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h. Incubate at 30° for 12-18 hr. 3. Selection of ts mutants a. Dilute each culture in nutrient broth (1:106) and plate 0.1 ml on nutrient broth plates. b. Incubate plates at 30° for 24-48 hr. c. Replica plate each plate twice and incubate one at 30° and the counterpart at 42 °. d. Select colonies which grow at 30° but not at 42°. e. Repeat the temperature selection for all positive colonies 2-3 times. 4. Screening of ts mutants for competence of DNA transfection a. Inoculate 2 ml of nutrient broth with single colonies and incubate the cultures at 30° overnight with gentle shaking. b. Remove 0.25 ml of culture into a sterile tube containing 0.25 ml of PAM + 0.48 sucrose (PAMI~). c. Incubate for 5 hr with gentle shaking at 42°. d. Add 0.5 ml of 4~x174 ss DNA solution containing 1/zg/ml DNA in 50 mM Tris HC1, pH 8.1. e. Incubate at 42° for 2.5 min and transfer the culture to 0-4 °. f. Titer the infective centers. 12 10 g casamino acids, 10 g nutrient broth, 1 g glucose, 2 g MgSO4 (per liter). G. D. Guthrie and R. L. Sinsheimer, Biochim. Biophys. Acta 72, 290 (1963).
[23] B a c i l l u s s u b t i l i s a s a H o s t f o r M o l e c u l a r C l o n i n g
By PAUL S. LOVETT and KATHLEEN M. KEGGINS Prior to the advent of molecular cloning technologyla the two methods most commonly used to isolate quantities of cellular DNA enriched for specific genes involved the generation of specialized transducing phages that harbor specific regions of the bacterial chromosome, or the isolation of specific regions of cellular DNA on the basis of their unique physical properties or their occurrence within a subcellular organelle. The advantages of molecular cloning over these methods include its application in the isolation, in principle, of any small fragment of cellular DNA and in maintaining the selected fragment joined to a small, easily isolated replicon. The principles of molecular cloning have been developed using Escherichia coli, its plasmids, and its bacteriophages. In recent years the 1 S. N. Cohen, A. C. Y. Chang, H. W. Boyer, and R. B. Helling, Proc. Natl. Acad. Sci. U.S.A. 70, 3240 (1973). 2 D. A. Jackson, R. H. Symons, and P. Berg, Proc. Natl, Acad. Sci. U.S.A. 69, 2904 (1972). METHODS IN ENZYMOLOGY, VOL, 68
Copyright O 1979by Academic Press, Inc. All rights of reproductionin any formreserved. ISBN 0-12-181968-X
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components of the E. coli cloning systems have been extensively modified by genetic and biochemical manipulation to provide a variety of sophisticated phages, plasmids, and recipient bacterial strains specifically useful for molecular cloning. The current E. coli cloning systems have been shown to be extremely reliable for the isolation, amplification and, in many instances, genetic expression of prokaryotic and certain eukaryotic DNA fragments. Development of easily manipulated cloning systems within nonenteric bacteria such as Bacillus subtllis, or eukaryotic cells such as Sacckarornyces cerevisiae, will expand the types of DNA fragments that can be selected directly on the basis of their genetic function. Such recipient cell lines also provide a means to analyze the role of cloned genes that determine specialized biological functions. As an example, it has been estimated that more than 40 loci (operons) participate specifically in the formation of spores by B. subtilis.a Identification of fragments that harbor specific spore genes is most directly achieved by cloning the fragments into a chosen sporulation-negative mutant ofB. subtilis and seeking complementation of the sporulation defect. Efforts to date to clone sporulation genes have used E. coli, a non-spore former, as the cloning recipient.4 These experiments are technically difficult because of the absence of a direct biological selection for the cloned fragment. Similarly, identification of DNA fragments harboring genes involved in such functions as yeast cell division is most readily approached by cloning fragments directly into S. cerevisiae. Plasmids occur naturally in certain strains of the related Bacillus species B. pumilus and B. subtilis. 5-7 The majority of these plasmids specify no known biological function and are therefore classed as cryptic. Two Bacillus plasmids, pPL108 and pPL7065, ° determine the production of bacteriocin-like activities, and a third plasmid, pPL576,1° reduces the ability of the host to form spores. These last-mentioned three plasmids have been transformed into B. subtilis 168, and each has been used as a vector for cloning DNA fragments. Unfortunately, the absence of an easily selected, plasmid-specified trait has made these experiments technically cumbersome. The recent development of easily manipulated plasmid cloning vectors 3 p. j. Piggot and J. G. Coote, Bacteriol. Rev. 40, 908 (1976). 4 j. Segall and R. Losick, Cell 11, 751 (1977). s p. S. Lovett and M. G. Bramucci, J. Bacteriol. 124, 484 (1975). 6 T. Tanaka, M. Kuroda, and K. Sakaguchi, J. Bacteriol. 129, 1487 (1977). r J.-C. LeHegarat and C. Anagnostopoulos, Mol. Gen. Genet. 157, 167 (1977). 8 p. S. Lovett, E. J. Duvall, and K. M. Keggins, J. Bacteriol. 127, 817 (1976). 9 p. S. Lovett, E. J. Duvall, M. G. Bramucci, and R. Taylor, Antimicrob. Agents & Ckemotker. 12, 435 (1977). 10 p. S. Lovett, J. Bacteriol. 115, 291 (1973).
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V E H I C L E S A N D HOSTS F O R C L O N I N G TABLE 1 SOME PLASMIDS INTRODUCED INTO Bacillus subtilis 168 BY TRANSFORMATION
Source of plasmid B. pumilus A T C C 12140
Plasmid designation pPL10
Molecular weight (Md)
Plasmid-specified trait
Reference
4.4
Bacteriocin-like
8
activity Bacteriocin-like activity
9
B. purnilus A T C C 7065
pPL7065
4.6
B. purnilus N R S 576 B. cereus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus Streptococcus sanguinis
pPL576 pBC16 pT127 pC194 pC221 pC223 pLkB112 p U B 110 pSA2100 pSA0501 pSC194 pS194 pC194 pAM77
28.0 2.8 2.9 1.8 3.0 3.0 3.0 2.9 4.7 2.8 4.8 3.0 2.0 4.5
Oligosporogenesis Tet R Tet a Cm a Cm R Cm R Cm a K a n R/Neo a C m R Sm a Sm a Sm R C m a Sm a Cm a Ery a
10 12 I1 11 11 11 11 14 14 14 15, 16 15, 16 15, 16 13
for B. subtil& resulted from the observation that certain small, highcopy-number, antibiotic resistance plasmids detected in Staphylococcus aureus could be transformed directly into B. subtilis where the plasmids were stably maintained and expressed the appropriate antibiotic resistance trait, la Since the initial observation other antibiotic resistance plasmids originating in Bacillus ~2 and Streptococcus, ~3 in addition to Staphylococcus, have been transformed into B. subtilis (Table I), a-le making available many potential plasmid cloning vectors. In this chapter we describe the use of one such plasmid, p U B l l 0 (Table II), as a vector for cloning DNA fragments in B. subtilis. Principle of the Method Plasmids are small, circular replicons easily isolated in high purity from bacterial cells as covalently closed, circular (CCC)duplex D N A molecules. 17 Reintroduction of purified plasmids, in the CCC configura1~ S. 12 K. ~3 D. ~4 T. ~5 S. le S. ~r T.
