Vol. 159, No. 1

Protoplast Transformation of Glutamate-Producing Bacteria withPlasmid DNA

RYOICHI KATSUMATA,* AKIO OZAKI, TETSUO OKA, AND AKIRA FURUYATokyo Research Laboratories, Kyowa Hakko Kogyo Company, Ltd., Machida, Tokyo, Japan 194

Received 11 January 1984/Accepted 23 April 1984

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria withplasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin bytreatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected theefficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regenera-tion of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeastextract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacteriumglutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacte-rium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurallyunchanged and stably maintained in new hosts. The transformation frequency of most competent protoplastswith pCG4 DNA isolated from primary transformants was high (ca. 106 transformants per ,ug of covalentlyclosed circular DNA) but was still two orders of magnitude below the frequency of transfection with modifiedDNA of the bacteriophage +CG1. The difference was ascribed to the involvement of regeneration intransformation.

The glutamate-producing bacteria, represented by Cory-nebacterium glutamicum, are industrially important micro-organisms, since they are used in fermentative production ofvarious amino acids, as well as glutamic acid. Althoughextensive studies on these bacteria have so far been devotedto biochemical analysis of the regulatory mechanisms inamino acid biosyntheses to improve their yields (16), therehave been few studies on the gene transfer system, exceptfor only two reports: one on protoplast fusion (12) and oneon phage-mediated transduction (15) in Brevibacteriium fla-vum, one of the typical glutamate-producing bacteria. Thedevelopment of molecular cloning systems in these bacteriawill not only enable us to study their genetic organization butalso should provide a powerful new tool for future strainimprovements. Despite the shortage of genetic informationavailable for this approach, interest in both academic andindustrial applications encouraged us to undertake the estab-lishment of a gene cloning system with C. glutamicum as

host.One prerequisite for any cloning system is to develop a

method for introducing DNA into the host organism. Sincegenetic transformations of polyethylene glycol-treated pro-toplasts have been successful in such diverse organisms asBacillus subtilis (5), other Bacillus species (4, 11, 14),Streptomyces species (3), and Saccharomyces cerevisiae (9),a similar system might also be applicable to C. glutamiclum.Actually, transfection of C. glutamicium protoplasts withphage DNAs occurred in the presence of polyethylene glycolplus divalent cations (A. Ozaki et al., submitted for publica-tion). However, this DNA uptake system does not necessari-ly allow transformation with plasmid DNA at a frequency as

high as that of transfection, because regeneration of proto-plasts is involved in transformation. Therefore, we estab-lished conditions for preparation of protoplasts which pos-

sess high DNA uiptake and regeneration abilities. This report

* Corresponding author.

306

provides the first plasmid transformation system availablefor glutamate-producing bacteria, including not only C.glutamicum but also other coryneform bacteria such as B.flavum and Microbacterium ammoniaphilum.

MATERIALS AND METHODSBacterial strains, plasmid, and phage. Eight independent

wild-type glutamate-producing bacteria were used. Thestrains obtained from the American Type Culture Collectionwere as follows: C. glutamicum ATCC 13032, Corynebacte-rium herculis ATCC 13868, B. flavum ATCC 14067, and M.ammoniaphilum ATCC 15354. The other strains (T250,T218, T106, and KY9005), identified as C. glutamicumaccording to the proposal of Abe et al. (1), were from ourcollection. C. glutamicum T250 (ATCC 31830) harbors aplasmid of 29 kilobases named pCG4, which specifies resist-ance determinants to streptomycin and spectinomycin. Thetemperate bacteriophage 4CG1 was originally isolated fromits lysogen, C. glutamicum T36, and was infective to C.glutamicum T106.Media and culture conditions. BY medium (0.3% NaCl,

0.5% yeast extract, 0.7% meat extract, 1% peptone) and BYagar (1.6%) medium were used for routine bacterial growth.MMYE medium [glucose, 20 g; (NH4)2SO4, 10 g; urea, 3 g;yeast extract, 1 g; K2HPO4, 1 g; MgSO4 * 7H2O, 0.4 g;FeSO4 * 7H20, 2 mg; MnSO4 *4HO to 6H,O, 2 mg; NaCl,

