0021-9193/87/031147-06$02.00/0
Transformation of Vegetative Cells of Bacillus thuringiensis by
Plasmid
DNA
AGNETAHEIERSON, RITVA LANDEN, ANN LOVGREN, GUNNEL DALHAMMAR, ANDHANS G. BOMAN*
Departmentof Microbiology, University of Stockholm, S-106 91 Stockholm, Sweden Received 7October 1986/Accepted 9 December 1986
Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the
following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris
hydrochloridebuffer (pH 8.9) containing 30% sucrose.Transformationfrequency wasstronglyinfluencedby
culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a
minimal mediumsupplemented with Casamino Acidsgave35 timesmoretransformantsthangrowth inarich
medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was
10-3
transformants perviable cellor104
transformants per ,ug of DNA. Cellspreviously frozen were also used as recipients in transformation experiments; such cells gave frequencies
similartothoseobtained with freshlygrown cells. The procedurewasoptimized forB. thuringiensis subsp.
gekchiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast
transformation,ourmethod ismuchfaster and 3ordersof magnitudemoreefficientpermicrogramof added
DNA.
Muchofthe recent literature onBacillusthuringiensis has
been focused on the crystalline 8-endotoxin and on the
plasmid patterns (for reviews see references 2 and 7). The
facts that the 8-endotoxin is plasmid encoded and that the genehas beenclonedin Escherichiacoli andBacillussubtilis create aneed for a method to reintroduce cloned material
back into B. thuringiensis. Previously, transfer of genetic material in B. thuringiensis has been achieved by
transduc-tion(3, 11, 13, 14, 18, 20), by transformation of protoplasts, and by a "conjugation-like system" (2, 7). Previously
de-scribed methods for transformation ofB. thuringiensis have been based on a method developed for protoplasts of B.
subtilis (8). Using plasmid pBC16 isolated from Bacillus cereus(4),Alikhanianet al. (1)transformed protoplasts of B.
thuringiensis subsp. galleriae. Plasmid pC194, which
origi-natedfrom Staphylococcus aureus, has beentransferredto
protoplasts ofB. thuringiensis subsp. kurstaki (15), as well
as to some additional subspecies (9). Another S. aureus
plasmid, pUB110, has also been used to transform protoplasts ofB. thuringiensis subsp. galleriae (16). How-ever, in all cases the numbers of transformants were very
low,and theprocedures were timeconsuming.Moreover, it hasbeen difficultboth to prepare protoplasts and to regen-erate such protoplasts. For this reason we developed a
plasmid transformation method which, compared with
pre-viously described procedures, is much more efficient and
saves time.
Inthispaper we reporttransformation ofvegetative cells
ofB. thuringiensis. We used
plasmids
pBC16 andpC194
with B. thuringiensis subsp. gelechiae as the recipient and with all parameters optimized obtained transformationfre-quencies ofup to
io-3
(corresponding
to 104transformants per ,ugofDNA).*Correspondingauthor.
MATERIALS ANDMETHODS
Bacterial strains, plasmids and media used. The B. thuringiensis strains used inthispaper have beendescribed previously (13). Strain Bt213 (B. thuringiensis subsp.
kurstaki) isaspontaneous streptomycin- and nalidixic acid-resistantmutantofBt2(13). This strainwasselected firston platescontaining100 ,ug of streptomycin per ml and then on
plates containing25
jig
of nalidixic acid per ml. B. subtilis151,containingplasmidpBC16coding for tetracycline
resist-ance, was provided by W. Goebel. B. subtilis containing plasmid pC194 expressing chloramphenicol resistance was
provided by P. Martin. Bacteria were grown in minimal medium (20) which was prepared withoutglucose and was
supplemented with 0.5% (wt/vol) Casamino Acids (Difco Laboratories, Detroit, Mich.). When glucose was used, it was added to the minimalmediumto afinalconcentration of
5g/liter. Other growth mediaused were LB broth(13)and, for some experiments, brain heart infusion medium
(Oxoid
Ltd.,London, England).LBPmediumwas amixture(1:1) of phosphate buffer (0.2 M sodium-potassium phosphate, pH 6.4) and double-strength LB medium. The plates used for selection of transformants were LA plates (11) containing
eithertetracycline (25 ,uglml) orchloramphenicol (5
,ug/ml).
