Vol. 169, No. 11

Identification of Self-Transmissible Plasmids in FourBacillus thuringiensis Subspecies

AMALA REDDY,t LAURIE BATTISTI, AND C. B. THORNE*Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003

Received 4 May 1987/Accepted 21 August 1987

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis andBacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr).Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency alongwith a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14,pXO15, and pXO16, originating from B. thuringiensis subsp. morisoni, B. thuringiensis subsp. toumanoffl, B.thuringiensis subsp. alesti, and B. thuringiensis subsp. israeknsis, respectively, were demonstrated to beresponsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability,while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids conferconjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained bythe isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of theseplasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively,from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridizationexperiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp.thuringiensis conjugative plasmids pXOll and pXO12. Matings performed between strains which eachcontained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in manyinstances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16transfer appeared to be enhanced.

The reports by Gonzalez and co-workers (10-12) of aBacillus mating system prompted us to investigate whetherwe could use it to facilitate genetic analyses of variousBacillus plasmids. Our laboratory strains of Bacillusthuringiensis were screened for their donor ability in matingswith Bacillus anthracis and Bacillus cereus recipients. Theplasmids present in the potential B. thuringiensis donorswere cryptic; hence, to monitor plasmid transfer duringconjugation, we introduced the selectable B. cereus plasmidpBC16 (2.8 megadaltons), which encodes tetracycline resis-tance (3), into each strain by CP-51-mediated transduction(29). Our survey showed that 6 of 12 B. thuringiensis strainsin our collection, representing five subspecies, were effec-tive donors of pBC16 when grown in mixed culture with B.anthracis or B. cereus recipients (2, 34). Two large plasmids,pXOll and pXO12, from B. thuringiensis subsp. thuringi-ensis 4042A were characterized as being able to mediateconjugal transfer of themselves and other plasmids at highfrequencies (2). pXO12 was also found to carry genesinvolved in crystal toxin production.The present study was undertaken with the idea of iden-

tifying and characterizing the plasmids which were respon-sible for donor activities of the other strains which wereeffective in our screening tests: B. thuringiensis subsp.morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringi-ensis subsp. alesti, and B. thuringiensis subsp. israelensis.When matings were performed with these strains as donors,each strain was found to harbor a unique large plasmid whichwas transmitted at a high frequency to recipients. Theseplasmids were named pXO13, pXO14, pXO15, and pXO16.Experiments presented here demonstrate an association

* Corresponding author.t Present address: Department of Biological Sciences, Wellesley

College, Wellesley, MA 02181.

between the presence of these plasmids in a strain and itscorresponding donor ability. The results indicate that theselarge B. thuringiensis plasmids are self-transmissible plas-mids which can also mobilize pBC16 and other plasmids.Several of the B. thuringiensis self-transmissible plasmidsisolated in our laboratory were examined for DNA homologyand entry exclusion effects, and evidence that the plasmidsare distinct but related is presented. The plasmid-encodedmating system provides a method for shuttling plasmidsamong strains of B. cereus, B. anthracis, and B. thuringi-ensis.

MATERIALS AND METHODS

Organisms. The strains of B. thuringiensis, B. cereus, andB. anthracis used in this study and their relevant character-istics and plasmids are listed in Table 1. Transcipient strainsgenerated in mating experiments are not included in the listof strains. They are identified throughout the paper byinclusion of the abbreviation tr in the strain designations.The bacteriophages that were used, CP-51 and CP-54, havebeen described previously (32, 33).

Media. L broth, brain heart infusion (BHI) broth, andpeptone diluent were prepared as described previously (2,33). The minimal medium (MinIC [2]) was supplemented as

appropriate with the required amino acids, purines, orpyrimidines at a concentration of 40 jig/ml. For solid media,15 g of agar (Difco Laboratories, Detroit, Mich.) was addedper liter of the appropriate broth. Streptomycin and nalidixicacid were used at final concentrations of 200 and 30 ,ug/ml,respectively. Tetracycline was used at a concentration of 5or 25 jig/ml as explained previously (2). Casamino Acids(CA) broth was prepared as described previously (35),except that activated charcoal was omitted from the me-dium. CA-agarose was prepared by adding the following

5263

JOURNAL OF BACTERIOLOGY, Nov. 1987, p. 5263-52700021-9193/87/115263-08$02.00/0Copyright X) 1987, American Society for Microbiology

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

5264 REDDY ET AL.

TABLE 1. Bacterial strains

Strain Relevant characteristicsa Relevant plasmid(s) Originb

B. thuringiensis subsp. morrisoni4049 pXO13 NRRL4049 UM1 Tcr pXO13, pBC16 Td of 4049

B. thuringiensis subsp. toumanoffi4059 pXO14 NRRL4059 UM1 Tcr pXO14, pBC16 Td of 4059

B. thuringiensis subsp. alestiYAL pXO15, pXO31 A. YoustenYAL UM1 Tcr pXO15, pXO31, pBC16 Td of YAL

B. thuringiensis subsp. israelensisBIS pXO16, pXO39 M. deBariacBIS UM1 Tcr pXO16, pXO39, pBC16 Td of BIS

B. cereus569 Wild type pXO3, pXO4, pXO5 NRRL569 UM20 Ant- pXO3 (pXO4, pXO5)- UV of 569569 UM20-1 Ant- Stef pXO3 UV of IJM2O

B. anthracisWeybridge Avirulent pXOl MREWeybridge UM44 Ind- pXOl UV of WeybridgeWeybridge UM44-1 Ind- Strr pXOl UV of UM44Weybridge A Colonial variant of Weybridge pXo0 C. B. ThorneWeybridge A UM23 Ura- pXOl UV of Weybridge AWeybridge A UM23C1 Ura- (pXOl)- C. B. ThorneWeybridge A UM23C1-1 Ura- Strr (pXOl)- UV of UM23C14229 Cap' pXO2 ATCC4229 UM12 Cap' Nalr pXO2 UV of 4229

a Abbreviations: Ant, anthranilic acid; Ura, uracil; Ind, indole, Cap, synthesis of capsule; NaIr, nalidixic acid resistant; Strr, streptomycin resistant; Tcr,pBC16-encoded tetracycline resistance.