50 mg; biotin, 50 ,ug; thiamine, 200 [ig; deionized water, 1liter (adjusted to pH 7.2)] was used for growth to prepareprotoplasts and isolate the plasmid. For lysozyme digestionof intact cells and dilution of protoplasts, hypertonic mediumRCG was used, which had the following composition: glu-cose, 5 g; Casamino Acids, 5 g; yeast extract, 2.5 g;K2HPO4, 3.5 g; MgCI2 6H14O, 0.4 g; biotin, 30 ,ug; thia-mine * HCI, 2 mg; polyvinylpyrrolidone K15 (average mo-lecular weight, 10,000; Tokyo Kasei Co., Tokyo, Japan), 30g; sodium succinate, 135 g; trace element solution, 1 ml;agar, 16 g; and deionized water, 1 liter (adjusted to pH 7.6).The trace element solution contained 88 mg of Na-2B407 * 7H20, 37 mg of (NH4)6Mo7027 4H,O, 8.8 mg of

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PLASMID TRANSFORMATION OF C. GLUTAMICUM 307

ZnSO4 4H,O, 270 mg of CUS04 5H,O, 7.2 mg ofMnCl2 4H,O, and 970 mg of FeCI3 * 6H2O in a liter ofdeionized water. RCGA medium (RCG medium solidifiedwith 1.6% agar, pH 7.2) was ordinarily used for regenerationof protoplasts. PS medium, twofold-diluted MMYE mediumcontaining 0.41 M sucrose and 10 mM MgCI2, was used asthe double-layer plating medium (1.6% agar for the bottomlayer and 0.7% agar for the top layer) for the assay oftransfection.

All cultures were incubated at 30°C both on agar platesand in liquid media with shaking. When necessary, bacterialgrowth was pursued turbidimetrically at 660 nm with aTokyo Koden photometer model 7A.

Isolation of plasmid and phage DNAs. Plasmid DNA wasisolated from cells sensitized to lysozyme by treatment withpenicillin, because glutamate-producing bacteria were insen-sitive to lysozyme, like Corvnebacteriirn diphtheriae (19).Overnight cultures were inoculated into 400 ml of MMYEmedium and grown with shaking. At the early logarithmicphase of growth (the optical density at 660 nm [OD660J was0.15 to 0.2), penicillin G (Pen G) (potassium salt; MeijiseikaCo., Tokyo, Japan) was added to a final concentration of 0.5U/ml. The culture was futher continued up to an OD660 of 0.9to 1.0. The cells were harvested by centrifugation, washedonce with 40 ml of TES buffer (10 mM Tris-hydrochloride, 10mM NaCl, 1 mM EDTA [pH 8.01), and suspended in 11 ml of50 mM Tris-hydrochloride buffer (pH 8.0) containing 12.5%sucrose, 100 mM NaCI, and 1 mg of lysozyme per ml. Themixture was incubated with gentle shaking at 37°C for 3 h.To the suspension, 0.6 ml of 0.5 M EDTA (pH 8.5), 2.4 ml of5 M NaCl, and 4.4 ml of4% sodium dodecyl sulfate (NakaraiChemicals, Kyoto, Japan)-0.7 M NaCl were added. Thesuspension was mixed gently and placed on ice for 18 h.After the lysate was centrifuged at 65,000 x g for 1 h at 40C,the supernatant solution was mixed with polyethylene glycol6000 (Nakarai Chemicals) (50% stock solution) at a finalconcentration of 10% (wt/vol) and kept on ice for at least 3 h.The precipitate was collected by centrifugation at 1,000 x gfor 5 min, dissolved in 5 ml of TES buffer, and transferred toa nitrocellulose tube, to which 7.5 g of CsCl and 2 ml ofethidium bromide solution (1.5 mg/ml) in TES buffer wereadded. The solution was centrifuged at 39,000 rpm in aHitachi RP55T rotor for 42 h at 18°C. The presence ofplasmid DNA was located by fluorescense under UV light.The lower band (covalently closed circular [CCC] plasmidDNA) was withdrawn with a syringe, extracted with CsCl-saturated isopropanol (90% [vol/vol]) to remove the ethidiumbromide, dialyzed against TES buffer, and stored at 4°C untiluse. This process was established on the basis of a combina-tion of several techniques described previously (8, 10, 18).Bacteriophage XCG1 DNA was extracted from the phageparticles propagated in C. glutamicum T106 as describedelsewhere (Ozaki et al., submitted for publication).