Trishydrochloride bufferwas prepared from Trizma
(Sigma
ChemicalCo., St. Louis, Mo.). Phosphate bufferanda40% polyethylene glycol 6000 solution (BDH Chemicals
Ltd.,
Poole, England)werepreparedasdescribed
previously (19).
Isolation ofplasmidDNAfrom B.subtilis. Cultures(200ml)
were grown overnight in LB mediumcontaining
eithertetracycline (25
jig/ml)
orchloramphenicol
(5jig/ml).
Cells wereharvestedbycentrifugation,washedoncewith TES(30
mM Trishydrochloride,
5 mM disodiumEDTA,
50 mMNaCl, pH 8.0), and suspended to a total volume of 8 ml in TES. Tothissuspensionwereadded 1.0 ml of TES
ing 5 mg offreshly dissolved lysozyme and 0.4 ml of TES
containing 0.8 mg of preboiled RNase. The mixture was
incubated for 30minat 37°C. The bacteriawere lysed bya
60-min incubationat37°C with 1.4 ml of 8%(vol/vol) Triton X-100 and 0.6 ml of TES containing 3 mg of pronase K
(predigested for 60 minat37°C). Ethidium bromide (0.6 ml; 10mg/ml) and solid CsClwereaddedtothelysate togivea
final refractive index of 1.391. The DNA was purified by ultracentrifugation, and the concentration was determined
by UV(260-nm) spectrophotometry. Generally,about100to 200 jigofpurified plasmid DNAwasobtained froma200-ml
culture.
PlasmidpBC16 DNAwascleaved with BamHI andligated
with T4ligase accordingto theinstructions of the manufac-turer(New EnglandBioLabs, Inc., Beverly, Mass.).
Standard procedure for transformation of B. thuringiensis by plasmid DNA. A 0.3-ml inoculumofanovernightculture was addedto 10 ml of minimal medium supplemented with 0.5%Casamino Acids. Thebacteriawere grownwith shak-ingat37°C until midorlatelogarithmic phase (80to175 Klett turbidity units, which corresponded to 1 x 107 to5 x 107
CFU/ml).Thebacteriawerecollected in a12-mlglass tube by centrifugation at3,700rpmfor 10 min atroom tempera-ture. All of the centrifugation procedures described below
were carried out in the same way. The cells were washed
with 5 ml of 50 mM Tris hydrochloride buffer (pH 7.5)and suspended in 50 mM Tris hydrochloride buffer containing 30% (wt/vol) sucrose (pH8.9) to adensity between 60 and 120 Klett turbidity units. A5-ml portionof this suspension
was transferred to a 50-ml Erlenmeyer flask and incubated
for 35minwith slowshakingat37°C.Thebacteriawerethen collectedby centrifugationandsuspendedin0.5 ml of LBP medium. Plasmid DNAdissolvedin 100,ul ofa 1:1 mixture of TE buffer (50 mM Tris hydrochloride, 20 mMdisodium EDTA, pH 8.0) and LBPmediumwasaddedtothis
suspen-sion. Aftertheaddition of1.5 ml ofpolyethylene glycol6000 (40%,wt/vol)andgentlemixing,thebacteriawereincubated for 10 min with slow shaking at 37°C. The cells were
centrifuged, suspendedin 1 mlof LBmedium,andincubated forphenotypic expressionat37°Cwith slowshakingfor 1.5 h. To isolate transformants, samples (0.02 to0.2 ml) were
spread in softagarontoLAplates containingeither tetracy-cline(25
jig/ml)
orchloramphenicol (5jxg/ml).
Ifphenotypic expressionwasdeterminedonregularLAplates,anoverlayofsoftagarcontaining the appropriate antibioticwasadded
afterincubation for 2.5 hat37°C. Colonieswerescored after
incubationfor 15 hat 37°C.