b NRRL, Agricultural Research Service, Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill.; Td, transduction by phageCP-51 (29); UV, mutagenesis by UV light (33); MRE, Microbiological Research Establishment, Porton, England; ATCC, American Type Culture Collection,Rockville, Md.

components to 100 ml of CA broth: 0.75 g of agarose(International Biotechnologies, Inc., New Haven, Conn.), 8ml of 9% NaHCO3, 6 ml of goat antiserum to B. anthracis,and 10 ml of horse serUm (GIBCO Laboratories, GrandIsland, N.Y.). The antiserumn, which was supplied by per-sonnel of the Bacteriology Division of the U.S. ArmyMedical Research Institute of Infectious Diseases, FortDetrick, Frederick, Md., was prepared in goats by theinjection of viable spores of the B. anthracis Weybridgestrain.Mating procedure. The cultural conditions and procedures

for broth matings with Bacillus strains were as describedpreviously (2). Mating mixtures were incubated at 30°C for16 to 20 h. The use of auxotrophic and drug-resistant strainsallowed unambiguous strain selection and identification inthe matings.

Replica plate mating. A replica plate mating technique wasdeveloped to test large numbers of Tcr B. thuringiensiscolonies for fertility. Colonies to be tested were picked toBHI agar to form master plates. These were incubated for 16h at 30°C, and the colonies were replica plated to 13HI agarplates that had been spread with 0.1 ml of spores (approx 108CFU) of the B. cereus 569 UM20-1 Strr recipient. The plateswere incubated for 16 h at 30°C, growth was replica plated toL agar plates containing streptomycin and tetracycline, andincubation at 30°C was continued. After 16 to 20 h, patchesof transcipient growth were present in areas correspondingto particular B. thuringiensis colonies on the master platethat had donor ability.

Detection of pXOl and pXO2 transfer. Transcipients con-taining pXO1 were detected by a screening test for synthesisof the protective antigen (PA) component of anthrax toxin(19). Colonies were picked to CA-agarose plates which were

incubated for 12 to 24 h in an atmosphere of 20% C02 (26).PA synthesis by a colony was indicated by the presence of ahalo.

Transcipients that inherited pXO2 and were thus Cap+were selected on the basis of their resistance to bacterio-phage CP-54 (14).

Plasmid-curing procedures. B. thuringiensis donor strainswere subjected to plasmid-curing procedures to eure them ofresident plasmids. A 250-ml Erlentneyer flask containing 25ml of NBY broth was inoculated with a loopful of spores andincubated with shaking at 42°C for 24 h. Growth wasstreaked on an NBY agar plate, which was then incubated at30°C overnight. Colonies were purified and screened forplasmid loss. Growing cells at 42°C was also very useful forcuring strains of pBC16.

Strains were also grown in the presence of novobiocin toobtain plasmid-cured derivatives different from those ob-tained by growing cells at 42°C. Spores were inoculated into250-ml flasks containing 50 ml of L broth with 1 ,ug ofnovobiocin per ml. The flasks were incubated at 37°Covernight and then at 30°C until the cultures sporulated.

Plasmid DNA extraction and analysis. Plasmid DNA wasextracted by a mtodification of the procedure of Kado andLiu (15) described previously (2). Cells for plasmid extrac-tion were grown in 250-ml Erlenmeyer flasks containing 25mnl of BHI broth supplemented with 0.1% glycerol for B.thuringiensis and B. cereus or with 10% (vol/vol) horseserum (GIBCO Laboratories) for B. anthracis strains. Ex-tracts were analyzed by agarose gel electrophoresis asdescribed previously (2).

Plasmid DNA extracts obtained by the above procedurewere not suitable for restriction endonuclease analysis orradiolabeling because they contained large atnounts of chro-

J. BACTERIOL.

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

SELF-TRANSMISSIBLE PLASMIDS OF B. THURINGIENSIS 5265

TABLE 2. Comparison of B. thuringiensis donors of pBC16 inmating mixtures with B. cereus and B. anthracis recipientsa

No. of Tcr transcipients (CFU/ml)with recipient:

Tcr donor strainbB. cereus 569 B. anthracis WeybridgeUM20-1 Strr UM44-1 Strr

4049 UM1 1.2 x 105 5.8 x 1044059 UM1 2.4 x 104 8.2 x 103YAL UM1 2.5 x 105 2.3 x 102BIS UM1 1.0 x 104 2.6 x 101a Transcipients were selected on L agar containing tetracycline and strep-

tomycin. Control tubes in which each strain was incubated with 1 ml of BHIbroth yielded no spontaneous Tcr Strr colonies. The numbers of transcipientsare averages of results from at least three experiments.

b Strains are identified in Table 1.

mosomal DNA. We eliminated this problem by modifyingthe published procedure (2) slightly. Cells were incubated inlysis buffer (12) for 1 h at 60°C, and any unlysed cells wereremoved by centrifugation at 10,000 rpm for 10 to 15 min.The supematant fluid was chilled on ice for 5 min, and thetreatment was continued as before.To obtain sufficient pXOll DNA for 32p labeling, the

modified procedure was scaled up and applied to a 400-mlculture of a B. anthracis transcipient cured of all plasmidsexcept pXO11. Plasmid DNA from the resulting lysate wasconcentrated by precipitating it with ethanol and dissolvingin 4 ml of 0.1 x SSC buffer (0.015 M NaCl, 0.0015 M sodiumcitrate, pH 7). The DNA was purified by isopycnic centrif-ugation in cesium chloride gradients.

Restriction endonucleases were purchased from Interna-tional Biotechnologies, Inc., and used as recommended bythe supplier. Digests were examined by electrophoresis on0.7% agarose gels.

