Vol. 55, No. 10

Transformation of Bacillus polymyxa with Plasmid DNADARRELL H. MALLONEEt* AND RAY A. SPECKMAN

Department ofFood Science, University of Illinois, Urbana, Illinois 61801

Received 13 January 1989/Accepted 18 July 1989

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of thisstrain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA.Low-frequency transformation (10-6 per recipient cell) of plasmids pC194, pBD64, and pBC16 was

accomplished with this method. Selection for the transformants was accomplished on both hypertonic andnonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extractmedium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring theirantibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure whichallowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on thetransformed protoplasts, and the transformants could not be subcultured from the original selective media.

Strains of Bacillus polymyxa can produce a number ofuseful and potentially useful compounds, including peptideantibiotics, proteases, and a wide variety of carbohydrate-utilizing enzymes, such as P-amylase, ,-D-xylanase, pullu-lanase, glucose isomerase, and polygalacturonate lyase (12,13, 35-37). This bacterium has also been extensively testedas a source of 2,3-butylene glycol (23, 26, 38), which can beconverted into the feedstock chemical 1,3-butadiene.For studies on the genetic control and enhanced produc-

tion of these compounds, it would be useful to have a systemfor the transfer of genetic information within this species.Numerous methods of plasmid transfer between and withinvarious Bacillus spp. have been reported. These includetransduction (20, 25, 34), spontaneous transfer by a conju-gationlike process (15, 21, 32), competent-cell plasmid trans-formation procedures for B. subtilis (10, 11), protoplasttransformation procedures (5, 7, 18, 19, 27, 28), and a

procedure developed for B. brevis involving exposing thecells to alkaline Tris hydrochloride (39). However, no usefulprocedure for plasmid transfer has been reported for B.polymyxa. This study was initiated to develop such a proce-dure.

MATERIALS AND METHODSBacterial strains and plasmids. Table 1 lists the bacterial

strains and plasmids used in this study.Plasmid isolation procedures. Plasmid DNA was isolated

from strains of B. subtilis by the procedure of Gryczan et al.(17) and by a modification of the procedure of Birnboim andDoly (4). In the Birnboim-Doly procedure, the plasmid-containing strain was grown overnight on brain heart infu-sion (Difco Laboratories, Detroit, Mich.) agar plates con-

taining the appropriate antibiotic to select for the plasmid.Growth from this plate was used to inoculate 2.5 ml of brainheart infusion, which was incubated with shaking for 3.5 h at35°C (mid to late log phase). A 0.75-ml portion of this culturewas transferred to a 1.5-ml microcentrifuge tube and centri-fuged for 15 s at room temperature. The cell pellet was

suspended in 100 ,ul of protoplasting reagent (50 mM glu-

* Corresponding author.t Present address: Department of Microbiology and Immunology,

Medical College of Virginia, Virginia Commonwealth University,Richmond, VA 23298.

cose, 0.2% lysozyme, 10 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid [CDTA], 25 mM Tris hydrochlo-ride [pH 8]) and kept for 30 min at 0°C before being takenthrough the rest of the Birnboim-Doly (4) procedure. For B.polymyxa the modified Birnboim-Doly procedure was usedwith the additional modification of substituting 200 U ofmutanolysin (Sigma Chemical Co., St. Louis, Mo.) per mlfor the lysozyme.

Transformation of penicillin-treated cells. For the transfor-mation of penicillin-treated cells, B. polymyxa ATCC 12321was grown overnight on a Trypticase soy agar (TSA; BBLMicrobiology Systems, Cockeysville, Md.) plate at 35°C.Three to five colonies from the plate were touched with an

inoculating loop and used to inoculate 12.5 ml of PGY (1.5%proteose peptone no. 3 [Difco], 0.5% glucose, 0.5% yeastextract). After growth with shaking for approximately 2.5 hat 35°C (A540, 0.25), 12.5 ml ofPGY containing 0.5 M sucroseand 10 U of penicillin per ml was added and incubation was