Freezingofbacteriatobeused intransformation. By using
a rotatory shaker at 37°C bacteria were grown to late logarithmic phase (140to 190 Klettturbidity units) in mini-malmediumsupplemented with 0.5% Casamino Acids. The culture was centrifuged for 10 min at 3,700 rpm, and the bacteriawere concentrated about five times inLB medium
containing 10% (vol/vol) glycerol. Samples (1.5 ml) were
frozen in liquid nitrogen and stored at -70°C. Before the bacteriawereused intransformationexperiments, theywere
washed once toremove the glycerol.
DetectionofplasmidDNA intransformants. Plasmid DNA
wasisolatedbyamodification of the protocol of Hansenand Olsen (10). Bacteriaweregrown in10 ml of LB medium at
37°Ctostationary phase, washedonce in TEbufferatroom
temperature, andsuspended in 0.3 ml of TE buffer contain-ing 25% (wt/vol) sucrose(pH 8.0). To these cells we added 0.2 ml ofTE buffer containing 1 mg offreshly dissolved
lysozyme and 50 U of mutanolysin (Sigma). The suspension
was incubated for 40 min at room temperature. After the
TABLE 1. EffectsofpHanddivalentcationson
transformationfrequency
pHof Tris Divalent cation Transformation
hydrochloride- addition frequencya
20 sucrose 7.50 None <1.0 x 10-8 8.00 None 5.0 x 10-7 8.45 None 1.6 x 10-6 8.75 None 2.8 x 10-6 8.90 None 5.1 x 10-6 9.50 None 2.3 x 10-6 8.90 20 mMMg2+ 2.1 x 10-8 8.90 20 mM
Ca2l
<4.0 x 10-8aTherecipient was strainBtl3,and the DNA used wasplasmid pBC16
codingfortetracyclineresistance. Thetransformationfrequencywas calcu-lated as the number of transformants perviable cell. In each transformation mixture there was 2 x 107 CFU, and 5 ,ug of pBC16 DNA was used. In experiments in which the pHwas7.50orthebufferwas supplemented with
Mg2+,108 viable cellswereused.
addition of 0.2 ml of 250mM disodium EDTA (pH 8.0), the mixture was put on icefor 10min. The bacteria werelysed
byadding 0.5 ml of 15% sodium dodecyl sulfate andheating
for 5 minin a55°C water bath with gentle inversions of the tube. After treatment with RNase (final concentration, 50
p.g/ml)for 5 minat roomtemperature,themembrane-bound chromosomal DNA was precipitated with 0.5 ml of 5 M NaCl, and the mixture was kept on ice overnight. The precipitated chromosomal DNA was spun down, and the supernatant, containingmainly plasmid DNA,was concen-tratedby addingpolyethylene glycol 6000(42%,wt/vol, in 10 mM phosphate buffer, pH 7.0) to a final concentration of 10%. The mixture was left on ice for at least 6 h, after which the DNAwas collected bycentrifugation and suspended in 25 ,ul of water. Plasmid DNA was analyzed on a 0.6% (wt/vol) agarose gel.
RESULTS
Needfor a Tris-sucrose treatment. Except when otherwise stated, in all transformation experiments we used plasmid
pBC16, which codes for tetracycline resistance. The
recipi-ent, B. thuringiensis strain Btl3, was first treated with Tris
hydrochloride buffer as describedby Takahashi etal. (19), but with a reduced incubation time. This procedure gave
only a few tetracycline-resistant transformants. When the pH wasvaried, thehighesttransformationfrequency, 5.1 x 10-6transformantper viable cell(transformationfrequency was always calculated as the number of transformants per
viable cell),was obtained atpH 8.9 (Table 1), a somewhat
higher pH than that used for transformation of Bacillus brevis (19). The increase in frequency was not due to a decrease in the numberof viable cells, whichwas the case for B. brevis. When the bacteria were treated with Tris hydrochloride buffer containing 20 mM MgCl2 or 20 mM
CaCl2, the transformationfrequencydecreasedby 2 orders ofmagnitude.
Sucrose added to the Tris hydrochloride buffer increased thetransformationfrequency from2.5 x 10-8toabout 2 x
10'
transformant per viable cell (Fig. 1). A plateau was reached when thefinalsucrose concentration was over 25%.Microscopic observations showed some protoplast forma-tion aftercentrifugation of theTris-sucrose-treatedcells. In 20% sucrose there were about 15% protoplasts, compared with 5%protoplasts in30% sucrose.