Hybridization procedures. pXOll DNA was radiolabeledin vitro by nick translation (28) with [a-32P]dCTP purchasedfrom New England Nuclear Corp., Boston, Mass., and a kitobtained from Bethesda Research Laboratories, Inc.,Gaithersburg, Md. The specific activity of the resultinglabeled DNA ranged from 1 x 107 to 3 x 107 cpm/,ug.Plasmid DNA restriction fragments separated on a 0.7%agarose gel were transferred to nylon membranes by theSouthern blotting technique (30). GeneScreen Plus nylonmembranes were obtained from New England Nuclear. Priorto DNA hybridization, the nylon membranes were wettedfor 6 to 12 h at 60°C in a prehybridization solution containingDenhardt solution (7), 0.05 M Tris hydrochloride (pH 7.5), 1M NaCl, 1% sodium dodecyl sulfate, and 250 ,ug of dena-tured calf thymus DNA (type V; Sigma Chemical Co., St.Louis, Mo.) per ml. DNA-DNA hybridization was per-formed at 60°C for 24 h with continuous agitation in asolution containing the same components as the prehybrid-ization solution plus ca. 0.5 jig of denatured 32P-labeledprobe DNA. Filters were then washed twice under each ofthe following conditions: 2x SSC for 5 min at room temper-ature; 2x SSC containing 1% (wt/vol) sodium dodecyl sul-fate for 30 min at 60°C; and 0.lx SSC for 15 min at roomtemperature. After the washes, filters were air dried andautoradiographed at -70°C for 4 h with Kodak XAR-5 filmand a Du Pont Cronex Lightning-Plus intensifying screen.

RESULTS

Transfer of pBC16 by the four B. thuringiensis donors. Theresults in Table 2 demonstrate the ability of four B. thuringi-

ensis strains to transfer the tetracycline resistance plasmidpBC16 to B. cereus and B. anthracis recipients. In thesematings the B. cereus recipients were anthranilic acidauxotrophs and the B. anthracis recipients were indoleauxotrophs. Thus, the identity of the tetracycline-resistanttranscipients obtained could be verified on the basis of theirauxotrophic markers. The possibilities that plasmid transferwas phage mediated or due to transformation were elimi-nated as described previously (2).The average number of B. thuringiensis donors at the time

of sampling was 5 x 107 CFU/ml, and that of the recipients,except in matings with B. thuringiensis subsp. israelensis,was 4 x 108 CFU/ml for B. cereus and 4 x 107 CFU/ml forB. anthracis. In matings with that subspecies as the donor,growth of recipients was inhibited; the average number wasreduced to about 107 CFU/ml for B. cereus and about 106CFU/ml for B. anthracis. Frequencies of Tcr transcipientsper donor ranged from 2 x 10-4 to 5 x 10-3 in matings withB. cereus as the recipient and from 5 x 10-7 to 1 x 10-' inmatings with B. anthracis as the recipient. Generally lowerfrequencies of pBC16 transfer were obtained with B. anthra-cis than with B. cereus as a recipient. This effect wasespecially marked with B. thuringiensis subsp. israelensisand B. thuringiensis subsp. alesti donors, for which therewas a 400- to 1,000-fold decrease in the number of Tcrtranscipients per milliliter with B. anthracis.

Plasmid analysis and fertility properties of transcipients.We examined the plasmid profiles and fertility properties ofnumerous Tcr transcipients obtained from matings betweenB. thuringiensis donors and B. cereus and B. anthracisrecipients. Figure 1 shows the plasmids present in eachdonor and some representative transcipients derived fromeach. All transcipients examined showed the presence ofplasmid DNA that had the same mobility as pBC16 presentin the donor strains. Thus, the occurrence of Tcr tran-scipients correlated precisely with the acquisition of pBC16from the donors. Plasmid profiles of B. cereus transcipientsall showed the presence of the resident plasmid, pXO3,which migrated below the broad diffuse band of chromo-somal DNA.

Transcipients inherited various plasmids from the B.thuringiensis donors. Distribution of the low-molecular-weight plasmids was fairly random. There was a particularhigh-molecular-weight plasmid in each B. thuringiensisstrain that was transferred at a high frequency to B. cereus orB. anthracis transcipients. These large plasmids, originatingfrom strains 4049, 4059, YAL, and BIS (Table 1), werenamed pXO13 (ca. 72 megadaltons), pXO14 (ca. 54 mega-daltons), pXO15 (ca. 50 megadaltons), and pXO16 (>120megadaltons), respectively. The frequencies of cotransfer ofpXO13, pXO14, or pXO16 with pBC16 were between 96 and100%. pXO15 was acquired by 67% of the B. cereus Tcrtranscipients. None of the B. anthracis Tcr transcipientsexamined had pXO15, although some of them did inheritother large plasmids from B. thuringiensis subsp. alesti (Fig.1B, lane 4).The B. anthracis recipient used in these matings (Fig. 1)

contained the toxin plasmid pXO1 (112 megadaltons). Whilemost of the B. anthracis transcipients retained pXO1, sev-eral contained a slightly larger plasmid (Fig. 1A, lane 8, andFig. 1B, lane 4) that did not comigrate with any of the parentB. thuringiensis plasmids. When transcipients containing thepresumed altered pXO1 plasmid were picked to CA-agaroseplates that contained antiserum to the anthrax toxin, allcolonies produced a halo indistinguishable from that pro-duced by strains harboring pXO1.

VOL. 169, 1987

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

5266 REDDY ET AL.

A

BT BC BC BAE. 1 _ XL

BT BC BA BA

B

BT BC BC BA B T BC BA

FIG. 1. Agarose gel electrophoresis of plasmid DNA from B. thuringiensis donors and B. cereus and B. anthracis transcipients. pXOplasmids are labeled with their appropriate isolation numbers, e.g., pXO13 is shown as 13. The large diffuse band in all lanes is chromosomalDNA. Fertility of the strains is indicated above the lanes (+, fertile; -, nonfertile). (A) Lanes: 1, B. thuringiensis subsp. morrisoni 4049 UM1,donor; 2, B. cereus 569 UM20-1 trll2A-21, transcipient; 3, B. cereus 569 UM20-1 trll2A-10, transcipient; 4, B. anthracis Weybridge UM44-1trl68A-2, transcipient; 5, B. thuringiensis subsp. toumanoffi 4059 UM1, donor; 6, B. cereus 569 UM20-1 trll3A-8, transcipient; 7, B. anthracisWeybridge UM44-1 trl69A-4, transcipient; 8, B. anthracis Weybridge UM44-1 trl69A-3, transcipient. (B) Lanes: 1, B. thuringiensis subsp.alesti YAL UM1, donor; 2, B. cereus 569 UM20-1 trll4A-8, transcipient; 3, B. cereus 569 UM20-1 trll4A-7, transcipient; 4, B. anthracisWeybridge UM44-1 trl72A-8, transcipient; 5, B. thuringiensis subsp. israelensis BIS UM1, donor; 6, B. cereus 569 UM20-1 trll5A-20,transcipient; 7, B. anthracis Weybridge UM44-1 trl73A-7, transcipient.