continued for an additional 2 h. The cells were collected bycentrifugation for 15 min at 3,000 x g and washed once in 10ml of SMMP (equal volumes of SMM and 2x PGY; SMMconsisted of 0.5 M sucrose, 0.02 M maleate, and 0.02 MMgCl2 [pH 6.5]) before being transformed with plasmidDNA by a modification of the procedure of Chang andCohen (7). The cells were gently suspended in 0.5 to 1 ml ofSMMP, and 0.5-ml portions of the suspension were mixedwith 0.1 ml of a plasmid preparation dissolved in equalvolumes of 2x SMM and TE buffer (10 mM Tris hydrochlo-ride, 1 mM EDTA [pH 8.0]). A 1.5-ml portion of 40%polyethylene glycol 8000 in SMM was added, and thesolution was gently mixed and kept for 2.5 min at 0°C,followed by a 5-min incubation period at 35°C. SMMP (5 ml)was added, and the protoplasts were pelleted by centrifuga-tion at 3,000 x g for 15 min. The protoplasts were suspendedin 1 ml of SMMP and incubated for 30 min at 30°C withgentle shaking. Appropriate dilutions were made in SMMPand plated on TSA, PGYA, PGYSA, or DM3 (7) regenera-tion medium. PGYA was PGY with 1.5% agar. PGYSA was

PGYA with added sucrose (0.25 M) and bovine serum

albumin (0.01%). DM3 medium contained 0.5 M sodiumsuccinate (pH 7.3), 0.5% Casamino Acids (Difco), 0.5%yeast extract, 0.5% glucose, 0.35% K2HPO4, 0.15%KH2PO4, 0.02 M MgCI2, 0.01% bovine serum albumin, and1% agar. Appropriate selective antibiotics were added in the

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2518 MALLONEE AND SPECKMAN

TABLE 1. Bacterial strains and plasmids

Strain Genotype or Plasmid Plasmid size Sourcephenotype (phenotype) (kilobases)

B. polymyxa ATCC 12321 Wild type ATCC"B. polymyxa U121 Nonmucoid strain This studyB. subtilis 168 trpC2 BGSCbB. subtilis BGSC 1E6 thr-5 trpC2 pUB110 (Kmr) 4.5 BGSCB. subtilis BGSC 1E8 thr-5 trpC2 pSA2100 (Cmr Smr) 7.1 BGSCB. subtilis BGSC 1E9 trpC2 pBC16 (Tcr) 4.2 BGSCB. subtilis BGSC lEll thr-5 trpC2 pC221 (Cmr) 4.5 BGSCB. subtilis BGSC 1E17 trpC2 pC194 (Cmr) 2.9 BGSCB. subtilis BGSC 1E18 thr-5 trpC2 pE194 (Emr) 3.6 BGSCB. subtilis BGSC 1E22 thr-5 trpC2 pBD64 (Cmr Kmr) 4.8 BGSCB. subtilis BGSC 1E29 thr-5 trpC2 pSA0501 (Smr) 4.2 BGSC

a American Type Culture Collection, Rockville, Md.b Bacillus Genetic Stock Center, Ohio State University, Columbus.

following concentrations to nonhypertonic media andPGYSA: 5 ,ug/ml for neomycin, kanamycin, erythromycin,and chloramphenicol; 15 ,ug/ml for tetracycline; and 35 ,ug/mlfor streptomycin. For DM3 medium chloramphenicol anderythromycin were added to 10 ,ug/ml; tetracycline wasadded to 30 ,ug/ml; and kanamycin, neomycin, and strepto-mycin were added to 100 ,ug/ml. The plates were incubatedat 35°C for 2 to 5 days.

Protoplast formation and transformation of protoplasts. B.polymyxa was protoplasted by using either mutanolysin or acombination of penicillin and lysozyme. In the penicillin-lysozyme procedure the cells were grown and treated withpenicillin as described above, collected by centrifugation,and suspended in 2.5 ml of SMMT (equal volumes of SMMand 2x Trypticase soy broth [BBL Microbiology Systems]).Lysozyme was added to 10 mg/ml, and the suspension wasincubated for 1 h at 35°C. The protoplasts were collected bycentrifugation for 15 min at 3,000 x g, gently suspended in 1to 2.5 ml of SMMT, and transformed with plasmid DNA asdescribed above. In the mutanolysin procedure 25 ml ofTrypticase soy broth was inoculated with an overnightgrowth of B. polymyxa and incubated for approximately 3.5h at 35-C (A540, 0.4). The cells were harvested by centrifu-gation at 10,000 x g for 10 min, and the pellet was gentlysuspended in 2.5 ml of SMMT. Mutanolysin was added to aconcentration of 200 U/ml, and the suspension was incu-bated for 2 h at 35°C. The protoplasts were collected bycentrifugation at 3,000 x g for 15 min, gently suspended in 1to 2.5 ml of SMMT, and transformed with plasmid DNA asdescribed above.