-2 I n a 0 c E 0 li a 10 -6 10 0 10 20 30 40 Concentrationof sucrose (%)
FIG. 1. Effect ofsucroseoninduction ofcompetence. Bacteria
were treated for 35 min with Tris hydrochloride buffer (pH 8.9)
containing different concentrations ofsucrose.The recipient, strain Btl3, had been grown in minimal medium contairiing 0.5% Casamino Acids. In each transformation mixture 5 jig ofp13C16 DNA, which conferred tetracycline resistance, was used. Each
valuerepresentstheaveragefromtwoexperiments.
The optimum time of incubation in Tris hydrochloride buffercontaining 30% sucrose was35 min (Fig. 2).
Experi-ments performed with bacteria previously frozen gave a
similaroptimum. '0, -4 ~i o 10 Go ..5. . 20 40>6 8 00~~~~~ E E ~ ~ o o 000
Incubation time in Tris-sucrose (mlin)
FIG. 2. Timedependencefor inductionofcompetenceby treat-ment withTris hydrochloride buffer (pH 8.9) containing 30% su-crose.TherecipientwasstrainBt13,whichwaseitherfreshlygrown in minimal mediumconitaining0.5%Casamino Acids(-0)orfrozen beforebeingtreated with Tris-sucrosebuffer(O). Ineach transfor-mation mixture 5jiwgofpBCl6 DNA,which conferredtetracycline resistance,wasused.
TABLE 2. Effect ofDNAconfigurationonthe transformation frequencya
pBC16 DNA No. of transformants Transformation
per 5 ,ug of DNA frequency
CCC 854 2.0 x 10-5
Multimers 114 2.7 x 10-6
Linear 8 1.9 x 10-7
No DNA 2 4.8 x 10-8
aTherecipient was strainBtl3,and the totalnumber of viable cells in each
transformation mixture was 4.2x10'. Thebacteria were grown in LBmedium and were treated with Trishydrochloride buffer (pH 8.9) containing 20% sucrose. The transformation frequency was calculated as the number of tetracycline-resistant transformants per viable cell. Plasmid pBC16 (CCC form) was linearized by cleavage with endonuclease BamHI.
Transformation frequency depends on DNA concentration and configuration. There was a linear dependence over a
20-fold concentrationrangeof added plasmid DNA.
Satura-tionwasslowly approachedwith 10 ,ugof pBC16 DNA (data not shown). Based on these results, we used in all
subse-quent transformation experiments 5 ,ug of DNA in a total
volume of0.5 ml. Table 2 shows that the highest transfor-mation frequency, 2.0 x 1i-0 transformant perviable cell, was obtained with covalently closed circular (CCC) DNA.
Linear plasmid pBC16 cleaved with BamHI endonuclease had almost no transformation activity. BamHI cleaves this
plasmid at only one site,wh'ichisoutside the gene coding for
tetracycline resistance (17). The religated plasmid DNA,
which contained a majority of multimeric forms (as judged
byagarosegel electrophoresis), showeda10-folddecrease in transformation frequency compared with the CCC DNA.
Transforrmation
experiments performed with chromosomal DNA in which we selected forresistance to streptomycin andrifampingave notransformants (datanotshown).Effect of growth medium on transformation frequency. When recipientstrainBt13 wascultivated inLBmediumor
brain heart infusion medium, the transformation frequency was
10-5
transformant per viable cell (Table 3). Minimalmedium supplemented with Casamino Acids gave a fre-quencywhichwas atleast 1 order of magnitudehigher. Not
onlythefrequenciesbut also the numbers of transformants were clearly higher (about 35 times) whenthe bacteria were grown in
minimal
medium. Addition ofglucose to minimalmedium decreased the frequency by about 2 orders of
magnitude compared with medium withoutglucose.