D. Ehrlich, Proc. Natl. Acad. Sci. U.S.A. 74, 1680 (1977). Bernhard, H. Schrempf, and W. Goebel, J. Bacteriol. 133, 897 (1978). Clewell, personal communication. J. Gryczan, S. Contente, and D. D u b n a u , J. Bacteriol. 134, 318 (1978). L f f d a h l , J.-E. Sj6str6m, and L. Philipson, Gene 3, 149 (1978). L6fdahl, J.-E. Sj6str6m, and L. Philipson, Gene 3, 161 (1978). F. Roth and D. R. Helinski, Proc. Natl. Acad. Sci. U.S.A. 58, 650 (1967).
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TABLE II PROPERTIESOF PLASMIDpUB110 Property Molecular weight Sedimentation velocity Plasmid-specified trait Endonuclease sensitivity EcoRI BamHI XbaI Bg/II HindlI HpalI HaelII
Reference 2.9 × 106 21 S Resistance to 5/zg/ml of neomycin sulfate
14, 18 18 14
One site One site One site One site Two sites Three sites Three sites
tion, into appropriate plasmid-negative recipient bacteria is readily accomplished by transformation. The transformation frequency of small antibiotic resistance plasmids in the B. subtilis system is on the order of 0 . 1 - 1 0 × l03 transformants per m i c r o g r a m of plasmid. 14-16,1s-2° Small fragments of D N A can be inserted into purified plasmids in vitro (see below). Plasmids containing such insertions m a y then be transformed into an appropriate bacterial recipient where the constructed replicon is replicated with fidelity. I f the genes present on the insertion are properly expressed at the levels of transcription and translation, the cell carrying the constructed plasmid m a y acquire a new biological trait. In vitro insertion of D N A fragments into plasmids can be accomplished by either of two general approaches, both of which are similar in principle, m In the technically simpler a p p r o a c h a v e c t o r plasmid in the CCC configuration is c o n v e r t e d to a linear molecule by cleavage with a site-specific endonuclease such as H i n d I I I , E c o R I , or B a m H I which fulfills the following criteria: The enzyme-sensitive site on the plasmid must occur in a region not essential to plasmid replication or to expression of a plasmid function (e.g., antibiotic resistance) that m a y be used as a selected trait; cleavage by the e n z y m e should generate short, singlestranded termini each of which is identical in base sequence, and therefore the single-stranded termini are self-complementary. 21 Cleavage o f a vector plasmid and D N A f r o m a different source with the same site-specific endonuclease, such as E c o R I , generates a populaIs K. M. Keggins, P. S. Lovett, and E. J. DuvaU, Proc. Natl. Acad. Sci. U.S.A. 75, 1423 (1978). 19T. J. Gryczan and D. Dubnau, Proc. Natl. Acad. Sci. U.S.A. 75, 1428 (1978). 2o S. D. Ehrfich, Proc. Natl. Acad. Sci. U.S.A. 75, 1433 (1978). 21 R. J. Roberts, in "Microbiology, 1978" (D. Schlessinger, ed.), p. 5. Arner. Soc. Microbiol., 1978.
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tion of molecules sharing the same cohesive termini. Annealing of such DNA fragments at low temperature (< 10°) facilitates the stability of the joining of the cohesive ends through hydrogen-bond formation between the complementary termini. 22 This annealing procedure favors the formation of intramolecular associations at low DNA concentrations, and the probability of intermolecular associations increases with increasing DNA concentration. 23 Thus, at moderately high DNA concentrations (e.g., 2 /xg/ml) intermolecular complexes are generated. Many of these intermolecular complexes are the result of joining DNA fragments from unrelated sources, i.e., the vector plasmid and a fragment of foreign DNA. Fragments joined through hydrogen-bond formation between cohesive ends can be covalently sealed through the action of the enzyme DNA ligase. 1 Therefore, fragments of a foreign DNA molecule that are incapable of autonomous replication in a bacterium, such as E. coli or B. subtilis, can be physically joined to a replicon (plasmid) capable of autonomous replication in the chosen recipient. Transformation of an appropriate bacterial recipient with such constructed plasmids produces a cell line carrying plasmid-linked genes that the cell might not otherwise be capable of stably maintaining. If the genes on the insertion are fully expressed at the levels of transcription and translation, the cell can acquire a new biological trait. Selection of a constructed plasmid carrying a specific cloned fragment is achieved either by applying biological selection for expression of a specific cloned gene (e.g., t r p C ) or by prepurifying the desired fragment on the basis of some physical or biological characteristic unique to the fragment to be joined to the plasmid. The second method of inserting fragments of DNA into plasmids in vitro is commonly referred to as the tailing method. T M In this method the purified vector plasmid is linearized with a site-specific endonuclease, chosen because insertions into its site of cleavage on the plasmid do not disrupt the replicating ability of the plasmid or expression of a desired plasmid-associated trait to be used for selection. Cleavage by the endonuclease may generate either flush ends or cohesive ends. The DNA fragments to be joined to the vector plasmid can be generated by endonuclease cleavage or by mechanical shearing. In either event, the cohesive termini that will allow the plasmid vector and the foreign DNA fragments to associate are synthesized on each by using the enzyme terminal transferase. As an example, terminal transferase can be used to generate single-stranded poly(dA) tails (10 to 30 bases in length) on the free ends of the vector, and poly(dT) tails on the fragments of the foreign DNA. ~2j. E. Mertz and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 69, 3370 (1972). za A. Dugaiczyk,H. W. Boyer, and H. M. Goodman,J. Mol. Biol. 96, 171 (1975). L. Clarke and J. Carbon, Proc. Natl. Acad. Sci. U.S.A. 72, 4361 (1975).
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Mixing of the two classes of DNA molecules permits hydrogen-bond formation to occur between the synthesized termini. Although the tailing method has not yet been tested in the B. subtilis cloning system, this approach has certain advantages for cloning over the use of only sitespecific endonucleases that generate cohesive termini. The ability to add tails to DNA fragments generated by random mechanical shearing ensures that virtually any desired gene can be isolated on a discrete fragment, and indeed controlled shearing can result in varied sizes of DNA fragments. Plasmid transformants resulting from cloning by the tailing method all must, in principle, contain insertions, since a plasmid with poly(dA) tails cannot readily recyclize. Materials and Reagents
Bacterial Strains Mutant strains ofB. subtilis 168:BR151 (trpC2 metBlO lys-3), BD224 (trpC2 thr-5 recE4), T24 (trpE), T22 (trpD), T5 (trpC), T12 (trpF), T20 (trpB), T4 (trpA ).
Growth Media Liquid media used include antibiotic medium No. 3 [penassay broth (PB) Difco] and Spizizen's minimal medium supplemented with 0.05% acid-hydrolyzed casein (MinCH). Solid media for plates include tryptose blood agar base (TBAB, Difco) and MinCH containing 0.5% acidhydrolyzed casein solidified with 1.9% noble agar (Difco). Media containing 5 tzg/ml of neomycin sulfate are indicated as TBAB-Neo, PB-Neo, etc.