Several B. cereus and B. anthracis transcipients were

tested for their ability to act as plasmid donors (Table 3).Transcipients containing only the B. thuringiensis plasmidsthat migrated below chromosomal DNA in agarose gels werenot fertile, e.g., B. cereus UM20-1 trll2A-10 (Fig. 1A, lane3). Tcr transcipients containing pXO13, pXO14, pXO15, orpXO16 were effective donors of pBC16 in mating mixtureswith other B. cereus or B. anthracis recipients. B. anthracistranscipients that inherited other large plasmids from B.thuringiensis subsp. alesti, e.g., B. anthracis WeybridgeUM44-1 trl72A-8 (Fig. 1B, lane 4), were not able to mobilizepBC16. Transcipients inheriting pXO16 were more efficientdonors than the parent strain, B. thuringiensis subsp.israelensis BIS UM1, in which pXO16 was originally found.However, this is probably a reflection of the fact that, unlikethe BIS UM1 strain, the pXO16-containing transcipients didnot inhibit the growth of recipients in mixed culture. Earlierobservations in our laboratory had confirmed that pBC16 byitself does not confer fertility on a host strain (2). We wereunable to cure B. cereus of its resident plasmid, pXO3.However, strains containing only pXO3 and pBC16 were notfertile (2).Donor ability of the various transcipients was stably

maintained during subsequent growth and sporulation, andplasmid analysis confirmed that Tcr transcipients generatedfrom further matings inherited pBC16 as well as otherplasmids derived from the original parent B. thuringiensis

donor strains. Plasmids pXO13, pXO14, and pXO16 were

transferred at high frequencies to both B. cereus and B.anthracis. pXO15 was transferred at a high frequency to B.cereus, but its presence in B. anthracis transcipients wasnever observed.Evidence for the self-transmissible nature of pXO13,

pXO14, pXO15, and pXO16. Based on the above observa-tions, there was a correlation between the donor ability of atranscipient and the presence of pXO13, pXO14, pXO15, orpXO16. To show that these plasmids were unquestionablyself-transmissible, it was necessary to demonstrate that thepresence of each of them individually, in the absence ofother B. thuringiensis plasmids, could confer conjugativefunctions. Since most of the transcipients we isolated con-tained lower-molecular-weight B. thuringiensis plasmids inaddition to the large transmissible plasmids, it was possiblethat the former encoded conjugative functions or that theirpresence in conjunction with pXO13, pXO14, pXO15, orpXO16 conferred fertility. We were also interested inwhether any of the four B. thuringiensis donors containedmore than one conjugative plasmid, as was the case with B.thuringiensis subsp. thuringiensis 4042A, which harboredtwo, pXOll and pXO12 (2). Because pXOll was transferredfrom strain 4042A at a higher frequency than pXO12, theconjugative ability of pXO12 initially escaped notice.We addressed both of these problems by subjecting the B.

thuringiensis donors to novobiocin treatment or growth at

StrainFertility

J. BACTERIOL.

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

SELF-TRANSMISSIBLE PLASMIDS OF B. THURINGIENSIS 5267

TABLE 3. Effectiveness of various B. cereus and B. anthracistranscipients as donors in the transfer of pBC16a

No. of Tcr transcipientsDonor strain derivative (CFU/ml) with

EXptb (plasmids) recipient:B. cereus B. anthracis

A B. cereus 569 UM20-1trll2A-10 (pXO3, pBC16) 0 0trll2A-21 (pXO3, pXO13, pBC16) 2.7 x 103 NTctrll3A-8 (pXO3, pXO14, pBC16) 4.6 x 103 NTtrll4A-7 (pXO3, pBC16) 0 0trll4A-8 (pXO3, pXOl5, pBC16) 1.3 x 104 NT

B. anthracis Weybridge UM44-1tr168A-2 (pXO1, pXO13, pBC16) 2.5 x 104 1.6 x 104trl69A-3 (pXO1,* pBC16)d 0 0tr169A-4 (pXO1, pXO14, pBC16) 1.3 x i05 6.9 x 104trl72A-8 (pX01,* pBC16, pX031) 0 0trl73A-7 (pXO1, pXO16, pBC16) NT 4.2 x 104

B B. cereus 569 UM20-1tr242A-1 (pXO3, pXO13, pBC16) 1.4 x 103 1.6 x 103tr300A-1 (pXO3, pXO14, pBC16) 1.3 x 103 3.0 x 102trl15A-20 (pXO3, pXO16, pBC16) 1.5 x 105 4.7 x 103

B. anthracis Weybridge A UM23Cl-ltr359A-1 (pXO13, pBC16) 1.6 x 104 NTtr338A-1 (pXO14, pBC16) 1.2 x 104 NTtr336A-4 (pXO16, pBC16) 3.5 x 103 NT

a Transcipients were selected on MinIC containing tetracycline and thespecific nutrient required by the recipient. The values are averages from atleast two experiments.

b Donor strains in section A of the table contained various unnamed B.thuringiensis plasmids in addition to the indicated plasmids. Those in sectionB contained only the indicated plasmids.

c NT, Not tested.d The asterisk denotes the altered plasmid in transcipients which synthe-

sized PA and contained a plasmid with a mobility close to, but not identical to,that of pXO1.