Strains of B. subtilis were protoplasted and transformedwith plasmid DNA by the procedure of Chang and Cohen(7).Development of a nonmucoid strain of B. polymyxa. A

nonmucoid strain of B. polymyxa was developed for use inthis study by mutagenesis with N-methyl-N'-nitro-N-nitro-soguanidine. B. polymyxa ATCC 12321 from an overnightgrowth on TSA was used to inoculate 100 ml of Trypticasesoy broth in a 300-ml nephelometer flask to an A540 of 0.02.Cells were grown with shaking at 35°C until an A540 of 0.2was reached. A 6.8-ml portion of the cell suspension wasmixed with 3.2 ml of 0.25-mg/ml N-methyl-N'-nitro-N-nitro-soguanidine in 0.05 M sodium acetate (pH 5.5). The mixturewas incubated without shaking for 30 min at 35°C beforecentrifugation at 3,000 x g for 8 min at room temperature.The pellet was washed once in 10 ml of 0.85% NaCl andsuspended in 1 ml of minimal medium (26). Dilutions (in0.85% NaCl) were plated on TSA with 0.25 M sucrose and

incubated at 35°C for 24 to 48 h. Colonies showing anonmucoid colony type on the plates were checked onminimal media for wild-type nutritional requirements.

RESULTS

Several methods of plasmid transfer were tested for usewith B. polymyxa ATCC 12321 and B. polymyxa U121, amutant strain displaying a nonmucoid phenotype whengrown on sucrose-containing media. Modifications of proce-dures for spontaneous transfer of plasmids (15, 31), plasmidtransformation of competent cells (9, 24, 33), and plasmidtransformation of cells treated with alkaline Tris hydrochlo-ride (39) were not successful with the B. polymyxa strains.

Transformation of protoplasted cells of B. polymyxa. Nu-merous attempts were also made to create a protoplasttransformation system for B. polymyxa. Lysozyme was usedin the prototype B. subtilis protoplast transformation proce-dure of Chang and Cohen (7) and in numerous other trans-formation procedures for Bacillus spp. However, lysozymewas ineffective when used with B. polymyxa, producingfewer than 5% protoplasts, as determined by microscopicanalysis.Two protoplasting procedures described in the Materials

and Methods section were developed for use with B. poly-myxa. The methods used either mutanolysin or a combina-tion of penicillin and lysozyme. More than 90% protoplasts,as determined by microscopic analysis and growth on hyper-tonic (DM3) and nonhypertonic (TSA) media, were pro-duced when the procedures were used with either strainATCC 12321 or strain U121. Small colonies could be ob-tained by plating the protoplasted cultures on the hypertonicDM3 medium. However, cell walls could not be regeneratedon the protoplasted cells, and the resulting colonies couldnot be transferred to any of several variations of hypertonicand nonhypertonic liquid or solid media, including theoriginal DM3 medium. Numerous modifications to the re-generation media that have been utilized with other organ-isms were tried without success, including the use of dif-ferent hypertonic agents, the use of agar overlays, the use ofdifferent incubation conditions, and the addition of cell wallprecursors, autoclaved B. polymyxa cells and cell wallpreparations, minerals, polyvinylpyrrolidone, gelatin, andnutritional growth factors (1, 2, 8, 14, 22, 29).The apparent transfer of several plasmids carrying antibi-

otic resistance markers was obtained with B. polymyxaATCC 12321 when we used either the mutanolysin or thepenicillin-lysozyme protoplasting method with plasmid

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TABLE 2. Summary of plasmid transfer experiments involvingB. polymyxa protoplasts

Sb c ~~~~No. ofRecipient Plasmid Recipients Transformants transformants.strain' (CFU/ml) (CFU/ml) recipientcerecipient cell

