 sensitive (S) variants of Li and L9 retained the characteristic nutritional property of the parent, and each of 24 S variants tested lacked detectable levels of CCC duplex DNA (7 60 20 40 77 SLICE NUMBER
FIG. 9. Agarose gel electrophoresis of EcoRl-digested pPL1O. Plasmid pPLIO from strain LIO was digested with EcoRI and subjected to electrophoresis as in Fig. 8, but for only 8 h.
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J. BACTERIOL.
600
0
20
40 SLICE NUMBER
60
72
FIG. 10. Agarose gel electrophoresis ofHind III-digested pPL1O and EcoRl-digested pPL1O. pPLIO labeled with [14C]thymidine was digested to completion with Hind III. pPL10 labeled with [3H]thymidine was digested to completion with EcoRI . Each were heat inactivated at 65°C for 10 min, mixed, and subjected to electrophoresis as in Fig. 8.
been shown to determine a positive host function, a killing activity. The host range of the plasmid-determined killing activity, assayed by cross-streaking, includes S variants of ATCC 12140 and B. pumilus NRS 576. Several other strains of Bacillus, including B. pumilus NRRL B-3275 (18) and B. subtilis 168, appear to be resistant to the killing activity. As shown in the present study, B. subtilis 168 can maintain pPL10 when the plasmid is introduced by transformation. Similarly, B. pumilus NRRL B-3275 can maintain pPL10 when the plasmid is introduced by transduction, using an indirect selection procedure (M. Bramucci and P. S. Lovett, manuscript in preparation). Therefore, apparent insensitivity of a strain to the plasmiddetermined killing activity does not indicate that the strain is unable to maintain pPL10. Although pPL10 was originally detected in B. pumilus, the plasmid appears to be stably inherited in B. subtilis 168. We have not yet encountered loss of pPL10 from B9 and B10 during over 100 successive single-colony transfers on rich media. Additionally, the plasmid is
present in each of the following types of derivatives of B9: spore+ and Phe+ transformants and transductants, 4105 lysogens, and derivates that contain the dna-i or divlV-BI mutations (Lovett, unpublished data; 22, 28). In all cases the plasmid was detected as CCC duplex molecules sedimenting at 25(± 1)S. Although the plasmid is maintained in B. subtilis 168, the plasmid copy number we determined in B9 and B10 (-10 per chromosome) is lower than the value of 20 copies per chromosome that we found when the plasmid was present inB. pumilus strains L10 and W20. At present, we do not know if the apparent reduced copy number of pPL10 in B. subtilis is real or an artifact possibly resulting from elevated levels of nucleases in B. subtilis relative to B. pumilus strains. Apparent similarities exist between the Bacillus pPL10 plasmid system and the plasmiddetermined bacteriocin systems detected in other species of bacteria (20, 21). For instance, since pPL10 determines the production of a killing activity, the plasmid must also confer on the host a form of resistance or immunity to the
BACILLUS PLASMID pPL10
VOL. 127, 1976
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This investigation was supported by Public Health Service grant AI-10331 from the National Institute of Allergy and Infectious Diseases and by a grant from the American Cancer Society, Maryland Division, Inc.
C-)
50 30 SLICE NUMBER FIG. 11. Agarose gel electrophoresis of EcoRI-digested pPL10 isolated from B9 and B. pumilus L10. Approximately 0.1 pg of ['4C]thymidine-labeled pPL10 isolated from B. pumilus L10 was combined with 0.1 pg of [3H]thymidine-labeled pPL10 isolated from B9. The mixture was digested with EcoRI and subjected to electrophoresis as in Fig. 8.
killing activity. In this respect pPL10 appears analogous to the colicinogenic factors (20). However, the chemical nature of the pPL10determined killing activity and the host range of the activity remained to be determined. Therefore, a more extensive comparison between pPL10 and the well-studied bacteriocin systems is not warranted at this time. The lack of a general method for performing genetic complementation in B. subtilis 168, coupled with recent developments in in vitro construction of plasmids (4, 6), suggests a potential use for pPL10 in the generation of diploid strains. The stability of pPL10 in B. subtilis and the small size of the plasmid suggest that, with further modification, pPL10 may be useful for such experiments. ACKNOWLEDGMENTS J. A. Hoch, N. Mendelson, N. Sueoka, and R. Yasbin generously provided mutant derivatives ofB. subtilis 168.
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