Materials and Reagents fi~r Plasmid Isolation 1. TES buffer: 0.02 M Tris-HCl, pH 7.5, 5 mM EDTA, 0.1 M NaC1 2. Lysozyme: 10 mg/ml of crystalline enzyme in TES 3. RNase: Pancreatic, 2 mg/ml in water, heat-shocked at 80° for 15 min 4. Pronase: 10 mg/ml in TES, predigested at 37° for 90 rain 5. Sarkosyl: NL30 (ICN Pharmaceuticals, Inc.) 6. Cesium chloride: High purity (Kawecki Berylco Industries, Inc.) 7. Polyallomer tubes:'~ × 3 inch tubes (Beckman) 8. [3H]thymidine: 1 mCi/ml (New England Nuclear) 9. Deoxyadenosine: 25 mg/ml in water 10. Isoamyl alcohol 11. Fraction collector
348
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12. Scintillation counter 13. Lysozyme buffer: 30 mM Tris, 50 mM EDTA, 50 mM NaCI, 25% sucrose, pH 8.0, with KOH 14. Sodium dodecyl sulfate (SDS) buffer: 2 ml of 10% SDS, 2 ml of 500 mM disodium EDTA, pH 8.0, 12 ml of TES buffer. 15. 5 M NaC1 in water 16. Polyethylene glycol (PEG): 50% type 6000 in TES 17. Ethidium bromide: 10 mg/ml in TES 18. Uv light source 19. Needle (18-gauge) and syringe 20. Ti50 rotor or equivalent 21. Ultracentrifuge capable of 44,000 rpm Materials and Reagents for Cellular D N A Isolation 1. Source of DNA: B. subtilis 168, B. pumilus N R R L B-3275, B. pumilus NRS576, B. licheniformis 9945A, B. licheniformis 749C 2. Lysozyme, RNase, pronase, and sarkosyl as in plasmid isolation 3. Phenol: Washed with TES buffer 4. Ethanol: 95%, cold 5. Recording spectrophotometer Reagents for Cloning 1. Endonucleases: EcoRI, BamHI, and HindIII, all commercially available from Bethesda Research Laboratories, Miles Research Products, and New England BioLabs 2. T4 DNA ligase: Available from New England BioLabs; and Bethesda Research Laboratories 3. EcoRI buffer: 0.1 M Tris-HCl, pH 7.5, 0.05 M NaCI, 0.01 M MgCI2 4. HindIII buffer: 6 mM Tris-HCl, pH 7.5, 50 mM NaC1, 6 mM MgCI2, 0.1 mg/ml of bovine serum albumin 5. BamHI buffer: 0.1 M Tris-HCl, pH 7.5, 0.01 M MgCI2 6. Dithiothreitol (DTT): 0.1 M in 0.1 M Tris-HC1, pH 7.5 7. Adenosine triphosphate (ATP): 0.01 M in 0.1 M Tris-HCl, pH 7.5 Method Plasmid Isolation Methods developed for isolating plasmid from E. coli can generally be applied, occasionally with slight modification, to the isolation of plasmids from B. subtilis and related species. One approach we have routinely used for isolating plasmids from B. subtilis and B. pumilus is as follows. Ba-
[23]
B. subtilis AS A HOST FOR CLONING
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cillus subtilis 168 (pUB 110) is grown to late log or early stationary phase in 200-500 ml MinCH, deoxyadenosine (250 p,g/ml), nutritional supplements required by the specific bacterial strain, and 1-20 ttCi of [aH]thymidine per milliliter. Cells are harvested by centrifugation, washed twice with TES buffer, and resuspended to approximately 1-%the original volume in TES. Lysozyme and RNase are added to 500 and 100/.~g/ml, respectively, and the suspension is incubated at 37° for 20-25 rain. The lysate is diluted twofold with TES, and pronase (to 500/zg/ml) and sarkosyl (to 0.8%) are added. After incubation for approximately 30 min at 37° the lysate, which is highly viscous and generally clear, is centrifuged at 12,500 rpm in the SS34 rotor of a Sorvall RC2B centrifdge at 4° for 25 min. The resulting supernatant fraction (cleared lysate) is carefully decanted. To 5 ml of the cleared lysate is added 7.1 g of CsC1. When the salt is completely dissolved, the volume is approximately 7.1 ml. This volume is divided equally between two -~ x 3 inch polyallomer tubes (3.5 ml per tube). Then, 1.5 ml of ethidium bromide solution (4 mg/ml in 0.1 M sodium phosphate buffer, pH 7) is added, the tubes are gently inverted, and paraffin oil is added to bring the tubes to volume. The tubes are centrifuged for 40 hr in a Ti50 rotor at 36,000 rpm, at 15°. The position of the plasmid peak in the gradient can be determined by fractioning the gradient into 15 portions of equal volume and counting 10-~d portions of each dried on 25-mm Whatman filter disks for radioactivity. Fractions containing the plasmid are pooled and extracted with equal volumes of isoamyl alcohol until all red color due to ethidium bromide has been visibly removed from the aqueous layer. The aqueous layer containing the plasmid is then dialyzed against 1000 volumes of TES at 4°. A convenient alternative we are currently using to isolate relatively large quantities of unlabeled plasmid is as follows. Bacillus subtilis 168 (pUBII0) is grown overnight (18-20 hr) in 250 ml of PB-Neo. The cells are chilled, concentrated by centrifugation, washed twice with TES, resuspended in 4 ml of lysozyme buffer, and incubated for 30 min at 37° with 500 ~g/ml of lysozyme. Sixteen milliliters of SDS buffer is added, and incubation is continued at 37° for 30 min. To this solution is added 5 ml of 5 M NaCI. After gentle mixing the solution is placed on ice for 20 hr and then centrifuged in the SS34 rotor at 15,000 rpm for 30 min. The resulting supernatant fraction is mixed in the cold with 5 ml of 50% PEG, held on ice for 3-5 hr, and then centrifuged at 10,000 rpm at 10° for 10 min in the SS34 rotor. The resulting pellet is resuspended in 2 ml of TES containing 200/xg/ml of heat-shocked RNase and held at 65° for 30 min. Ethidium bromide is added to 500 ~g/ml, and the final volume is brought to 4.0 ml with TES. Exactly 3.65 g of CsCI is then dissolved in the lysate, and the solution is transferred to a ~ x 3 inch polyallomer tube and centrifuged at 44,000 rpm in the Ti50 rotor for 40 hr. Illumination of the tube
350
VEHICLES AND HOSTS FOR CLONING
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with uv light shows two bands that fluoresce. The lower band is plasmid. This band is removed through the side of the tube with a needle and syringe. The plasmid is extracted with isoamyl alcohol and dialyzed as above.
Isolation of Bulk Cellular DNA Strains o f B . pumilus, B. subtilis, or B. licheniformis are grown at 37° in 10 ml of PB for 18-20 hr. The cells are washed twice with T E S buffer, resuspended in 2.5 ml of TES, and treated sequentially with lysozyme, RNase, pronase, and sarkosyl as described for plasmid isolation. The lysate is shaken gently with an equal volume of TES-saturated phenol at room temperature for 10 min. After centrifugation at room temperature for 10 min the aqueous phase is removed with a large-bore pipette and extracted with phenol twice again. The final aqueous phase ( - 3 ml) is transferred to 2 volumes of cold 95% ethanol and held at 4 ° for 30 min. The precipitate is resuspended in 2 ml of T E S and dialyzed against 1000 volumes of T E S at 4 ° for 12-18 hr. A dilution of the DNA-containing solution is scanned in a Cary recording spectrophotometer over the range of 240-290 nm to estimate D N A concentration (OD of 1 at 260 nm 50/~g/ml of DNA) and to determine OD~6o/OD2so ratios.