Preparation and regeneration of protoplasts. Protoplastswere prepared by the method of Kaneko and Sakaguchi (12)with some modifications. A 1% inoculum from an overnightculture in BY medium was inoculated into MMYE mediumand grown with aeration. When the OD660 reached 0.15, PenG was added to a final concentration of 0.5 U/ml unlessotherwise indicated. The culture was further incubated to anOD660 of 0.5 to 0.6, at which point cells were harvested bycentrifugation and suspended in RCG medium (pH 7.6)containing 1 mg of lysozyme per ml in one-fifth volume ofthe original culture. The suspension was incubated at 30°Cwithout shaking for 16 h to generate protoplasts. The proto-plasts were harvested and washed once with TSMC buffer

(50 mM Tris-hydrochloride [pH 7.5], 0.5 M sodium succi-nate, 10 mM MgCl2, 20 mM CaCI2) and resuspended in halfthe volume of the same buffer. The formation of protoplastswas monitored under a phase-contrast microscope by count-ing spherical cells. The number of osmotically stable surviv-ors (nonprotoplasted cells) was determined by diluting thesuspension in deionized water and plating on BY agar plates.The ratios of the later counts to the CFU before treatmentwith lysozyme were used as another estimate of the efficien-cy of protoplast formation. The regenerable population of aprotoplast suspension was measured by plating on RCGAmedium after appropriate dilution and counting CFU afterincubation for 10 to 14 days, in which the fraction ofregenerated cells reached a maximum. The regenerationefficiency was calculated by dividing the number of regener-ated colonies by the number of initially input intact cells.

Transfection and transformation. Introduction of DNAinto protoplasts was performed under the conditions whichallowed the most efficient transfection with phage DNA(Ozaki et al., submitted for publication). One millititer ofprotoplast suspension (ca. 2 x 109 cells per ml) was trans-ferred to a tube, centrifuged, and suspended in 0.1 ml ofcooled TSMC buffer. Plasmid or phage DNA in 100 [tI of amixture (1:1) ofTES and two-fold-strength TSMC buffer wasmixed with protoplasts, which was followed by addition of1.8 ml of 20% polyethylene glycol 6,000 dissolved in TSMCbuffer. The mixture was gently mixed, incubated on ice for 5min, and then subjected to a heat pulse at 37°C for 3 min.For transfection, serial dilutions of the above protoplasts

were plated in PS soft-agar overlays together with intactindicator cells. Transfection efficiency was determined bythe PFU per milliliter after overnight incubation. In transfor-mation with pCG4, the protoplasts were harvested, washedonce with 3 ml of TSMC buffer, and resuspended in 1 ml ofRCG medium (pH 7.2). The suspension was incubated for 2h to allow phenotypic expression of genetic determinants onthe plasmid. Diluted or concentrated protoplasts were platedon RCGA medium containing 400 vLg of spectinomycin perml. After incubation for 10 to 14 days, spectinomycin-resistant colonies were scored and subsequently identified aspCG4 transformants by checking for resistance to spectino-mycin (100 p.g/ml) and streptomycin (12.5 Fg/ml) on BY agarplates.

Analysis of plasmids. Digestion of plasmid DNA withrestriction endonuclease BamHI (Takara Shuzo Co., Kyoto,Japan) was performed according to the specifications of thesupplier. Agarose gel electrophoresis was carried out in ahorizontal slab gel of 1.0% agarose in 40 mM Tris-acetate(pH 8.0)-20 mM sodium acetate-2 mM EDTA-0.6 mg ofethidium bromide per ml at 100 V for 3 h. Plasmids and theirrestricted fragments were detected under UV light andphotographed.