Influenceof culture density and growth phaseon the trans-formation frequency. The culture density was varied be-tween 15 and 300 Klett turbidity units during the Tris-sucrose treatment (Fig. 3). Early and late logarithmically growing bacteria were used. When these cultures were
TABLE 3. Effect of thegrowth mediumonthe transformation frequency
No. of
Mediu
Additiontransformants
Transformationper5,ugof frequencya
DNA
Minimal 0.5% Casamino Acids 10,500 1.5 x 1O-4 Minimal 0.5% Casamino Acids 830 4.4 x 10-6
and 0.5%glucose
Brain heart None 318 1.0 x 10-5
infusion
LB None 288 9.0 x 10-6
aTherecipientwas strainBtl3, and the plasmidused waspBC16. The
transformation frequency was calculated as the number of
tetracycline-resistant transformantsperviable cell.
10-71
CV t-Go c co E 0 c D .O-0 az z c 3 0 -0 (a 0 3 0 'I' CDcn o 0 z 10 30 100 300 10 30 100 300
Culturedensity during Tris treatment (Klettunits)
FIG. 3. (A) Effect of culture density on induction of competence by the Tris-sucrose treatment. The recipient was strain Btl3. Early-log-phase bacteria (0)wereharvestedat60Klettturbidityunits.Late-log-phase bacteria (A)wereharvested between 120 and 175 Klett
turbidity units. The density of stationary-phase bacteria (A)was240 Klettturbidity units.(B) Number of tetracycline-resistanttransformants per5,ug of pBC16 DNA. Eachvalue is theaveragefrom threeexperiments.
adjustedtothe different densities, asharp peakwasobtained with a maximum transformation frequency of 2 x 10-3
transformant per viable cell at 35 Klett turbidity units. If stationary-phase bacteriaweretreatedinthe sameway,the maximum was around 60 Klett turbidity units. The highest
number of transformants(1.3 x
104
transformantsper5,ug of DNA)wasobtained iflate-log-phase bacteriawereadjustedtodensities between35 and 120 Klettturbidity units. Trans-formation ofan overnight culture of strain Btl3 gave less than 10 transformants per p.gofpBC16 DNA.
Importanceof low culturedensity during DNA uptake. We determined whether the culturedensity wasimportant
dur-ing the Tris-sucrose treatment orduring some later step in the transformation protocol (Table 4). Regardless of the densities usedduring the Tris-sucrose treatment,thehighest transformation frequencies were obtained if the culture
density during DNA uptake waslow. Higher densities
de-creased the frequency by 2 orders of magnitude. These experimentswereperformed with cells thatwerepreviously
frozen. Compared with freshlygrown cells, where a maxi-mum transformation frequency was obtained at a density
correspondingto35Klettturbidityunits(asshown inFig. 3),
no such maximum was found for frozen cells. Instead, a
plateauwasreached when the culturedensitywasdecreased
further(datanotshown). Varying the culture density during phenotypic expression had no effect on the transformation
frequency (datanotshown).
If thepolyethylene glycol wasomitted, notransformants were obtained (data not shown). The length oftreatment with polyethylene glycol was not critical. Similar transfor-mationfrequencieswereobtained with bacteria incubatedin polyethylene glycol from5 up to30min.
Phenotypic expression onLA plates. The selected marker onpBC16 was tetracycline resistance, which requires time
for phenotypic expression. For this purpose we normally
usedliquid medium in which the shortest time for obtaining optimal transformation frequency was 1 h (Fig. 4).
Trans-formantscould also be obtained afterphenotypic expression
on regular LA plates. In this case the time needed for
expression of antibiotic resistancewas2 h (Fig. 4).
Transformation with pC194 DNA and transfer of pBC16 DNA to different B. thuringiensis subspecies. We also
per-TABLE 4. Transformation frequencyas a function of culture densityduring Tris-sucrose treatment and during uptake of DNA'
Culturedensity during Transformationfrequencyas afunction ofculturedensity duringDNAuptakeb
Tris-sucrose treatment
(Klettunits) 12 Klett units 60 Klett units 300 Klett units
12 9.3 x 10-4(495)C NTd NT
60 1.3 x 10-3 (918) 8.9 x 10-5(340) 3.0 x 10-6(48)
300 1.6x 10-3 (835) 3.4 x 10-5(583) 5.1 x 10-6(108)
aThe recipient, strainBtl3,was frozen before Tris-sucrose treatment. Thetransformationfrequency was calculated as the number of tetracycline-resistant
transformantsperviablecell.
