Vol. 42, No. 3INFECTION AND IMMUNITY, Dec. 1983, P. 973-9790019-9567/83/120973-07$02.00/0Copyright C 1983, American Society for Microbiology

Mutagenesis of Extrachromosomal Genetic Determinants forExfoliative Toxin B and Bacteriocin R1 Synthesis in

Staphylococcus aureus After Plasmid Transfer by ProtoplastFusion

ROBERT MASTERSON,1 WILLIAM VON DAVID,1 BILL B. WILEY,2 AND MARVIN ROGOLSKYI*Department ofBiology and School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri

64110,1 and University of Utah School of Medicine, Salt Lake City, Utah 841322

Received 11 July 1983/Accepted 12 September 1983

In previous studies, we have shown that a 27-megadalton plasmid (pRWO02) inStaphylococcus aureus contains genetic determinants for exfoliative toxin B (ETB) and bacteriocin (Bac Rj) synthesis and Bac R, resistance. Attempts totransform or transduce this plasmid to S. aureus or Bacillus subtilis recipientswere not successful. However, genetic transfer of the plasmid was possible afterpolyethylene glycol-induced fusion of S. aureus protoplasts containing pRWO02and S. aureus protoplasts lacking this plasmid. Some of the resulting fusants lostthe ability to make ET B, Bac Rl, or both products. Fusants that were Bac Rl-,Bac Ris, ET B- all lacked the 27-megadalton pRWO02 plasmid. The largest classof fusants was Bac Rl+, Bac R, , ET B. Immunodiffusion analyses of ET Bextracts from 28 fusants showed that four ET B+ strains were cross-reactingmutants that produced ET B protein that was serologically related to, but notidentical to, the wild-type toxin. Results indicated that genetic transfer ofpRWO02after protoplast fusion induced molecular rearrangements that resulted in muta-tion of the genetic determinants for ET B and Bac R, synthesis. Recombination ofchromosomal genes was enhanced after CaCl2 was added to the protoplast-fusionmixture.

Staphylococcal exfoliative toxin (ET) is re-sponsible for an overlapping spectrum of clinicalmanifestations called the staphylococcal scaldedskin syndrome (6). The disease is characterizedby a scarlatiniform rash, bullae, and a general-ized exfoliation which results from intraepider-mal cleavage through the stratum granulosum(6). Both neonatal humans and newborn miceare extremely susceptible to ET (6). In paststudies, our laboratory (6, 14) discovered thatphage group II staphylococci synthesize twoserologically distinct types of ET which weredesignated as ET A and ET B. The geneticdeterminant of ET A synthesis was found to bechromosomal, and the genetic locus for ET Bsynthesis was found to reside on a large 56 S, 27-megadalton (Mdal) virulence plasmid (9, 12, 14).The 27-Mdal virulence plasmid was also foundto contain genes for the production of a bacterio-cin (Bac R1) and resistance to Bac R, (8, 13).Phage group II strains which made one or bothserological types of ET were identified. Warren(12) studied the molecular relationships of theplasmid for ET B synthesis in six different ET'strains. Analyses of the DNA from these strainsby agarose gel electrophoresis showed that they

all contained a 27-Mdal plasmid. EcoRI cleavedfive of these plasmids into four fragments withmolecular masses of 10.7, 6.7, 5.2, and 3.09Mdal. EcoRI digestion of the ET B+ plasmid instrain UT0002(pRWO02) showed that pRWO02differed from the five other ET B+ plasmids inthat it contained small deletions within the 10.7-and 6.7-Mdal fragments.