Assay of streptomycin-inactivating enzyme. Streptomycin-inactivating activity was assayed by the microbiologicalmethod of Kawabe and Mitsuhashi (13). To facilitate disrup-tion of cells, crude extracts were prepared from protoplastsas follows. After being washed with TSMC buffer, theprotoplasts were suspended in 1/10 volume of the originalculture by the addition of TMK buffer (100 mM Tris-hydrochloride [pH 7.8], 60 mM KCI, 10 mM magnesiumacetate, 6 mM 2-mercaptoethanol) and sonicated briefly.The extract was clarified by centrifugation at 30,000 x g for30 min. The supernatant fluid was used as crude enzyme.The reaction mixture consisted of the following materials:0.2 ml of crude enzyme solution (10 mg of protein per ml),0.1 ml of 0.1 M ATP-Na, solution, 0.6 ml of TMK buffer,

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308 KATSUMATA ET AL.

and 0.1 ml of dihydrostreptomycin solution. The reactionwas carried out at 35°C for 60 min and then stopped byheating at 80°C for 3 min. The activity of the antibioticremaining in the reaction mixture was determined by bioas-say with B. subtilis as the test organism.

RESULTSEffect of penicillin pretreatment on protoplast formation.

trhe effect of Pen G on protoplast formation was investigatedwith C. glutamicum T106 (Table 1). Pen G inhibited growthat higher concentrations than 0.25 U/ml. Cells grown in thepresence of Pen G were more or less converted to sphericalforms by the subsequent lysozyme digestion in hypertonicRCG medium, whereas cells grown in the absence of Pen Gremained rod shaped with the same treatment. Efficientconversion to spherical forms was observed in the cellstreated with Pen G at concentrations over 0.5 U/ml. As thelysozyme digestion proceeded, the number of sphericalforms gradually increased and reached a maximum (85 to90%) by 8 to 10 h of incubation and then remained fairlyconstant until at least 24 h. The rest exhibited irregularlyswollen shapes different from the original rod shape (data notshown). The proportion of osmotically stable cells fell to lessthan 10-3. These observations indicated that the sphericalcells were true protoplasts and that the irregularly swollencells were presumably spheroplasts. Only the exposure to1.0 U of Pen G per ml, the highest concentration tested,appeared to cause partial lysis during lysozyme digestion.

Similar protoplast formations depending on Pen G treat-ment were also observed in other glutamate-producingstrains (data not shown). When some strains were digestedwith lysozyme in a hypertonic solution containing 0.41 Msucrose, as was described by Kaneko and Sakaguchi (12),irreversible agglutination of cells occurred and disturbedfurther operations. The replacement of sucrose with 0.5 Msodium succinate eliminated the agglutination.

EDNA uptake and regeneration abilities of protoplasts fromcells treated with various concentrations of penicillin. Becausetransformants have to be recovered through regeneration of

TABLE 1. Formation and characterization of C. glutamicumT106 protoplasts

Growth condition Protoplast formation"

Pen G Doubling Spherical Proportion Regeneration Transfectiongadded" time" cells of osmoti- frqec~ (PFU/ml)(U/ml)(min) formed" cally stable

(U/mI) (min) (%) cellse

0 96 0 9.8 x 10-1 97.4 <100.1 96 21 1.7 x 10-2 53.7 8.7 x 1040.25 98 66 3.1 x 10-3 10.2 2.1 x 1060.5 103 87 4.2 x 10-4 6.4 1.4 x 1070.75 116 90 4.5 x 10-4 1.7 1.2 x 1071.0 114 81 3.8 x 10-4 0.1 6.2 x 106

Cells fromn each culture were suspended in RCG medium containinglysozyme (1 mg/ml) and incubated at 30°C for 16 h.

b Added to MMYE culture early in the logarithmic phase of growth.' The time required for doubling of ODw.d Percentage of spherical cells counted under a phase-contrast microscope.e Osmotically stable cells per initial viable cell subjected to the treatment

with lysozyme.f Percentage of colonies regenerated on RCGA plates to the initial viable

cells subjected to the treatment with lysozyme.X Protoplast preparation containing 1.5 x 109 cells was used in each

transfection with 4CG1 DNA (0.05 ,ug) extracted from the phage grown on C.glutamicum T106. Transfection was determined by counting PFU on theindicator lawn with intact cells of C. glutamicum T106, as described in thetext.