Cloning Method The level of endonuclease used to digest plasmid or phenol-purified cellular D N A is determined empirically as twice the activity of a specific enzyme, such as EcoRI, required to convert a fixed concentration of DNA, such as pUB110, to a linear form in 30 rain at 37 ° as monitored by electrophoresis of the intact and digested plasmid through 0.7% agarose gels. TM pUB110 (0.5 txg) and approximately 3/zg of cellular D N A (e.g., B. subtilis, B. pumilus, or B. licheniformis) are combined in 200 pJ of T E S buffer and dialyzed against 1000 volumes of EcoRI buffer for 4 hr at 4 °. The solution is then incubated at 37 ° with the appropriate activity of EcoRI to ensure complete cleavage within 30 min. After cleavage the enzyme activity is terminated by placing the digest at 65 ° for 15 min. Annealing is achieved by incubating the D N A at 0 ° for 18 hr (in an ice bath in a 4 ° cold room). D T T is added to 10 mM, and A T P to 0.1 mM. One unit of T4 DNA ligase is then added, and the solution is held at 4 ° for 8 - 1 2 hr and then moved to 15° for 10 hr. The resulting D N A preparation is dialyzed against 1000 volumes o f T E S buffer, and approximately 2 txg of D N A is shaken at 37 ° with 5 × l0 s competent B. subtilis trpC2 cells (e.g., strain BR151) in 1 ml for 1 hr. Approximately 0.9 ml of the transformation reaction mixture is added to 20 ml of PB containing 5 / z g / m l of neomycin sulfate which is shaken at 37 ° for 2 0 - 2 4 hr. The remainder of the trans-
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351
formed cells are diluted and plated on T B A B - N e o to estimate transformation frequency. Transformation controls, including cells and D N A incubated separately, are routinely run, and each is treated as described for the transformed cells. Afl:er overnight incubation the transformed cells in 20 ml of PB-Neo have grown to saturation ( - 5 × 10S/ml) and there is no growth in the cell or D N A control flasks. The transformed cells are washed with Spizizen's minimal medium twice, and the culture is diluted 10-fold in MinCH (no tryptophan) containing 5 /xg/ml of neomycin sulfate. This culture is shaken at 37 ° for 20 hr at which time the cells have grown to 2 - 5 × 10S/ml. A loopful o f the culture is streaked onto a MinCH-Neo plate, or serial dilutions of the culture are plated on MinCH-Neo and the plates are incubated at 37 ° overnight. A single resulting colony is purified by restreaking, and the plasmid DNA is isolated and used to transform a trpC2 mutant ofB. subtilis to Neo a. Each of 50 transformants is tested for the Trp phenotype. Frequently we find that the plasmid preparations at this stage contain both a constructed trp plasmid and pUB110. Therefore, only 40-45 of the N e o R clones are Trp ÷. One of these Trp + Neo rt transformant is then used as the source of the particular constructed trp plasmid. Two types of recipients have been used for cloning, depending on the properties o f the DNA to be cloned. If the source of the D N A to be cloned is a bacterial species sufficiently closely related to B. subtilis 168 such that there exists extensive homology between donor and recipient genomes, a recombination-deficient recipient is used, e.g., a recipient carrying the recE4 mutation. 25,26 If the donor DNA shares little homology with the B. subtilis c h r o m o s o m e (e.g., D N A extracted from B. pumilus or B. lichen# formis) a wild-type (Rec +) transformation recipient is used. Identification of the genetic constitution o f cloned trp segments is rapidly ac,hieved by complementation analysis. The trp biosynthetic genes in B. subtilis appear contiguous and map as a cluster in the order: trpE D C F B A. 27 The ability of a given plasmid to complement point mutations in each o f the B. subtilis trp genes is determined by transforming the plasmid into six mutant derivatives o f B . subtilis each carrying a mutation in a different trp gene 2s (see Materials and Reagents). Neomycin-resistant transformants are selected, and the Trp phenotype of 50-200 of the transformants is determined by patching the colonies into MinCH. 2s D. Dubnau and C. Cirigliano,J. Bacteriol. 117, 488 (1974). 2s K. M. Keggins, E. J. DuvaU, and P. S. Lovett, J. Bacteriol. 134, 514 (1978). 2~I. P. Crawford, Bacteriol. Rev. 39, 87 (1975). ~s S. O. Hoch, C. Anagnostopoulos, and I. P. Crawford, Biochem. Biophys. Res. Commun. 35, 8."t8 (1969).
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Genetic and Biochemical Properties of Cloned EcoRl and BamHl trp Segments
pUB 110 is sensitive to several site-specific endonucleases (Table II). Both the single BamHI site and the single EcoRI site on p U B l l 0 appear available for cloning. EcoRI-generated trp segments we have cloned using pUB110 as a vector have molecular weights in the range of 1.5-2.5 Md. This size is sufficient to specify two to four genes, and complementation analysis of each of the EcoRI trp plasmids shows that each complements point mutations in two, three, or four trp genes (Tables III and IV). Digestion of the majority of the EcoRI-generated trp plasmids (e.g., pSL103) with EcoRI yields two linear fragments whose combined molecular weight (e.g., 2.3 + 2.8 Md) approximates the molecular weight of the intact plasmid (e.g., 5.0 Md). If EcoRI-digested pSL103 is subjected to the annealing and ligation procedure and used to transform B. subtilis 168 to neomycin resistance, the original vector plasmid can be removed from the constructed trp derivative. Occasionally trp plasmids constructed using EcoRI retain only a single EcoRI site. Thus digestion of one such plasmid, pSL101, with EcoRI generates a single linear modecule of the same molecular weight as intact pSL101. The reason for the occurrence of constructed plasmids retaining only a single EcoRI site is not known. Construction of trp plasmids using BamHI endonuclease has in our hands always yielded trp plasmids that retain only a single Bam HI site (Table V). Comparison of the inferred molecular weights of BamHIgenerated trp segments with the molecular weights oftrp segments cloned using EcoRI suggests that BamHI-generated fragments are larger. Plasmid pUB 110 and each of the trp derivatives are maintained in B. subtilis at approximately 50 copies per chromosome equivalent. Comparison of the Trp enzyme levels in B. subtilis cells carrying each of five constructed trp plasmids with the levels of the chromosomally specified Trp enzymes in strains harboring no plasmids generally confirms the genetic constitution of each plasmid initially deduced from complementation analysis 2~ (Tables III and IV). Plasmids that complement point mutations in individual trp genes specify the corresponding enzyme activities. The specific activities of plasmid-specified Trp enzymes in B. subtilis are significantly greater than the repressed levels of the chromosomally specified enzymes and equal to or below the derepressed levels of the chromosomally specified enzymes. Each of the constructed trp plasmids was initially selected for ability to complement a specific mutation in trpC (i.e., trpC2), and each of five trp plasmids tested contained a single HindIII-sensitive site. pUBI10 is z9K. M. Keggins, P. S. Lovett, R. Marrero, and S. O. Hoch, J. Bacteriol. 139, 1001 (1979).