B. polymyxa pC194 1.0 X 107 2.8 x 104 2.8 x 10-3B. polymyxa pBD64 2.7 x 107 1.8 x 104 6.7 x 10-4B. polymyxa pBC16 3.4 x 107 2.1 X 103 6.2 x 10-5B. polymyxa pSA0501 2.9 x 107 2.0 x 103 6.9 x 10-5B. polymyxa pSA2100 3.5 x 107 2.3 X 102 6.6 x 10-6B. polymyxa pE194 2.2 x 107 3.0 x 101 1.4 x 10-6B. polymyxa pC221 2.0 x 107 2.0 x 101 1.0 X 10-6B. subtilis pBD64 4.6 x 108 6.3 X 105 1.4 x 10-3

aB. polymyxa ATCC 12321 was prepared by either a mutanolysin or apenicillin-lysozyme protoplasting procedure. B. subtilis 168 was transformedby the procedure of Chang and Cohen (7).

b Determined by growth on DM3 medium.c Transformants were selected for on DM3 medium containing 10 p.g of

chloramphenicol per ml for pBD64, pC194, pC221, and pSA2100; 10 ,ug oferythromycin per ml for pE194; 30 ,ug of tetracycline per ml for pBC16; and100 1Lg of streptomycin per ml for pSA0501. Approximately 1 ,ug of plasmidDNA was used in all transformations. Controls run without plasmid DNAproduced 4.0 x 101 CFU/ml on media selective for pSA0501. All transfor-mants were protoplasted cells and could not be transferred from the originalhypertonic selective media.

transformation procedures and selection on DM3 mediumcontaining the appropriate selective antibiotic (Table 2). Thesmall size of the transformant colonies and the impossibilityof subculturing prevented further characterization or confir-mation of plasmid transfer.Transformation of penicillin-treated cells. The lack of a cell

wall regeneration technique for use with the B. polymyxaprotoplasts prompted the study of potential injured-celltransformation techniques, in which the cell wall of theorganism is damaged but not removed. Microscopic obser-vation of different treatments suggested the use of eitherlysozyme or penicillin alone instead of in combination as inthe protoplasting technique. Treatments with either penicil-lin or lysozyme produced fewer than 5% protoplasts, asdetermined by microscopic analysis. Plasmid transformationof lysozyme-treated cells produced no transformant colonieson hypertonic or nonhypertonic media.However, transformant colonies were obtained when pen-

icillin-treated cells of B. polymyxa UI21 were exposed toDNA from plasmid pC194. Microscopic analysis of cellsfrom the selective plates and transfers of the colonies tononhypertonic selective media showed that the transfor-mants were intact cells. We tested the effect of includingdivalent cations in the SMM buffer used during the plasmiduptake step of the transformation procedure. Transforma-tions were done with B. polymyxa U121 as the recipient forapproximately 1 ,ug of plasmid pC194 DNA. Selection wasdone on PGYA plus 5 ,ug of chloramphenicol per ml. Thenumber of transformants per recipient cell was based on 4.2x 107 CFU/ml of recipient cells. The original formulation ofSMM, which included 20 mM MgCl2, produced more trans-formant colonies (90 CFU/ml, or 3.8 x 10-6/recipient cell)on PGYA selective plates than did SMM containing 20 mMCaCl2 instead (10 CFU/ml, or 4.2 x 10' recipient cell).Controls run without plasmid DNA produced no colonies onselective media. When neither MgCl2 nor CaCl2 was addedno transformant colonies were obtained. Subsequent exper-iments were run with the original SMM formulation. Wetested different formulations of selective media. Transforma-tions were done with B. polymyxa UI21 as the recipient forapproximately 1 ,ug of plasmid pC194 DNA. When PGYA,

4-3-2

FIG. 1. Agarose gel (0.7%) of modified Birnboim-Doly plasmidDNA preparations. The preparations were additionally treated withRNase A (1 RlI of a 2-mg/ml solution; Sigma) and 3 U of HindIIIrestriction endonuclease for 1 h at 37"C. The preparations were fromB. subtilis 168 (lane A), B. subtilis BGSC 1E17(pC194) (lane B), andB. polymyxa U121 before (lane C) and after (lane D) transformationwith plasmid pC194. All samples were 50 p.l. The arrow points to the2.9-kilobase linear form of plasmid pC194. Molecular size markersin kilobases are given to the left of the gel. Transformation was bythe penicillin-treated-cell procedure, with selection on DM3 mediumplus 5 jig of chloramphenicol per ml.