42°C to cure them of particular plasmids. We looked for acorrelation between the fertility of a particular cured strainand its specific plasmid content. The replica plate matingmethod enabled us to screen large numbers of cured isolatesfor those which had totally lost their donor ability. PlasmidDNA extracts from such strains lacked any detectablepXO13, pXO14, pXO15, or pXO16 DNA. The results sug-gested that pXO13, pXO14, pXO15, and pXO16 were theonly B. thuringiensis plasmids in their respective donorstrains that were self-transmissible. Strains containing vari-ous combinations of other B. thuringiensis plasmids werenot fertile. For example, Fig. 2 shows the plasmids presentin B. thuringiensis subsp. alesti YAL UM1 (lane 8) whichwas cured of pXO15 and was no longer able to transferpBC16 to recipients. The isolation of this cured strain whichretained all the B. thuringiensis subsp. alesti YAL plasmidsexcept pXO15 also provided evidence that only pXO15 isresponsible for conjugation functions in B. thuringiensissubsp. alesti.By using B. thuringiensis donors which were cured of

specific low-molecular-weight plasmids, we were able toisolate B. cereus transcipients which contained only pXO3,pBC16, and one of the implicated conjugative plasmids andB. anthracis transcipients which contained only pBC16 andone of the implicated conjugative plasmids. In Fig. 2, lanes2, 4, and 6 show the plasmid profiles of B. cereustranscipients which contained pXO13, pXO14, or pXO16.We were unable to isolate a transcipient that contained only

pXO15 and pBC16. Notwithstanding the absence of other B.thuringiensis plasmids, these transcipients were able totransfer pBC16 and the respective conjugative plasmids torecipients (Table 3, footnote b). The fact that B. anthracistranscipients which contained only pBC16 and one of the B.thuringiensis conjugative plasmids were fertile confirmedthat pXO3 was not responsible for the donor ability of B.cereus transcipients.

Correlation of parasporal crystal production with specificplasmids. In many B. thuringiensis strains, the formation ofparasporal crystals is associated with specific plasmids (1, 9,12, 13, 16, 17). None of the self-transmissible plasmidsidentified in this study encoded crystal toxin synthesis, asdemonstrated by the absence of crystals in B. cereus and B.anthracis transcipients during sporulation. B. thuringiensisstrains cured of their resident conjugative plasmids were stillCry', indicating that pXO13, pXO14, pXO15, or pXO16 wasnot required for crystal formation. This is in contrast to thefinding (2) that the conjugative plasmid pXO12 does encodecrystal formation, as evidenced by the fact that B. cereusand B. anthracis transcipients which inherited pXO12 pro-duced parasporal crystals.When cured B. thuringiensis subsp. israelensis BIS UM1

isolates were examined for production of parasporal inclu-

FIG. 2. Agarose gel electrophoresis of plasmid DNA from vari-ous B. cereus strains which were isolated to demonstrate thatpXO13, pXO14, pXO15, and pXO16 are self-transmissible plasmids.Extracts of the B. thuringiensis strains from which the self-transmissible plasmids were derived are shown also for purposes ofcomparison. Plasmid designations are the same as in Fig. 1. Lanes:1, B. thuringiensis subsp. morrisoni 4049 UM1; 2, B. cereus 569UM20-1 tr242A-1(pXO13, pXO3, pBC16); 3, B. thuringiensis subsp.toumanoffi 4059 UM1; 4, B. cereus 569 UM20-1 tr300A-1(pXO3,pXO14, pBC16); 5, B. thuringiensis subsp. israelensis BIS UM1; 6,B. cereus 569 UM20-1 trll5A-20(pXO3, pXO16, pBC16); 7, B.thuringiensis subsp. alesti YAL UM1; 8, B. thuringiensis subsp.alesti YAL UM1 (pXO15)-.

VOL. 169, 1987

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

5268 REDDY ET AL.

FIG. 3. Agarose gel electrophoresis of plasmid DNA from therelevant strains used to demonstrate mobilization of pXOl andpXO2 by pXOl4. Plasmid designations are the same as in Fig. 1.Lanes 1 to 4 show plasmids in strains used to demonstrate transferof pXOl, and lanes 5 to 7 show plasmids in strains used todemonstrate transfer of pXO2. Lanes: 1, B. thuringiensis subsp.t~oumanoffi 4059 UM1(pXOl4, pBC16), donor; 2, B. anthracisWeybridge UM44-1(pXOl), recipient; 3, B. anthracis WeybridgeUM44-1 trl69A-4(pXOl, pXO14, pBC16), transcipient derived froma mating between the strains in lanes 1 and 2; 4, B. anthracisWeybridge A UM23C1-1 tr357A-16(pXOl, pXO14, pBC16), tran-scipient derived from a mating between the strain in lane 3 as donorand the plasmid-cured strain B. anthracis Weybridge A UM23C1-1;5, B. anthracis 4229 UM12(pXO2), recipient; 6, B. anthracis 4229UM12 tr4O7A-7(pXO2, pXOl4, pBC16), transcipient derived from amating between the strains in lanes 1 and 5; 7, B. cereus 569 UM20-1tr4l2A-1(pXOl4, pXO2, pXO3, pBC16), transcipient derived from amating between the strain in lane 6 as donor and B. cereus 569UM20-1.

sions during sporulation, we noticed that derivatives whichwere cured of the third largest plasmid, named pXO39 (ca.80 megadaltons), differed from the parent strain. The curedstrains showed the presence of very small inclusions in thecytoplasm of sporulating cells, while pXO39-containingstrains produced large polymorphic crystalline inclusions inaddition to the smaller ones. pXO39 appeared to be a fairlyunstable plasmid, since spontaneously cured variants weredetected frequently by their altered colony morphology.