bDifferentcelldensitieswere obtained by either diluting or concentrating the culture to the Klett unit values indicated.
cThe numbers in parentheses are numbers of transformants per 5 ,ug of pBC16 DNA.
formed transformation experiments with plasmid pC194 by using5 ,ug ofpC194DNA andstrain Bt13astherecipient.In one experiment 1.5 x 104 transformants were obtained,
whichcorrespondedto afrequency of 1.8 x
10-4
transform-ant per viable cell. Preparation oftotal plasmid DNA fromsix of these
chloramphenicol-resistant
transformants and electrophoretic analysis on agarose gels revealed the2.7-kilobase band corresponding to pC194. The plasmid was stably maintained and could be detected in transformants
grown in the absence ofchloramphenicol (datanot shown).
Transformation frequencieswere alsodeterminedfor four
other subspeciesof B. thuringiensis (B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis,and B. thuringiensis subsp. israelensis)
(Table
5).
The frequency obtained for strain Btl3 (B.thuringiensis subsp. gelechiae) was 2 to 3 orders of
magni-tude higher than the frequencies obtained for the other
subspecies tested. Strain Bt213 (B. thuringiensis subsp.
kurstaki) gave the highest transformation frequency' with Trishydrochloride buffer containing35% sucrose.StrainBt3
(B. thuringiensis subsp. galleriae) was grown in minimal medium supplemented with both Casamino Acids and 2%
(vol/vol) LB
medium.
To demonstrate the presence ofpBC16 in transformants fromthedifferent subspecies, plasmid-enrichedDNAs were
isolated from four different strains and compared with'a sampleof purified pBC16DNAbyusing agarose gel
electro-phoresis
(data
not shown). The recipient strains of B.thuringiensissubsp. gelechiae, B. thuringiensis subsp.
gal-leriae, B. thuringiensis subsp. israelensis, and B. thuringi-ensissubsp.kurstaki lacked the 4.2-kilobase band of pBC16, whereasin the four transformants fromthese
subspecies
thisbandwas clearlypresent. Plasmid pBC16was stably
main-tained and was also detected in transformants which were grownwithout'tetracycline (datanot shown).
DISCUSSION
The method which we describe for transformation of
vegetativecellsof B.thuringiensisrequires
optimization
of anumberof different parameters.One of these is the induction
-4 10 cultures .0 0E -6 r-0 10 -7 I 10 I 0 30 60 g0 120 150 180
Time forphenotypic expression (mmn)
FIG. 4. Transformation frequency as a function of time for phenotypic expressioninliquidLBmedium(0)or onLAplates(A). Therecipient strainwasBtl3, andin eachtransformationmixture 5
jig
ofpBC16DNA wasused.TABLE 5. Transformation of different subspecies ofB. thuringiensisa
Strain Subspecies Transformation
frequency
Bt13 B. thuringiensis subsp. gelechiae 1.5 x 1O-3 Bt213 B. thuringiensis subsp. kurstaki 7.3 x 10-5 Bt3 B. thuringiensis subsp. galleriae 3.8 x 10-5
Bt4 B. thuringiensis subsp. thuringiensis 4.1 x
iO-Btal B. thuringiensissubsp. israelensis 6.7 x 10-7
aThetransformationfrequencywascalculated asthe number of
tetracy-cline-resistant transformants per viable cell. In each transformation mixture 5 ,ugof pBC16 DNA was used.
of competence by treatment of the bacteria with
Tris-sucrose. In B. brevis treatment with Tris buffer without sucrose induces competence due to the removal of two
crystalline protein layers (S layers) fromthe outside ofthe
cell wall(21). However, in B.
thuringiensis
no S layers arefound (12). Thus, the mechanisms by which Tris treatment induces competence are differentinthe two species.