B. subtilis AS A HOST FOR CLONING
[23]
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V E H I C L E S A N D HOSTS FOR C L O N I N G
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h. Incubate at 30° for 12-18 hr. 3. Selection of ts mutants a. Dilute each culture in nutrient broth (1:106) and plate 0.1 ml on nutrient broth plates. b. Incubate plates at 30° for 24-48 hr. c. Replica plate each plate twice and incubate one at 30° and the counterpart at 42 °. d. Select colonies which grow at 30° but not at 42°. e. Repeat the temperature selection for all positive colonies 2-3 times. 4. Screening of ts mutants for competence of DNA transfection a. Inoculate 2 ml of nutrient broth with single colonies and incubate the cultures at 30° overnight with gentle shaking. b. Remove 0.25 ml of culture into a sterile tube containing 0.25 ml of PAM + 0.48 sucrose (PAMI~). c. Incubate for 5 hr with gentle shaking at 42°. d. Add 0.5 ml of 4~x174 ss DNA solution containing 1/zg/ml DNA in 50 mM Tris HC1, pH 8.1. e. Incubate at 42° for 2.5 min and transfer the culture to 0-4 °. f. Titer the infective centers. 12 10 g casamino acids, 10 g nutrient broth, 1 g glucose, 2 g MgSO4 (per liter). G. D. Guthrie and R. L. Sinsheimer, Biochim. Biophys. Acta 72, 290 (1963).
[23] B a c i l l u s s u b t i l i s a s a H o s t f o r M o l e c u l a r C l o n i n g
By PAUL S. LOVETT and KATHLEEN M. KEGGINS Prior to the advent of molecular cloning technologyla the two methods most commonly used to isolate quantities of cellular DNA enriched for specific genes involved the generation of specialized transducing phages that harbor specific regions of the bacterial chromosome, or the isolation of specific regions of cellular DNA on the basis of their unique physical properties or their occurrence within a subcellular organelle. The advantages of molecular cloning over these methods include its application in the isolation, in principle, of any small fragment of cellular DNA and in maintaining the selected fragment joined to a small, easily isolated replicon. The principles of molecular cloning have been developed using Escherichia coli, its plasmids, and its bacteriophages. In recent years the 1 S. N. Cohen, A. C. Y. Chang, H. W. Boyer, and R. B. Helling, Proc. Natl. Acad. Sci. U.S.A. 70, 3240 (1973). 2 D. A. Jackson, R. H. Symons, and P. Berg, Proc. Natl, Acad. Sci. U.S.A. 69, 2904 (1972). METHODS IN ENZYMOLOGY, VOL, 68
Copyright O 1979by Academic Press, Inc. All rights of reproductionin any formreserved. ISBN 0-12-181968-X
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components of the E. coli cloning systems have been extensively modified by genetic and biochemical manipulation to provide a variety of sophisticated phages, plasmids, and recipient bacterial strains specifically useful for molecular cloning. The current E. coli cloning systems have been shown to be extremely reliable for the isolation, amplification and, in many instances, genetic expression of prokaryotic and certain eukaryotic DNA fragments. Development of easily manipulated cloning systems within nonenteric bacteria such as Bacillus subtllis, or eukaryotic cells such as Sacckarornyces cerevisiae, will expand the types of DNA fragments that can be selected directly on the basis of their genetic function. Such recipient cell lines also provide a means to analyze the role of cloned genes that determine specialized biological functions. As an example, it has been estimated that more than 40 loci (operons) participate specifically in the formation of spores by B. subtilis.a Identification of fragments that harbor specific spore genes is most directly achieved by cloning the fragments into a chosen sporulation-negative mutant ofB. subtilis and seeking complementation of the sporulation defect. Efforts to date to clone sporulation genes have used E. coli, a non-spore former, as the cloning recipient.4 These experiments are technically difficult because of the absence of a direct biological selection for the cloned fragment. Similarly, identification of DNA fragments harboring genes involved in such functions as yeast cell division is most readily approached by cloning fragments directly into S. cerevisiae. Plasmids occur naturally in certain strains of the related Bacillus species B. pumilus and B. subtilis. 5-7 The majority of these plasmids specify no known biological function and are therefore classed as cryptic. Two Bacillus plasmids, pPL108 and pPL7065, ° determine the production of bacteriocin-like activities, and a third plasmid, pPL576,1° reduces the ability of the host to form spores. These last-mentioned three plasmids have been transformed into B. subtilis 168, and each has been used as a vector for cloning DNA fragments. Unfortunately, the absence of an easily selected, plasmid-specified trait has made these experiments technically cumbersome. The recent development of easily manipulated plasmid cloning vectors 3 p. j. Piggot and J. G. Coote, Bacteriol. Rev. 40, 908 (1976). 4 j. Segall and R. Losick, Cell 11, 751 (1977). s p. S. Lovett and M. G. Bramucci, J. Bacteriol. 124, 484 (1975). 6 T. Tanaka, M. Kuroda, and K. Sakaguchi, J. Bacteriol. 129, 1487 (1977). r J.-C. LeHegarat and C. Anagnostopoulos, Mol. Gen. Genet. 157, 167 (1977). 8 p. S. Lovett, E. J. Duvall, and K. M. Keggins, J. Bacteriol. 127, 817 (1976). 9 p. S. Lovett, E. J. Duvall, M. G. Bramucci, and R. Taylor, Antimicrob. Agents & Ckemotker. 12, 435 (1977). 10 p. S. Lovett, J. Bacteriol. 115, 291 (1973).
344
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V E H I C L E S A N D HOSTS F O R C L O N I N G TABLE 1 SOME PLASMIDS INTRODUCED INTO Bacillus subtilis 168 BY TRANSFORMATION
Source of plasmid B. pumilus A T C C 12140
Plasmid designation pPL10
Molecular weight (Md)
Plasmid-specified trait
Reference
4.4
Bacteriocin-like
8
activity Bacteriocin-like activity
9
B. purnilus A T C C 7065
pPL7065
4.6
B. purnilus N R S 576 B. cereus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus Streptococcus sanguinis
pPL576 pBC16 pT127 pC194 pC221 pC223 pLkB112 p U B 110 pSA2100 pSA0501 pSC194 pS194 pC194 pAM77
28.0 2.8 2.9 1.8 3.0 3.0 3.0 2.9 4.7 2.8 4.8 3.0 2.0 4.5
Oligosporogenesis Tet R Tet a Cm a Cm R Cm R Cm a K a n R/Neo a C m R Sm a Sm a Sm R C m a Sm a Cm a Ery a
10 12 I1 11 11 11 11 14 14 14 15, 16 15, 16 15, 16 13
for B. subtil& resulted from the observation that certain small, highcopy-number, antibiotic resistance plasmids detected in Staphylococcus aureus could be transformed directly into B. subtilis where the plasmids were stably maintained and expressed the appropriate antibiotic resistance trait, la Since the initial observation other antibiotic resistance plasmids originating in Bacillus ~2 and Streptococcus, ~3 in addition to Staphylococcus, have been transformed into B. subtilis (Table I), a-le making available many potential plasmid cloning vectors. In this chapter we describe the use of one such plasmid, p U B l l 0 (Table II), as a vector for cloning DNA fragments in B. subtilis. Principle of the Method Plasmids are small, circular replicons easily isolated in high purity from bacterial cells as covalently closed, circular (CCC)duplex D N A molecules. 17 Reintroduction of purified plasmids, in the CCC configura1~ S. 12 K. ~3 D. ~4 T. ~5 S. le S. ~r T.