Mobilization of B. anthracis plasmids by pXOl4. As previ-ously reported, the B. thuringiensis subsp. thuringiensisconjugative plasmid pXOl2 can mobilize the B. anthracistoxin and capsule plasmids pXOl and pXO2 (2, 14, 34). Wehave now demonstrated that the B. thuringiensis subsp.toumanoffi 4059 plasmid pXOl4 can transfer pXOl andpXO2 from B. anthracis to plasmid-cured B. anthracis or B.cereus recipients more efficiently than pXOl2. Figure 3shows the plasmid profiles of the relevant strains used to

effect the mobilization of pXO1 and pXO2 by pXO14. Whenthe pXO14-containing B. anthracis donor, UM44-1 trl69A-4(Fig. 3, lane 3), was used, 6% of the Tcr transcipientsresulting from a mating with a plasmid-cured B. anthracisrecipient acquired pXO1. A representative transcipient isshown in lane 4 of Fig. 3.By mating a pXO14-containing donor with B. anthracis

4229 UM12 (Fig. 3, lane 5), we generated a transcipientwhich contained pXO14, pXO2, and pBC16 (lane 6). Whenthis strain was used as a donor to B. cereus 569 UM20-1, 1%of the transcipients displayed the Cap' phenotype charac-teristic of cells containing pXO2. Plasmid analysis confirmedthat such transcipients had acquired pXO2 (lane 7). Cellscarrying pXO16 were also able to transfer pXO2 to Cap-recipients, but at a lower frequency; only 0.25% of the Tcrtranscipients examined inherited pXO2. Tests for mobiliza-tion of pXO1 by pXO16 gave negative results. Similarly,pXO13 could not be demonstrated to mobilize pXO1 orpXO2. pXO15 was not tested for the ability to mobilize thetwo plasmids.DNA homology among self-transmissible B. thuringiensis

plasmids. Our laboratory has identified six different self-transmissible B. thuringiensis plasmids. Since these plas-mids are related in terms of their conjugative functions, wedecided to examine some of them for the presence ofhomologous DNA.

In tests for homology among plasmids pXO11, pXO12,pXO13, and pXO14, the plasmid DNAs were digested withrestriction endonuclease PstIl and transferred after electro-phoresis to a nylon membrane. The immobilized DNA wasprobed for hybridization with 32P-labeled pXOll DNA.Figures 4A and B show an agarose gel of restriction digestsand the corresponding autoradiograph. The results suggestthat DNA homology exists among the four self-transmissibleplasmids, but each displayed certain unique restriction frag-ments. The restriction patterns indicated that these plasmidswere not simple derivatives of each other. Preliminaryresults (L. Battisti and C. B. Thorne, unpublished data)indicate that for each plasmid, part of the homology can be

A B

1 2 3 4

a

S

U-

_ 4_

.d.o

FIG. 4. Demonstration of DNA homology among some of theself-transmissible B. thuringiensis plasmids. (A) Agarose gel elec-trophoresis of Pstl-cut plasmid DNAs. Lanes 1 to 4 have pXO13,pXO14, pXO12, and pXOll DNAs, respectively. (B) Autoradi-ograph of 32P-labeled pXO1l DNA hybridized to the restrictiondigests shown in panel A. Lanes 1 to 4 correspond to those shownin panel A.

J. BACTERIOL.

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

SELF-TRANSMISSIBLE PLASMIDS OF B. THURINGIENSIS 5269

attributed to the presence of the recently discovered B.thuringiensis transposon, Tn4430 (21).pBC16 transfer from donor to recipient strains when both

carry conjugative plasmids. It was of interest to determinewhether entry exclusion occurred between two strains car-rying the same or different conjugative plasmids. Strainscarrying the appropriate plasmids were mated, and thenumbers of Tcr transcipients per milliliter were scored. Forcomparison with the results from the various matings, therelative efficiency of transfer from a particular donor isexpressed as the number of Tcr transcipients obtained with arecipient containing a conjugative plasmid divided by thenumber obtained with the same recipient strain containingno conjugative plasmid. All five plasmids tested demon-strated entry exclusion when donor and recipient containedthe same conjugative plasmid (Table 4). The effect was mostpronounced with pXOl5. In matings with a pXO15-containing donor, there were 1,000-fold fewer Tcr transcip-ients when the recipient also contained pXO15 than whenthe recipient contained no conjugative plasmid. The inhibi-tion was obvious but less pronounced with plasmids pXO14,pXO16, pXO13, and pXO12, in order of decreasing effect.pXO1l was not tested for entry exclusion.

Also of interest in Table 4 is the demonstration that inseveral instances the presence of different conjugative plas-mids in the donor and recipient appeared to enhance theefficiency of plC16 transfer. It should be emphasized thateach of the values in Table 4 is based on averages of resultsfrom two or more independent experiments. The numbers ofdonor and recipient cells did not vary significantly from oneexperiment to another, nor was the number of donor orrecipient cells affected by their plasmid content. Therefore,the range of values for relative efficiency of transfer is not areflection of differences in the numbers of donor or recipientcells in the various mating mixtures.When several transcipients derived from matings between

pXO13- and pXO14-containing strains were examined forplasmid content, none was found that contained both pXO13and pXO14. However, from the other matings represented inTable 4, transcipients were found in which both parentalplasmids were present after at least 60 generations of growthin the absence of selection for their maintenance. Furtherexperiments to assess the compatibility of the fertility plas-mids have not been done.

DISCUSSIONA self-transmissible plasmid has been defined as one

which carries genes that determine the formation of specific

TABLE 4. Influence of conjugative plasmids in recipientcells on the transfer of pBC16

Relative efficiency of transfera whenConjugative plasmid recipient cells contained:

in Tcr donorpXO12 pXO13 pXO14 pXO15 pXO16

pXO12 0.2 11.6 1.7 NTb 4.8pXO13 3.9 0.05 2.8 1.0 5.8pXO14 1.4 3.6 0.01 1.5 3.5pXO15 0.7 NT 1.4 0.001 NTpXO16 2.4 1.8 1.5 1.4 0.03

a Reiative efficiency of transfer is expressed as the number of Tcrtranscipients obtained with a recipient containing the indicated plasmiddivided by the number obtained with the same recipient strain containing nofertility plasmid. Numbers in boldface type reflect reduced efficiency of BC16transfer when donor and recipient contained the same conjugative plasmid.

b NT, not tested.