TABLE 2. Influence of nutrients and osmotic stabilizers onregeneration of protoplastsa

Compounds added to basal mediumbRegeneration

Yeast Casamino frequencyextract Acids (0H2%) Stabilizer (%)(0.25%) (0.5%)

+ + - None 0.02+ + - 0.6 M sorbitol 0.02+ + - 0.8 M sorbitol 0.02+ + - 0.3 M sucrose 0.02+ + - 0.5 M sucrose 0.23+ + - 0.3 M sodium succinate 1.8+ + - 0.5 M sodium succinate 5.7+ + - 0.7 M sodium succinate 5.2+ - - 0.5 M sodium succinate 4.8- + - 0.5 M sodium succinate 0.03- - + 0.5 M sodium succinate 0.01

a Protoplasts prepared from C. glutamicum T106 cells grown in thepresence of 0.5 U of Pen G per ml were used.

b Basal medium: glucose, 5 g; K,HPO4, 3.5 g; KH,PO4, 1.5 g;MgCI. * 6H,O, 0.4 g; biotin, 30 ,ug; thiamine * HCI, 2 mg; polyvinylpyrroli-done K15, 30 g; trace element solution, 1 ml; agar, 16 g; and deionized water,1 liter (adjusted to pH 7.2). +, Added; -, not added.

protoplasts after uptake of DNA, transformation efficiencyis affected by both DNA uptake and regeneration processes.Therefore, we assessed the above protoplasts of strain T106with respect to their ability to take up DNA by transfectionand regenerate on RCGA plates (Table 1). With increasingPen G concentrations in pretreatment, the regenerationfrequency of subsequently induced protoplasts decreased.The protoplasts from cells treated with 0.5 U of Pen G per mlgave the maximum transfection frequency (107 PFU/ml),indicating the highest DNA uptake ability. These resultssuggest that treatment of cells with 0.5 U of Pen G per mlyielded the most favorable protoplasts for transformation.This concentration of Pen G was used throughout thesubsequent experiments described herein.Requirements for regeneration of protoplasts. Protoplast

regeneration is also affected by the composition of a hyper-tonic medium. Therefore, we tested regeneration media forprotoplasts of C. glutamicium T106, with special reference tothose developed for B. siubtilis (5) and B. flavum (12) (Table2). The effect of osmotic stabilizers on protoplast regenera-tion was initially examined by using a semisynthetic medi-um. Sorbitol failed to support regeneration at 0.6 and 0.8 M.Sucrose with a concentration over 0.5 M gave rise to unusualtranslucent colonies with a frequency about 10-fold higherthan that of osmotically resistant cells. These coloniesappeared to grow in a protoplastic state, because they couldnot grow upon transfer to hypotonic plates. The addition of0.3 to 0.7 M sodium succinate increased remarkably theefficiency of regeneration (2 to 6%). Colonies formed onplates with 0.3 M sodium succinate were osmotically sensi-tive, as was the case with sucrose, but those formned atconcentrations higher than 0.5 M were osmotically resistantand judged to be regenerated intact cells. Colonies devel-oped faster at 0.5 M than at 0.7 M sodium succinate.Therefore, we added 0.5 M sodium succinate as the osmoticstabilizer.Although strain T106 was prototrophic, its protoplasts

could not regenerate on the chemically defined mediumwhich contained NH4CI as the sole nitrogen source. Thesubstitution of yeast extract for the nitrogen source resultedin efficient regeneration, and the sizes of regenerated colo-nies depended on its concentration. Casamino Acids alone

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PLASMID TRANSFORMATION OF C. GLUTAMICUM 309

TABLE 3. Transformation of various glutamate-producing bacteria with pCG4 DNA

DNA Regeneration Coinheri- TransformationExpt source Recipient protoplasts" frequency Spc' clones"/ml tanec of frequencyd(%) ~~~~~~~~~~SMrt%Y