D. Ehrlich, Proc. Natl. Acad. Sci. U.S.A. 74, 1680 (1977). Bernhard, H. Schrempf, and W. Goebel, J. Bacteriol. 133, 897 (1978). Clewell, personal communication. J. Gryczan, S. Contente, and D. D u b n a u , J. Bacteriol. 134, 318 (1978). L f f d a h l , J.-E. Sj6str6m, and L. Philipson, Gene 3, 149 (1978). L6fdahl, J.-E. Sj6str6m, and L. Philipson, Gene 3, 161 (1978). F. Roth and D. R. Helinski, Proc. Natl. Acad. Sci. U.S.A. 58, 650 (1967).
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TABLE II PROPERTIESOF PLASMIDpUB110 Property Molecular weight Sedimentation velocity Plasmid-specified trait Endonuclease sensitivity EcoRI BamHI XbaI Bg/II HindlI HpalI HaelII
Reference 2.9 × 106 21 S Resistance to 5/zg/ml of neomycin sulfate
14, 18 18 14
One site One site One site One site Two sites Three sites Three sites
tion, into appropriate plasmid-negative recipient bacteria is readily accomplished by transformation. The transformation frequency of small antibiotic resistance plasmids in the B. subtilis system is on the order of 0 . 1 - 1 0 × l03 transformants per m i c r o g r a m of plasmid. 14-16,1s-2° Small fragments of D N A can be inserted into purified plasmids in vitro (see below). Plasmids containing such insertions m a y then be transformed into an appropriate bacterial recipient where the constructed replicon is replicated with fidelity. I f the genes present on the insertion are properly expressed at the levels of transcription and translation, the cell carrying the constructed plasmid m a y acquire a new biological trait. In vitro insertion of D N A fragments into plasmids can be accomplished by either of two general approaches, both of which are similar in principle, m In the technically simpler a p p r o a c h a v e c t o r plasmid in the CCC configuration is c o n v e r t e d to a linear molecule by cleavage with a site-specific endonuclease such as H i n d I I I , E c o R I , or B a m H I which fulfills the following criteria: The enzyme-sensitive site on the plasmid must occur in a region not essential to plasmid replication or to expression of a plasmid function (e.g., antibiotic resistance) that m a y be used as a selected trait; cleavage by the e n z y m e should generate short, singlestranded termini each of which is identical in base sequence, and therefore the single-stranded termini are self-complementary. 21 Cleavage o f a vector plasmid and D N A f r o m a different source with the same site-specific endonuclease, such as E c o R I , generates a populaIs K. M. Keggins, P. S. Lovett, and E. J. DuvaU, Proc. Natl. Acad. Sci. U.S.A. 75, 1423 (1978). 19T. J. Gryczan and D. Dubnau, Proc. Natl. Acad. Sci. U.S.A. 75, 1428 (1978). 2o S. D. Ehrfich, Proc. Natl. Acad. Sci. U.S.A. 75, 1433 (1978). 21 R. J. Roberts, in "Microbiology, 1978" (D. Schlessinger, ed.), p. 5. Arner. Soc. Microbiol., 1978.
346
VEHICLES AND HOSTS FOR CLONING
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tion of molecules sharing the same cohesive termini. Annealing of such DNA fragments at low temperature (< 10°) facilitates the stability of the joining of the cohesive ends through hydrogen-bond formation between the complementary termini. 22 This annealing procedure favors the formation of intramolecular associations at low DNA concentrations, and the probability of intermolecular associations increases with increasing DNA concentration. 23 Thus, at moderately high DNA concentrations (e.g., 2 /xg/ml) intermolecular complexes are generated. Many of these intermolecular complexes are the result of joining DNA fragments from unrelated sources, i.e., the vector plasmid and a fragment of foreign DNA. Fragments joined through hydrogen-bond formation between cohesive ends can be covalently sealed through the action of the enzyme DNA ligase. 1 Therefore, fragments of a foreign DNA molecule that are incapable of autonomous replication in a bacterium, such as E. coli or B. subtilis, can be physically joined to a replicon (plasmid) capable of autonomous replication in the chosen recipient. Transformation of an appropriate bacterial recipient with such constructed plasmids produces a cell line carrying plasmid-linked genes that the cell might not otherwise be capable of stably maintaining. If the genes on the insertion are fully expressed at the levels of transcription and translation, the cell can acquire a new biological trait. Selection of a constructed plasmid carrying a specific cloned fragment is achieved either by applying biological selection for expression of a specific cloned gene (e.g., t r p C ) or by prepurifying the desired fragment on the basis of some physical or biological characteristic unique to the fragment to be joined to the plasmid. The second method of inserting fragments of DNA into plasmids in vitro is commonly referred to as the tailing method. T M In this method the purified vector plasmid is linearized with a site-specific endonuclease, chosen because insertions into its site of cleavage on the plasmid do not disrupt the replicating ability of the plasmid or expression of a desired plasmid-associated trait to be used for selection. Cleavage by the endonuclease may generate either flush ends or cohesive ends. The DNA fragments to be joined to the vector plasmid can be generated by endonuclease cleavage or by mechanical shearing. In either event, the cohesive termini that will allow the plasmid vector and the foreign DNA fragments to associate are synthesized on each by using the enzyme terminal transferase. As an example, terminal transferase can be used to generate single-stranded poly(dA) tails (10 to 30 bases in length) on the free ends of the vector, and poly(dT) tails on the fragments of the foreign DNA. ~2j. E. Mertz and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 69, 3370 (1972). za A. Dugaiczyk,H. W. Boyer, and H. M. Goodman,J. Mol. Biol. 96, 171 (1975). L. Clarke and J. Carbon, Proc. Natl. Acad. Sci. U.S.A. 72, 4361 (1975).
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B. subtilis AS A HOST FOR CLONING
347
Mixing of the two classes of DNA molecules permits hydrogen-bond formation to occur between the synthesized termini. Although the tailing method has not yet been tested in the B. subtilis cloning system, this approach has certain advantages for cloning over the use of only sitespecific endonucleases that generate cohesive termini. The ability to add tails to DNA fragments generated by random mechanical shearing ensures that virtually any desired gene can be isolated on a discrete fragment, and indeed controlled shearing can result in varied sizes of DNA fragments. Plasmid transformants resulting from cloning by the tailing method all must, in principle, contain insertions, since a plasmid with poly(dA) tails cannot readily recyclize. Materials and Reagents
Bacterial Strains Mutant strains ofB. subtilis 168:BR151 (trpC2 metBlO lys-3), BD224 (trpC2 thr-5 recE4), T24 (trpE), T22 (trpD), T5 (trpC), T12 (trpF), T20 (trpB), T4 (trpA ).
Growth Media Liquid media used include antibiotic medium No. 3 [penassay broth (PB) Difco] and Spizizen's minimal medium supplemented with 0.05% acid-hydrolyzed casein (MinCH). Solid media for plates include tryptose blood agar base (TBAB, Difco) and MinCH containing 0.5% acidhydrolyzed casein solidified with 1.9% noble agar (Difco). Media containing 5 tzg/ml of neomycin sulfate are indicated as TBAB-Neo, PB-Neo, etc.