High concentrations ofsucrose duringtheTristreatment are necessary for a
high
transformation frequency (Fig'. 1).Sinceincubation of bacteria inanisotonicmedium has been reported to result in formation of protoplasts (2), we
sus-pectedthat we might have protoplast transformation. How-ever, this possibilitywasruledoutforthereasonsdescribed below. (i) Optimal transformation frequency was obtained with 30% sucrose, despite the fact that 20% sucrose gave
somewhat more protoplasts. (ii) There was no increase in
transformation frequency if experiments were performed with an osmotic support during the uptake of DNA and
during phenotypic expression (data not shown). (iii) We
obtained efficient transformation after phenotypic
expres-siononregularLAplates (Fig. 4), conditions which do not
allow protoplast regeneration. (iv) Transformants on LA
plates
could
be scoredafter7 to 8 hof incubation, whereasprotoplast regeneration
normally
requires 2 to 3 days(15).
For the reasons described above we are confident that wetransformedvegetative cells.
The addition ofglucosetothe minimalmedium decreased the transformation frequency by 2 orders of magnitude compared with medium without glucose. This suggests that
inB. thuringiensisthesynthesis ofsomefactor(s)of impor-tance for competence is repressed by glucose. The occur-rence of glucose in the LB medium could alsoexplain why this mediumwasless efficientthanminimalmedium contain-ing Casamino Acids.
Table 4 shows that the same degree ofcompetence was
induced
regardless
of cell density during the Tris-sucrose treatment. These dataalso showthat itwascrucialto use alowculturedensityduringtheuptake ofDNA. The decrease in number oftransformants with higher cell densities could havebeen dueto anobserved
aggregation
ofbacteria, which could have influencedtheuptake
of DNA.In B. subtiliscompetence can onlybe inducedduringlate
log phase(5), whereasinB. thuringiensistherewasabroad
optimum between
early
and latelogatithmic phases
(Fig.
3A). However, the
highest
number of transformants wasobtainedifa
late-log-phase
culturewas used(Fig. 3B).
Transformation ofB. subtilis by
plasmid
DNArequired
multimeric forms, and CCC DNA was inefficient
(6).
We found that theCCCformofplasmid
DNAwas moreefficient than linear forms ofDNA(Table
2). ChromosomalDNA,
which is always linear, gave notransformants. The 10-fold decrease in frequency for the multimeric DNA probably
reflectsthe lowernumber ofmoleculescompared with CCC DNA. The transformation protocol worked well for two
differentplasmids, and the methodispotentially suitable for
cloning experiments.
Thetransformation ofvegetative cells of B. thuringiensis was optimized for B. thuringiensis subsp. gelechiae. For B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. gal-leriae,and B. thuringiensis subsp. thuringiensis we obtained
frequenciesbetween 4 x
10-5
and 7 x10-5
transformantperviable cell, (Table 5), results which probably can be
im-proved by further study of the transformation protocol.
These results were all obtained with cells which had been frozen. We observed that B. thuringiensis subsp. kurstaki frozencells survivedtheTris-sucrosetreatment betterthan freshlygrowncells.
Protoplasttransformation ofB. thuringiensis isvery
inef-ficient, with about 10 transformants per ,g ofplasmid DNA (1, 9, 15, 16). With our method for transformation of vege-tative cells, we obtained about 1,000 times more transform-antspermicrogram of DNA, whichmeansthatour frequen-ciesaresimilartothose obtained for B. subtilis and B. brevis (6, 19). In additiontothe highfrequencies, ourmethodhas
the advantage of being time saving because transformants canbe scored after only 7 to 8 h and because frozen cells can be used.
ACKNOWLEDGMENT
This work was supported by grant BU2453 from the Swedish NaturalScience ResearchCouncil.
ADDENDUMINPROOF
An efficient transformation ofprotoplasts of B.
thuringi-ensis subsp. israelensis was recentlyreportedby Loprasert
etal.(J. Invert. Pathol. 48:325-334, 1986). LITERATURE CITED
1. Alikhanian,S.I.,N.F.Ryabchenko,N.0.Bukanov,andV. A. Sakanyan. 1981.Transformation of Bacillusthuringiensis subsp. galleriae protoplasts byplasmidpBC16.J.Bacteriol. 146:7-9. 2. Aronson, A. I., W. Beckman, and P. Dunn. 1986. Bacillus
thuringiensis and related insectpathogens. Microbiol.Rev. 50: 1-24.