I T250 C. gI,ataitticim T106 5.3 9.2 x 10' 100 1.2 x 10-6C. glitalninwun ATCC 13032 3.5 2.4 x 10' 83 3.8 x 10 7

C. glitamin-um T218 6.7 1.9 x 102 100 1.8 x 10-6C. glutaili(uin KY9005 3.1 9.0 x 10' 98 1.9 X 10-6C. heru-ldis ATCC 13868 7.1 4.1 x 102 100 3.8 x 10--6B. flavum ATCC 14067 4.3 3.2 x 10' 94 5.0 x 10-7M. al/ltltoliiOpIlililfl ATCC 15354 4.5 2.6 x 10' 69 2.7 x 10 -7

II T250 C. glutarnicuin T106 6.2 1.1 X 102 100 1.2 x 10-6T106 C. glautaiCulin T106 6.2 3.5 x 104 100 4.6 x 10-4

Protoplasts were induced from cells pretreated with 0.5 U of Pen G per ml. A protoplast preparation containing aibout 1.5 x 10' input cells was used initially.Spc' clones were selected directly on RCGA plates containing 400 ,ug of spectinomycin per ml.All or 50 randomly selected Spc' clones were scored for coinheritance of streptomycin resistance (Sm').

"Number of Spc' Sm' clones per regenerated cell.

did not support regeneration, but their addition to theregeneration medium containing low concentrations of yeastextract increased the regeneration rate. Polyvinylpyrroli-done tended to make regeneration more reproducible. Basedon these results, RCGA medium was established as aregeneration medium.

Regeneration of protoplasts was slow and asynchronous.Colonies became visible after 3 days and increased innumber until day 10. Protoplasts of strain T106 and otherglutamate-producing strains regenerated on RCGA mediumat frequencies of 3 to 7% (Tables 2 and 3).

Transformation of protoplasts with plasmid pCG4. Thesuccess in efficient transfection and regeneration of proto-plasts prompted us to try transformation with plasmid pCG4,which is indigenous to C. gliutainiciin T250. The plasmidencodes resistance to spectinomycin and streptomycin,which has enabled us to identify transformants easily.

Protoplasts of glutamate-producing bacteria were trans-formed with 0.1 p.g of pCG4 DNA prepared from C. gllutami-culnl T250. After incubation for phenotypic expression,protoplasts were plated on RCGA agar medium containing400 p.g of spectinomycin per ml. Spectinomycin-resistant(Spcr) colonies developed in all the strains with frequenciesof 10-6 to 10-7 (Table 3, experiment I). Each protoplastpreparation plated on the same selective plates after thesame treatment in the absence of pCG4 gave spontaneousSpcr colonies at frequencies of 10-7 to 10-8. These colonieswere all sensitive to streptomycin, whereas a substantial part

or all of the Spcr colonies arising from pCG4-treated proto-plasts of each strain were resistant to streptomycin. All thedouble-resistant colonies derived from each strain showedthe same MICs against spectinomycin and streptomycin asthose for C. glittarnicuin T250, the original pCG4-carryingstrain (Table 4). The streptomycin-inactivating enzyme ofthese clones had activity comparable with that found in C.glutamicum T250, but untransformed recipient cells did notshow any activity. These results suggest that the double-resistant clones were pCG4 transformants.The presumptive pCG4 transformants were examined for

the presence of CCC DNA. All yielded CCC DNA bands incesium chloride-ethidium bromide density gradient. Whensubjected to electrophoresis, CCC DNA preparations fromdouble-resistant clones of M. ammoniaphilurn ATCC 15345,C. gllutamicum KY9005 and C. glittamicitin T218 gave twoplasmids bands, one corresponding to the CCC pCG4 DNAand the other indigenous to each host (Fig. 1). Completedigestion of these plasmids with the BamHI restrictionendonuclease gave seven common fragments which werefound in the BamHI digest of pCG4. Judging from theamount of plasmid DNA preparation loaded and the intensi-ty of the bands corresponding to the BamnHI fragments ofpCG4, pCG4 appeared to exist in all the transformants testedwith a copy number similar to that in the original host. Thestability of pCG4 in the transformants was checked bygrowth for about 10 generations in the absence of spectino-mycin. No sensitive colonies were detected among 300