cell contacts necessary for conjugation and whose DNA canbe prepared for transfer to a recipient cell (5). The resultspresented here demonstrate the existence of a self-transmissible plasmid in B. thuringiensis subsp. morrisoni4049, B. thuringiensis subsp. toumanoffi 4059, B. thuringi-ensis subsp. alesti YAL, and B. thuringiensis subsp.israelensis BIS. The plasmids have been named pXO13,pXO14, pXOl5, and pXO16, respectively. These plasmidswere identified by their ability to promote transfer of pBC16to B. cereus and B. anthracis recipients. The isolation ofnonfertile derivatives of each B. thuringiensis strain whichlacked the implicated self-transmissible plasmid is evidencethat these plasmids are responsible for eliciting the cellularcontacts required for transfer to occur. In addition, the factthat Tcr B. cereus or B. anthracis transcipients were fertilewhen they inherited only one B. thuringiensis plasmid, eitherpXO13, pXO14, or pXO16, indicates that these plasmids areconjugative.The mechanisms involved in producing effective cell-to-

cell contact between donors and recipients and the eventsleading to transmission of genetic material in Bacillusmatings are unknown. Neither donor nor recipient cellfiltrates had an effect on plasmid transfer by conjugationamong Bacillus strains (4), differentiating this mating processfrom that observed among some streptococci (6). In ourexperiments mating ability was monitored indirectly bymeasuring the transmission of the selectable plasmid pBC16.The fact that the transfer frequencies of pBC16 were veryreproducible suggests that this is an adequate measure of theexpression of functions related to conjugative plasmid trans-fer until a more direct technique is feasible.Many transcipients derived from mnatings performed with

a pXO1-containing B. anthracis recipient contained a plas-mid which encoded PA production but did not correspond toeither pXO1 or the donor plasmids. There are reports in theliterature presenting evidence of repetitive sequences andtransposonlike elements present on B. thuringiensis plas-mids (18, 20-24). Therefore, the variant plasmids mightresult from transposition of sequences from B. thuringiensisplasmids to pXO1. Brian Green in our laboratory has shownthat following mobilization by pXO12, pXO1 was altered andcontained some pXO12 sequences (personal communica-tion).None of the self-transmissible plasmids identified in this

study encodes crystal toxin synthesis. No property otherthan the ability to confer fertility has been assigned to any ofthem. However, the influence of another plasmid, pXO39,on crystal production by B. thuringiensis subsp. israelensisBIS was observed. There was a variation in the morphologyof crystals produced according to whether pXO39 waspresent in the strain. This result is consistent with thoseobtained by other investigators who have linked two dif-ferent plasmids with production of toxic crystalline inclu-sions and satellite inclusions in B. thuringiensis subsp.israelensis (12, 31).One of the most interesting questions arising from the

identification of six different self-transmissible plasmids re-gards their extent of relatedness. Their differences in mobil-ity in agarose gels show that they are not identical plasmidssimply isolated from different subspecies of B. thuringiensis,and examination of their DNA restriction patterns has led tothe conclusion that they are not related on the basis of mereduplications or deletions of DNA at one site. Althoughhybridization experiments suggested that DNA homologyexists among some of the plasmids, the presence of nonho-mologous DNA restriction fragments for each plasmid con-

VOL. 169, 1987

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

5270 REDDY ET AL.

firmed their nonidentity. The plasmids need to be tested forhybridization with specific DNA fragments associated withfertility functions before any conclusions may be made as towhether the presence of conserved sequences encodingconjugative functions contribute to their DNA homology.Research in this direction is in progress in our laboratory.We have isolated various transfer-deficient mutants ofpXO12 and are now in a good position to identify particularfragments involved in plasmid transmission.

Entry exclusion has been defined as interference by aresident plasmid with the entry of genetic material viaconjugation (27) and has been shown to occur in othermating systems (8, 25). The results presented here indicatethat entry exclusion can occur in Bacillus matings. Therewas a 20- to 1,000-fold decrease in the frequency of pBC16transfer between strains containing the same self-transmis-sible plasmid. However, in several instances the efficiency ofpBC16 transfer appeared to be enhanced when dornor andrecipient strains contained different conjugative plasmids.

ACKNOWLEDGMENT

This work was supported by contract DAMD17-85-C-5212 fromthe U.S. Army Medical Research Acquisition Activity.

LITERATURE CITED1. Aronson, A. I., W. Beckman, and P. Dunn. 1986. Bacillus

thuringiensis and related insect pathogens. Microbiol. Rev.50:1-24.

2. Battisti, L., B. D. Green, and C. B. Thorne. 1985. Mating systemfor transfer of plasmids among Bacillus anthracis, Bacilluscereus, and Bacillus thuringiensis. J. Bacteriol. 162:543-550.

3. Bernhard, K., H. Schrempf, and W. Goebel. 1978. Bacteriocinand antibiotic resistance plasmids in Bacillus cereus and Bacil-lus subtilis. J. Bacteriol. 133:897-903.

4. Chapman, J. S., and B. C. Carlton. 1985. Conjugal plasmidtransfer in Bacillus thuringiensis, p. 453-467. In D. R. Helinski,S. N. Cohen, D. B. Clewell, D. A. Jackson, and A. Hollaender(ed.), Plasmids in bacteria. Basic life sciences, vol. 30. PlenumPublishing Corp., New York.

5. Clark, A. J., and G. J. Warren. 1979. Conjugal transmission ofplasmids. Annu. Rev. Genet. 13:99-125.

6. Clewell, D. B. 1981. Conjugation and resistance transfer instreptococci and other Gram positive species: plasmids, sexpheromones and "conjugative transposons" (a review), p.191-205. In S. B. Levy, R. C. Clowes, and E. L. Koenig (ed.),Molecular biology, pathogenicity, and ecology of bacterial plas-mids. Plenum Publishing Corp., New York.

7. Denhardt, D. T. 1966. A membrane-filter technique for thedetection of complementary DNA. Biochem. Biophys. Res.Commun. 23:641-646.