Materials and Reagents fi~r Plasmid Isolation 1. TES buffer: 0.02 M Tris-HCl, pH 7.5, 5 mM EDTA, 0.1 M NaC1 2. Lysozyme: 10 mg/ml of crystalline enzyme in TES 3. RNase: Pancreatic, 2 mg/ml in water, heat-shocked at 80° for 15 min 4. Pronase: 10 mg/ml in TES, predigested at 37° for 90 rain 5. Sarkosyl: NL30 (ICN Pharmaceuticals, Inc.) 6. Cesium chloride: High purity (Kawecki Berylco Industries, Inc.) 7. Polyallomer tubes:'~ × 3 inch tubes (Beckman) 8. [3H]thymidine: 1 mCi/ml (New England Nuclear) 9. Deoxyadenosine: 25 mg/ml in water 10. Isoamyl alcohol 11. Fraction collector
348
V E H I C L E S A N D HOSTS FOR C L O N I N G
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12. Scintillation counter 13. Lysozyme buffer: 30 mM Tris, 50 mM EDTA, 50 mM NaCI, 25% sucrose, pH 8.0, with KOH 14. Sodium dodecyl sulfate (SDS) buffer: 2 ml of 10% SDS, 2 ml of 500 mM disodium EDTA, pH 8.0, 12 ml of TES buffer. 15. 5 M NaC1 in water 16. Polyethylene glycol (PEG): 50% type 6000 in TES 17. Ethidium bromide: 10 mg/ml in TES 18. Uv light source 19. Needle (18-gauge) and syringe 20. Ti50 rotor or equivalent 21. Ultracentrifuge capable of 44,000 rpm Materials and Reagents for Cellular D N A Isolation 1. Source of DNA: B. subtilis 168, B. pumilus N R R L B-3275, B. pumilus NRS576, B. licheniformis 9945A, B. licheniformis 749C 2. Lysozyme, RNase, pronase, and sarkosyl as in plasmid isolation 3. Phenol: Washed with TES buffer 4. Ethanol: 95%, cold 5. Recording spectrophotometer Reagents for Cloning 1. Endonucleases: EcoRI, BamHI, and HindIII, all commercially available from Bethesda Research Laboratories, Miles Research Products, and New England BioLabs 2. T4 DNA ligase: Available from New England BioLabs; and Bethesda Research Laboratories 3. EcoRI buffer: 0.1 M Tris-HCl, pH 7.5, 0.05 M NaCI, 0.01 M MgCI2 4. HindIII buffer: 6 mM Tris-HCl, pH 7.5, 50 mM NaC1, 6 mM MgCI2, 0.1 mg/ml of bovine serum albumin 5. BamHI buffer: 0.1 M Tris-HCl, pH 7.5, 0.01 M MgCI2 6. Dithiothreitol (DTT): 0.1 M in 0.1 M Tris-HC1, pH 7.5 7. Adenosine triphosphate (ATP): 0.01 M in 0.1 M Tris-HCl, pH 7.5 Method Plasmid Isolation Methods developed for isolating plasmid from E. coli can generally be applied, occasionally with slight modification, to the isolation of plasmids from B. subtilis and related species. One approach we have routinely used for isolating plasmids from B. subtilis and B. pumilus is as follows. Ba-
[23]
B. subtilis AS A HOST FOR CLONING
349
cillus subtilis 168 (pUB 110) is grown to late log or early stationary phase in 200-500 ml MinCH, deoxyadenosine (250 p,g/ml), nutritional supplements required by the specific bacterial strain, and 1-20 ttCi of [aH]thymidine per milliliter. Cells are harvested by centrifugation, washed twice with TES buffer, and resuspended to approximately 1-%the original volume in TES. Lysozyme and RNase are added to 500 and 100/.~g/ml, respectively, and the suspension is incubated at 37° for 20-25 rain. The lysate is diluted twofold with TES, and pronase (to 500/zg/ml) and sarkosyl (to 0.8%) are added. After incubation for approximately 30 min at 37° the lysate, which is highly viscous and generally clear, is centrifuged at 12,500 rpm in the SS34 rotor of a Sorvall RC2B centrifdge at 4° for 25 min. The resulting supernatant fraction (cleared lysate) is carefully decanted. To 5 ml of the cleared lysate is added 7.1 g of CsC1. When the salt is completely dissolved, the volume is approximately 7.1 ml. This volume is divided equally between two -~ x 3 inch polyallomer tubes (3.5 ml per tube). Then, 1.5 ml of ethidium bromide solution (4 mg/ml in 0.1 M sodium phosphate buffer, pH 7) is added, the tubes are gently inverted, and paraffin oil is added to bring the tubes to volume. The tubes are centrifuged for 40 hr in a Ti50 rotor at 36,000 rpm, at 15°. The position of the plasmid peak in the gradient can be determined by fractioning the gradient into 15 portions of equal volume and counting 10-~d portions of each dried on 25-mm Whatman filter disks for radioactivity. Fractions containing the plasmid are pooled and extracted with equal volumes of isoamyl alcohol until all red color due to ethidium bromide has been visibly removed from the aqueous layer. The aqueous layer containing the plasmid is then dialyzed against 1000 volumes of TES at 4°. A convenient alternative we are currently using to isolate relatively large quantities of unlabeled plasmid is as follows. Bacillus subtilis 168 (pUBII0) is grown overnight (18-20 hr) in 250 ml of PB-Neo. The cells are chilled, concentrated by centrifugation, washed twice with TES, resuspended in 4 ml of lysozyme buffer, and incubated for 30 min at 37° with 500 ~g/ml of lysozyme. Sixteen milliliters of SDS buffer is added, and incubation is continued at 37° for 30 min. To this solution is added 5 ml of 5 M NaCI. After gentle mixing the solution is placed on ice for 20 hr and then centrifuged in the SS34 rotor at 15,000 rpm for 30 min. The resulting supernatant fraction is mixed in the cold with 5 ml of 50% PEG, held on ice for 3-5 hr, and then centrifuged at 10,000 rpm at 10° for 10 min in the SS34 rotor. The resulting pellet is resuspended in 2 ml of TES containing 200/xg/ml of heat-shocked RNase and held at 65° for 30 min. Ethidium bromide is added to 500 ~g/ml, and the final volume is brought to 4.0 ml with TES. Exactly 3.65 g of CsCI is then dissolved in the lysate, and the solution is transferred to a ~ x 3 inch polyallomer tube and centrifuged at 44,000 rpm in the Ti50 rotor for 40 hr. Illumination of the tube
350
VEHICLES AND HOSTS FOR CLONING
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with uv light shows two bands that fluoresce. The lower band is plasmid. This band is removed through the side of the tube with a needle and syringe. The plasmid is extracted with isoamyl alcohol and dialyzed as above.
Isolation of Bulk Cellular DNA Strains o f B . pumilus, B. subtilis, or B. licheniformis are grown at 37° in 10 ml of PB for 18-20 hr. The cells are washed twice with T E S buffer, resuspended in 2.5 ml of TES, and treated sequentially with lysozyme, RNase, pronase, and sarkosyl as described for plasmid isolation. The lysate is shaken gently with an equal volume of TES-saturated phenol at room temperature for 10 min. After centrifugation at room temperature for 10 min the aqueous phase is removed with a large-bore pipette and extracted with phenol twice again. The final aqueous phase ( - 3 ml) is transferred to 2 volumes of cold 95% ethanol and held at 4 ° for 30 min. The precipitate is resuspended in 2 ml of T E S and dialyzed against 1000 volumes of T E S at 4 ° for 12-18 hr. A dilution of the DNA-containing solution is scanned in a Cary recording spectrophotometer over the range of 240-290 nm to estimate D N A concentration (OD of 1 at 260 nm 50/~g/ml of DNA) and to determine OD~6o/OD2so ratios.