TABLE 4. Antibiotic resistance conferred by pCG4 in glutamate-producing bacteria

MIC" (pkg/ml) Streptomycin-Strain inactivating

Spectinomycin Str-cptomycin activity ()C. glhtainicitin T250 >1.600 2')0 92C. gliutatinicium T106 25 3.2 0C. hercidlis ATCC 13868 25 1.6 0B. flaviun ATCC 14067 25 1.6 0M. ammoniaphiliuiin ATCC 15354 25 3.2 0C. glutamincum T106(pCG4) >1. 600 20() 100C. herclilis ATCC 13868(pCG4) >1.600 20)0 95B. flavuin ATCC 14067(pCG4) >1 600 200 100M. aininoniaplhliuin ATCC 15354(pCG4) >1.600 200 96

a One loopful of each cell suspension diluted to ca. 106 cells was inoculated onto BY agar plates containing a series of twofold dilutions of the drug. MICs weredetermined after 24 h of incubation.

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(1t

r; . i A.(.1

*..:I

FIG. 1. Presence of plasmid pCG4 in Spc' Smr transformants ofvarious glutamate-producing bacteria. CCC plasmid DNA was iso-lated from each Spcr Smr transformant. Undigested DNAs (lanes 1,3, 5, 7, 9, and 11) and BamHl-digested DNAs (lanes 2, 4. 6, 8, 10,and 12) were subjected to electrophoresis in 1% agarose gel. Lanes:1 and 2, M. ammoniaphilum transformant; 3 and 4, B. flawi'mntransformant; 5 and 6, C. glutamicum T106 transformant: 7 and 8, C.glutamicum KY9005 transformant; 9 and 10, C. glittaiicum T218transformant; 11 and 12, original pCG4-carrying strain C. glutaini-cum T250.

colonies tested for each strain. These results show thatpCG4 was transferred, expressed, and stably maintained in avariety of glutamate-producing bacteria.The transformation frequency of C. glhtamicllmn T106

protoplasts with pCG4 prepared from C. glittamicirm T250was much lower than the transfection frequency with modi-fied 4CG1 DNA. This suggests the involvement of a restric-tion modification system in C. glltamictim T106. To testthis, the T106 protoplasts were transformed with pCG4prepared from it and T250 (Table 3, experiment II). Thetransformation frequency with pCG4 from strain T106 wastwo orders of magnitude higher than that with pCG4 fromstrain T250, indicating the presence of a restriction modifica-tion system in C. glutamicum T106.The effect of DNA concentration on transfection and

transformation of C. gllutamicum T106 protoplasts was ex-amined with modified 4CG1 and pCG4 DNA. The frequen-cies of both transfection and transformation were linearlyincreased in response to increasing DNA concentration (0.1to 10 ng) and saturated at around 50 ng/ml. The transforma-tion frequency was two orders of magnitude lower than thatof transfection at any concentration tested. Within a linearrange, the frequencies of transfection and transformation per

jig of DNA were about 108 PFU and 5 x 105 transformants,respectively. The difference is explained by the additionalrequirement of the regeneration process for transformation.

DISCUSSION

Kaneko and Sakaguchi have reported that B. flaviuin cellsgrown below inhibitory concentrations of penicillin becamesensitive to lysozyme and were converted to protoplastsunder hypertonic conditions. These protoplasts were fusedand regenerated to give fusants (12). Protoplasts of variousglutamate-producing bacteria prepared by this method havebeen found to be transfected by phage DNA (Ozaki et al.,

submitted for publication). Extending the transfection proce-dure, we established a plasmid transformation system forthese bacteria by using plasmid pCG4, which specifiesresistances to spectinomycin and streptomycin. Althoughbasically similar to those developed for protoplast transfor-mation in B. subtilis and Streptomyces species (3), thissystem needs penicillin pretreatment, which was indispens-able to protoplast formation, as noted by Kaneko andSakaguchi (12), and critically affected the ability of resultantprotoplasts to take up DNA and to regenerate, as demon-strated in this study.Transformation requires both DNA uptake and regenera-