8. Dunny, G. M., D. L. Zimmerman, and M. L. Tortorello. 1985.Induction of surface exclusion (entry exclusion) by Streptococ-cus faecalis sex pheromones: use of monoclonal antibodies toidentify an inducible surface antigen involved in the exclusionprocess. Proc. Natl. Acad. Sci. USA 82:8582-8586.

9. Faust, R. M., K. Abe, G. A. Held, T. Iizuka, L. A. Bulla, andC. L. Meyers. 1983. Evidence for plasmid-associated crystaltoxin production in Bacillus thuringiensis subsp. israelensis.Plasmid 9:98-103.

10. Gonzalez, J. M., Jr., B. J. Brown, and B. C. Carlton. 1982.Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains ofB. thuringiensis and B. cereus. Proc.Natl. Acad. Sci. USA 79:6951-6955.

11. Gonzalez, J. M., Jr., and B. C. Carlton. 1982. Plasmid transferin Bacillus thuringiensis, p. 85-95. In U. N. Streips, S. H.Goodgal, W. R. Guild, and G. A. Wilson (ed.), Genetic ex-change: a celebration and a new generation. Marcel Dekker,Inc., New York.

12. Gonzalez, J. M., Jr., and B. C. Carlton. 1984. A large transmis-sible plasmid is required for crystal toxin production in Bacillus

thuringiensis variety israelensis. Plasmid 11:28-38.13. Gonzalez, J. M., Jr., H. T. Dulmage, and B. C. Carlton. 1981.

Correlation between specific plasmids and delta-endotoxin pro-duction in Bacillus thuringiensis. Plasmid 5:351-365.

14. Green, B. D., L. Battisti, T. M. Koehler, C. B. Thorne, and B. E.Ivins. 1985. Demonstration of a capsule plasmid in Bacillusanthracis. Infect. Immun. 49:291-297.

15. Kado, C. I., and S.-T. Liu. 1981. Rapid procedure for detectionand isolation of large and small plasmids. J. Bacteriol. 145:1365-1373.

16. Klier, A., F. Fargette, J. Ribier, and G. Rapoport. 1982. Cloningand expression of the crystal protein genes from Bacillusthuringiensis strain berliner 1715. EMBO J. 1:791-799.

17. Kronstad, J. W., H. E. Schnepf, and H. R. Whiteley. 1983.Diversity of locations for Bacillus thuringiensis crystal proteingenes. J. Bacteriol. 154:419-428.

18. Kronstad, J. W., and H. R. Whiteley. 1984. Inverted repeatsequences flank a Bacillus thuringiensis crystal protein gene. J.Bacteriol. 160:95-102.

19. Leppla, S. H., B. E. Ivins, and J. W. Ezzell, Jr. 1985. Anthraxtoxin, p. 63-66. In L. Leive (ed.), Microbiology-1985. Amer-ican Society for Microbiology, Washington, D.C.

20. Lereclus, D., M.-M. Lecadet, J. Ribier, and R. Dedonder. 1982.Molecular relationships among plasmids of Bacillus thuringi-ensis: conserved sequences through 11 crystalliferous strains.Mol. Gen. Genet. 186:391-398.

21. Lereclus, D., J. Mahillon, G. Menou, and M.-M. Lecadet. 1986.Identification of Tn4430, a transposon of Bacillus thuringiensisfunctional in Escherichia coli. Mol. Gen. Genet. 204:52-57.

22. Lereclus, D., G. Menou, and M.-M. Lecadet. 1983. Isolation of aDNA sequence related to several plasmids from Bacillusthuringiensis after a mating involving the Streptococcusfaecalisplasmid pAM31. Mol. Gen. Genet. 191:307-313.

23. Lereclus, D., J. Ribier, A. Klier, G. Menou, and M.-M. Lecadet.1984. A transposon-like structure related to the delta-endotoxingene of Bacillus thuringiensis. EMBO J. 3:2561-2567.

24. Mahillon, J., J. Seurinck, L. V. Rompuy, J. Delcour, and M.Zabeau. 1985. Nucleotide sequence and structural organizationof an insertion sequence element (IS231) from Bacillus thuringi-ensis strain berliner 1715. EMBO J. 4:3895-3899.

25. Manning, P. A., and M. Achtman. 1979. Cell-to-cell interactionsin conjugating Escherichia coli: the involvement of the cellenvelope, p. 409-447. In M. Inouye (ed.), Bacterial outermembranes. John Wiley & Sons, Inc., New York.

26. Mikesell, P., B. E. Ivins, J. D. Ristroph, and T. M. Dreier. 1983.Evidence for plasmid-mediated toxin production in Bacillusanthracis. Infect. Immun. 39:371-376.

27. Novick, R. P. 1969. Extrachromosomal inheritance in bacteria.Bacteriol. Rev. 33:210-263.

28. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1980.Labelling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

29. Ruhfel, R. E., N. J. Robillard, and C. B. Thorne. 1984. Interspe-cies transduction of plasmids among Bacillus anthracis, B.cereus, and B. thuringiensis. J. Bacteriol. 157:708-711.

30. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

31. Tam, A., and P. Fitz-James. 1986. Plasmids associated with aphage-like particle and with a satellite inclusion in Bacillusthuringiensis ssp. israelensis. Can. J. Microbiol. 32:382-388.

32. Thorne, C. B. 1968. Transduction in Bacillus cereus and Bacil-lus anthracis. Bacteriol. Rev. 32:358-361.

33. Thorne, C. B. 1978. Transduction in Bacillus thuringiensis.AppI. Environ. Microbiol. 35:1109-1115.

34. Thorne, C. B. 1985. Genetics of Bacillus anthracis, p. 56-62. InL. Leive (ed.), Microbiology-1985. American Society for Mi-crobiology, Washington, D.C.

35. Thorne, C. B., and F. C. Belton. 1957. An agar-diffusion methodfor titrating Bacillus anthracis immunizing antigen and its appli-cation to a study of antigen production. J. Gen. Microbiol.17:505-516.

J. BACTERIOL.

on Decem

ber 25, 2019 by guesthttp://jb.asm

.org/D

ownloaded from