DM3, and TSA (all containing 5 ,ug of chloramphenicol perml) were tested, 100, 25, and 10 CFU of transformants per ml(1.9 x 10-6, 4.8 x 10-7, and 1.9 x 10-7 transformants perrecipient cell, respectively, based on 5.2 x 107 CFU/ml ofrecipient cells) were produced, respectively. When PGYSAand PGYA (both containing 5 ,ug of chloramphenicol per ml)were tested, 506 and 123 CFU of transformants per ml (4.2 x

10-6 and 1.0 x 10-6 transformants per recipient cell, respec-tively, based on 1.2 x 108 CFU/ml of recipient cells) wereproduced, respectively. Controls run without plasmid DNAproduced no colonies on selective media.

Plasmid DNA bands could be detected in B. polymyxaUI21 cells that had been transformed with plasmid pC194 bythe penicillin-treated-cell technique (Fig. 1). The plasmidDNA bands appeared to have the same molecular weight as

plasmid pC194 DNA obtained from B. subtilis BGSC 1E22.Plasmids pBD64 and pBC16 could also be used to transformB. polymyxa U121 by the penicillin-treated-cell technique(data not shown). No detectable transformants were ob-tained with pSA2100 or pE194 DNA. The use of plasmidDNA in excess of 1 ,ug had no effect on the transformationresults. Plasmid pC194 DNA collected from both B. subtilisand B. polymyxa could be used to transform B. polymyxa.Transformation results for wild-type B. polymyxa ATCC12321 were similar to those for strain U121.

All of the plasmids transferred to the B. polymyxa strainswere relatively unstable. Plasmid-containing strains thatwere grown overnight on nonselective media could not betransferred back to selective media. Frequent transfers ofthe plasmid-containing strains on selective media were nec-

essary for viability.

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2520 MALLONEE AND SPECKMAN

DISCUSSION

This paper describes the transformation of B. polymyxacells with plasmid DNA. A transformation system for B.polymyxa was developed by using a cell wall injury treat-ment with penicillin. This penicillin-treated-cell transforma-tion procedure was used to transfer plasmid DNA to B.polymyxa strains, with selection on either hypertonic ornonhypertonic media. Cells transformed by this procedurecould be subcultured and used as donor strains for furtherplasmid transformation procedures. Plasmids pC194 andpBD64 were transferred by this technique, with selection forchloramphenicol resistance, and plasmid pBC16 was trans-ferred by this technique, with selection for tetracyclineresistance. Plasmid pC194 was originally isolated fromStaphylococcus aureus (11), and plasmid pBC16 was iso-lated from B. cereus (3). Plasmid pBD64 is a chimericplasmid developed from the S. aureus plasmids pC194 andpUB110 (16).

Several additional plasmids originally isolated from S.aureus could apparently be transferred to B. polymyxaprotoplasts. This procedure provided a higher transforma-tion frequency than did the penicillin-injured-cell procedure,but cell walls could not be regenerated on the transformedcells and the colonies could not be subcultured. The com-plexity of the B. polymyxa cell wall (6, 30) and the highlyefficient cell wall removal obtained with the mutanolysin andpenicillin-lysozyme protoplasting procedures may help toexplain the inability to develop a cell wall regenerationprocedure. Regeneration of the cell wall may require thepresence of some cell wall material, although purified B.polymyxa cell wall preparations had no effect on regenera-tion.The penicillin-treated-cell transformation procedure pro-

vides a method for transferring plasmid DNA to B. polymyxaat a very low frequency (_10-6 transformants per recipientcell) and cannot be used effectively with selection techniquesthat produce spontaneous mutant colonies above this thresh-old. However, the technique should be useful in transferringto B. polymyxa plasmids that have been developed bycloning techniques in Escherichia coli or B. subtilis. Thetechnique might also be useful in other species of gram-positive organisms that have been resistant to other plasmidtransfer techniques. Somewhat more efficient protoplasttransformation methods showed that several additional plas-mids could be transferred to B. polymyxa. Additional proto-plast regeneration studies may prove useful with this bacte-rium.

ACKNOWLEDGMENTS

This study was supported by U.S. Department of AgricultureHatch grant ILLU-50-0315 and by a University of Illinois GraduateCollege dissertation research grant awarded to D.H.M.

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