tion, whereas transfection requires only the uptake. There-fore, we tried to maximize the product of the two successiveevents (DNA uptake and regeneration) by adjusting theconcentration of penicillin in the pretreatment and the com-position of the regeneration medium. The resultant proto-plasts, however, still regenerated less efficiently comparedwith protoplasts of B. subtilis (5, 7) and Streptomycesspecies (2, 17). The possibility that colonies appearing fasterinterfere with the regeneration of nearby protoplasts, asobserved with B. subtilis (6) and Streptomyces species (2),was ruled out, because the regeneration frequency did notchange upon plating dilutions of transformation mixtures.Another possibility, that the regeneration medium was stillinappropriate, cannot be denied completely but seems un-likely, because further manipulations of the medium, includ-ing increasing the amount of yeast extract (which was themost effective factor in it), failed to improve the frequency.Thus, we consider it more likely that pretreatment withpenicillin resulted in its covalent binding to membraneproteins involved in cell wall synthesis, as found in alleubacteria examined (22), thereby impairing protoplast re-generation. This may also explain the relatively slow regen-eration. In contrast, protoplasts of B. subtilis and Streptomy-ces species can be prepared without penicillin treatment andthus regenerate more efficiently.The transfection frequency of protoplasts of C. glutami-

cum T106 with PCG1 DNA (10-2 per input cell) is compara-ble to the transformation frequency with plasmid DNAs in B.subtilis and Streptomyces protoplasts (3, 5). This indicatesthat C. gliitainicuin T106 protoplasts are as competent asother protoplasts in DNA uptake. The transformation fre-quency of C. gluitatnicuin T106 with pCG4, however, is morethan two orders of magnitude less than the transfectionfrequency, even if the modified pCG4 was used. The possi-bility that 2 h of incubation is not sufficient for the expres-sion of drug resistance is unlikely, because the transforma-tion frequency in the direct selection used throughout thisstudy was almost identical to that of the indirect selection, inwhich transformed protoplasts were first allowed to regener-ate on a nonselective medium and then selected for spectino-mycin resistance (data not shown). This lower frequency oftransformation is probably due to the fact that it requiresregeneration, which occurred with an efficiency just corre-sponding to the ratio between transformation and transfec-tion. The poor regeneration inherent in the penicillin treat-ment is serious but still allows us to obtain the desiredtransformants at a reasonable frequency. To circumvent thisdefect, we obtained a mutant which can be converted toprotoplasts without penicillin treatment (manuscript in prep-aration).The transformation frequencies differed widely according

to the source of pCG4 DNA. The frequency of C. glitami-cuim T106 with pCG4 isolated from the same host was over100 times higher than that with pCG4 from C. glutamicum

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PLASMID TRANSFORMATION OF C. GLUTAMICUM 311

T250, the native host, indicating the presence of a restrictionmodification system in C. glhutanicuin T106. The relativelylow transformation frequencies of other strains with pCG4from C. glhtaminilin T250 may also be due to their restrictionmodification system.To construct a recombinant DNA technology, we need a

transformation procedure and vectors. The protoplast-medi-ated transformation system presented here will facilitate theconstruction of a plasmid-based cloning system for thisindustrially important group of glutamate-producing bacte-ria, which will eventually be used for gene cloning, as hasbeen successfully done with a similar transformation systemin Streptony(ves species (20, 21). For cloning, a plasmidvector with a selectable marker is desirable, but suchplasmids have not been reported for these bacteria. PlasmidpCG4 can stably replicate and express its spectinomycinand streptomycin resistance genes in these bacteria.Because of its large size (29 kilobases) and multiple sites forvarious restriction endonucleases (unpublished data), pCG4itself is not a practical cloning vector. Instead, a smallerderivative of pCG4 and recombinant plasmids containing apart of pCG4, especially a fragment coding for spectinomy-cin or streptomycin resistance, or both, is more suitable as acloning vector. The construction of these vectors will bereported.

ACKNOWLEDGMENTS

We thank K. Nakayama and T. Suzuki for encouragementthroughout this work and M. Mochizuki for capable technicalassistance.

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