3. Barsomian, G. D., N. J. Robillard, and C. B. Thorne. 1984. Chromosomal mapping of Bacillus thuringiensis by transduc-tion.J. Bacteriol. 157:746-750.
4. Bernhard, K.,H. Schrempf, and W.Goebel. 1978.Bacteriocin andantibiotic resistanceplasmids in Bacillus cereus and Bacil-lus subtilis. J. Bacteriol. 133:897-903.
5. Bott, K. F., and G. A. Wilson. 1967. Development of compe-tence in the Bacillus subtilistransformation system. J. Bacte-riol.94:562-570.
6. Canosi, U., G. Moreili,andT. A. Trautner. 1978. The relation-ship between molecular structure and transformationefficiency of some S.aureus plasmids isolated from B. subtilis. Mol. Gen. Genet. 166:259-267.
7. Carlton,B.C.,andJ.M.
Gonzalez,
Jr.1985. Thegenetics and molecularbiology of Bacillus thuringiensis, p. 211-249. In D. A.Dubnau (ed.), The molecularbiology of the bacilli. Academic Press, Inc., Orlando, Fla.
8. Chang, S.,andS. N. Cohen. 1979. Highfrequency transforma-tionofBacillussubtilisprotoplastsbyplasmidJ)NA.Mol. Gen. Genet. 168:111-115.
9. Fischer,H.-M.,P.Luthy,andS. Schweitzer. 1984.Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer. Arch. Microbiol. 139:213-217.
10. Hansen, J. B., andR.H.Olsen. 1978.Isolationof largebacterial plasmids and characterization of theP2 incompatibility group plasmids pMG1 and pMG5. J. Bacteriol. 135:227-238. 11. Heierson, A., R. Landen, and H. G. Boman. 1983.
Trans-ductional mapping of ninelinked chromosomal genes inBacillus
thuringiensis. Mol. Gen. Genet. 192:118-123.
12. Heierson, A., I. Siden, A. Kivaisi, and H.G, Boman. 1986. Bacteriophage-resistant mutants of Bacillus thuringiensis with a decreasedvirulence in pupaeofHyalophora cecropia. J. Bac-teriol. 167:18-24.
13.
Landin,'R1.,
A.Heierson,andH.G. Boman. 1981. A phage for generalizedtransduction inBacillus thuringiensis andmapping of four genes forantibiotic resistance. J. Gen.Microbiol. 123: 49-59.14. Lecadet, M.-M., M.-O. Blondel, andJ. Ribier. 1980. General-ized transduction in Bacillus thuringiensis var. berliner 1715 using bacteriophage CP-54 Ber. J. Gen. Microbiol. 121:203-212. 15. Martin, P., J.R.Lohr,and D.H. Dean. 1981.Transformation of Bacillus thuringiensisprotoplasts by plasmiddeoxyribonucleic acid. J. Bacteriol. 145:980-983.
16. Miteva, V. I., N. I. Shivarova, and R. T. Grigorova. 1981. Transformationof Bacillusthuringiensisprotoplasts byplasmid DNA.FEMS Microbiol. Lett. 12:253-256.
17. Perkins, J. B.,andP. Youngman. 1983. Streptococcus plasmid pAMal isacompositeoftwoseparablereplicons,oneof which is closely relatedtoBacillusplasmidpBC16. J. Bacterioj. 155: 607-615.
18. Perlak, F. J., C. L. Mendelsohn, and C. B. Thorne. 1979. Convertingbacteriophage forsporulationandcrystalformation inBacillus thuringiensis. J. Bacteriol. 140:699-706.
19.
Tskahashi,
W.,H. Yamagata,K. Yamaguchi,N.Tskukagoshi, andS. Udaka. 1983. Genetic transformation of Bacillus brevis 47, aprotein secreting bacterium, by plasmid DNA. J. Bacte-riol. 156:1130-1134.20. Thorne, C. B. 1978. Transduction in Bacillus thuringiensis. Appl. Environ. Microbiol. 35:1109-1115.
21. Yamuada, H.,N.Tsukagoshi,andS.Udaka.1981.Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47. J. Bacteriol. 148:322-332.