Cloning Method The level of endonuclease used to digest plasmid or phenol-purified cellular D N A is determined empirically as twice the activity of a specific enzyme, such as EcoRI, required to convert a fixed concentration of DNA, such as pUB110, to a linear form in 30 rain at 37 ° as monitored by electrophoresis of the intact and digested plasmid through 0.7% agarose gels. TM pUB110 (0.5 txg) and approximately 3/zg of cellular D N A (e.g., B. subtilis, B. pumilus, or B. licheniformis) are combined in 200 pJ of T E S buffer and dialyzed against 1000 volumes of EcoRI buffer for 4 hr at 4 °. The solution is then incubated at 37 ° with the appropriate activity of EcoRI to ensure complete cleavage within 30 min. After cleavage the enzyme activity is terminated by placing the digest at 65 ° for 15 min. Annealing is achieved by incubating the D N A at 0 ° for 18 hr (in an ice bath in a 4 ° cold room). D T T is added to 10 mM, and A T P to 0.1 mM. One unit of T4 DNA ligase is then added, and the solution is held at 4 ° for 8 - 1 2 hr and then moved to 15° for 10 hr. The resulting D N A preparation is dialyzed against 1000 volumes o f T E S buffer, and approximately 2 txg of D N A is shaken at 37 ° with 5 × l0 s competent B. subtilis trpC2 cells (e.g., strain BR151) in 1 ml for 1 hr. Approximately 0.9 ml of the transformation reaction mixture is added to 20 ml of PB containing 5 / z g / m l of neomycin sulfate which is shaken at 37 ° for 2 0 - 2 4 hr. The remainder of the trans-
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B. subtilis AS A HOST FOR CLONING
351
formed cells are diluted and plated on T B A B - N e o to estimate transformation frequency. Transformation controls, including cells and D N A incubated separately, are routinely run, and each is treated as described for the transformed cells. Afl:er overnight incubation the transformed cells in 20 ml of PB-Neo have grown to saturation ( - 5 × 10S/ml) and there is no growth in the cell or D N A control flasks. The transformed cells are washed with Spizizen's minimal medium twice, and the culture is diluted 10-fold in MinCH (no tryptophan) containing 5 /xg/ml of neomycin sulfate. This culture is shaken at 37 ° for 20 hr at which time the cells have grown to 2 - 5 × 10S/ml. A loopful o f the culture is streaked onto a MinCH-Neo plate, or serial dilutions of the culture are plated on MinCH-Neo and the plates are incubated at 37 ° overnight. A single resulting colony is purified by restreaking, and the plasmid DNA is isolated and used to transform a trpC2 mutant ofB. subtilis to Neo a. Each of 50 transformants is tested for the Trp phenotype. Frequently we find that the plasmid preparations at this stage contain both a constructed trp plasmid and pUB110. Therefore, only 40-45 of the N e o R clones are Trp ÷. One of these Trp + Neo rt transformant is then used as the source of the particular constructed trp plasmid. Two types of recipients have been used for cloning, depending on the properties o f the DNA to be cloned. If the source of the D N A to be cloned is a bacterial species sufficiently closely related to B. subtilis 168 such that there exists extensive homology between donor and recipient genomes, a recombination-deficient recipient is used, e.g., a recipient carrying the recE4 mutation. 25,26 If the donor DNA shares little homology with the B. subtilis c h r o m o s o m e (e.g., D N A extracted from B. pumilus or B. lichen# formis) a wild-type (Rec +) transformation recipient is used. Identification of the genetic constitution o f cloned trp segments is rapidly ac,hieved by complementation analysis. The trp biosynthetic genes in B. subtilis appear contiguous and map as a cluster in the order: trpE D C F B A. 27 The ability of a given plasmid to complement point mutations in each o f the B. subtilis trp genes is determined by transforming the plasmid into six mutant derivatives o f B . subtilis each carrying a mutation in a different trp gene 2s (see Materials and Reagents). Neomycin-resistant transformants are selected, and the Trp phenotype of 50-200 of the transformants is determined by patching the colonies into MinCH. 2s D. Dubnau and C. Cirigliano,J. Bacteriol. 117, 488 (1974). 2s K. M. Keggins, E. J. DuvaU, and P. S. Lovett, J. Bacteriol. 134, 514 (1978). 2~I. P. Crawford, Bacteriol. Rev. 39, 87 (1975). ~s S. O. Hoch, C. Anagnostopoulos, and I. P. Crawford, Biochem. Biophys. Res. Commun. 35, 8."t8 (1969).
352
VEHICLES AND HOSTS FOR CLONING
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Genetic and Biochemical Properties of Cloned EcoRl and BamHl trp Segments
pUB 110 is sensitive to several site-specific endonucleases (Table II). Both the single BamHI site and the single EcoRI site on p U B l l 0 appear available for cloning. EcoRI-generated trp segments we have cloned using pUB110 as a vector have molecular weights in the range of 1.5-2.5 Md. This size is sufficient to specify two to four genes, and complementation analysis of each of the EcoRI trp plasmids shows that each complements point mutations in two, three, or four trp genes (Tables III and IV). Digestion of the majority of the EcoRI-generated trp plasmids (e.g., pSL103) with EcoRI yields two linear fragments whose combined molecular weight (e.g., 2.3 + 2.8 Md) approximates the molecular weight of the intact plasmid (e.g., 5.0 Md). If EcoRI-digested pSL103 is subjected to the annealing and ligation procedure and used to transform B. subtilis 168 to neomycin resistance, the original vector plasmid can be removed from the constructed trp derivative. Occasionally trp plasmids constructed using EcoRI retain only a single EcoRI site. Thus digestion of one such plasmid, pSL101, with EcoRI generates a single linear modecule of the same molecular weight as intact pSL101. The reason for the occurrence of constructed plasmids retaining only a single EcoRI site is not known. Construction of trp plasmids using BamHI endonuclease has in our hands always yielded trp plasmids that retain only a single Bam HI site (Table V). Comparison of the inferred molecular weights of BamHIgenerated trp segments with the molecular weights oftrp segments cloned using EcoRI suggests that BamHI-generated fragments are larger. Plasmid pUB 110 and each of the trp derivatives are maintained in B. subtilis at approximately 50 copies per chromosome equivalent. Comparison of the Trp enzyme levels in B. subtilis cells carrying each of five constructed trp plasmids with the levels of the chromosomally specified Trp enzymes in strains harboring no plasmids generally confirms the genetic constitution of each plasmid initially deduced from complementation analysis 2~ (Tables III and IV). Plasmids that complement point mutations in individual trp genes specify the corresponding enzyme activities. The specific activities of plasmid-specified Trp enzymes in B. subtilis are significantly greater than the repressed levels of the chromosomally specified enzymes and equal to or below the derepressed levels of the chromosomally specified enzymes. Each of the constructed trp plasmids was initially selected for ability to complement a specific mutation in trpC (i.e., trpC2), and each of five trp plasmids tested contained a single HindIII-sensitive site. pUBI10 is z9K. M. Keggins, P. S. Lovett, R. Marrero, and S. O. Hoch, J. Bacteriol. 139, 1001 (1979).
B. subtilis AS A HOST FOR CLONING
[23]
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