Previous attempts by our research group totransfer the 27-Mdal ET B+, Bac RI' plasmid byeither DNA-mediated transformation or trans-duction were unsuccessful. A locus on this plas-mid for conjugal-like transfer has not been re-ported. The pT169, 2.7-Mdal plasmid fortetracycline resistance, but not the ET B+, BacRl+ plasmid, could be transformed to proto-plasts of Bacillus subtilis (J. Bradley, and M.Rogolsky, Abstr. Annu. Meet. Am. Soc. Micro-biol. 1981, H116, p. 133). It was then thoughtthat transfer of the ET B+, Bac RI' plasmidmight occur after protoplast fusion. Experimen-tal conditions for the transfer of chromosomaland extrachromosomal genetic traits after fusionof staphylococcal protoplasts was recently re-ported by Gotz et al. (1). Staphylococcal proto-plasts were prepared by treatment with lysosta-

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phin and lysozyme. Treatment of a mixedprotoplast suspension with polyethylene glycol(PEG) resulted in efficient transfer of antibioticresistance plasmids. Recombination of chromo-somal genes was not detected. However, addi-tion of CaCI2 to the fusion mixture resulted inrecombination of chromosomal segments withinthe fused protoplasts.

Stahl and Pattee (10) also obtained geneticrecombinants after protoplast fusion of staphy-lococcal cells. In these studies, chromosomalrecombination in fused strain 8325 protoplastswas achieved by using high concentrations ofPEG in the absence of CaCl2. An excellentreview of genetic studies with bacterial proto-plasts was recently published by Hopwood (2).The major objectives of the work for this articlewere to transfer the pRW002 plasmid for ET Band Bac R1 synthesis by PEG-induced proto-plast fusion and to determine the fate of thisplasmid after transfer.

MATERIALS AND METHODS

Staphylococcal strains. The genetic strains and theirrelevant genetic traits are listed in Table 1. All strainsare phage group II except strain 502A. Strains UT0002and UT0007 are clinical isolates of Staphylococcusaureus (7). Both strains have a 27-Mdal virulenceplasmid for ET B and Bac R1 synthesis (9, 12, 14).Strain UT0002, but not strain UT0007, has both thechromosomal genetic determinant for ET A synthesisand a 2.4-Mdal plasmid (pRWO10) with a gene forcadmium resistance (12, 14). Strain UT0100 is a sub-strain of UT0007 that has been cured of the 27-Mdalplasmid and is therefore not an ET producer (13).Strain UT0017 makes neither ET A nor ET B andcontains no plasmids (7).

Preparation of protoplasts. Strains were grown over-night on heart infusion agar (HIA) (Difco Labora-tories, Detroit, Mich.). Cells from HIA were taken upin 0.85% saline and suspended into 50-ml volumes ofheart infusion broth (Difco) in nephelometer flasks.The suspensions were adjusted to 2 x 108 CFU per mlor 100 Klett units (green filter) and grown to a concen-tration of ca. 5 x 108 CFU per ml or 250 Klett units. Infusion experiments involving strains UT0100 andUT0002, strain UT0100 was inoculated to twice theconcentration of strain UT0002 since strain UT0100grew more slowly than strain UT0002. Cells of eachparent strain from 100 ml of heart infusion broth werethen harvested, washed, and suspended in 10 ml ofsucrose magnesium medium (SMM), which consistedof a final concentration of 100 mM Tris (hydroxy-methyl) aminomethane, 20 mM MgSO4 * 7 H20, and0.8 M sucrose in H20. SMM was adjusted to pH 7.6.Lysostaphin (Sigma Chemical Co., St. Louis, Mo.)was added to a final concentration of 30 jig/ml, and thesuspension was incubated at 37°C for ca. 50 min.Approximately 10% of the strain UT0100 cells and 1%of the strain UT0002 cells were protoplasted. InDNase control experiments, 0.05 ml of DNase I (10mg/ml) (Sigma) was added to the cell suspensionssimultaneously with lysostaphin. The protoplast sus-pensions were centrifuged at 2,500 x g for 8 min topellet whole cells, and the supernatant fluid was takenand centrifuged again at 16,000 x g for 10 min to pelletthe protoplasts. The protoplast pellets were then sus-pended in 0.6 ml of SMM.PEG. PEG (molecular weight 400) (Sigma) and

SMM were mixed to a ratio of 2.5:1.1 (vol/vol),respectively.

Protoplast fusion. To obtain protoplast fusion, 0.1 mlof each protoplast suspension was added to a test tubeat room temperature, and then 1.8 ml of the PEGsolution was added and mixed gently. This mixturewas allowed to incubate at room temperature for atleast 1 min. To isolate chromosomal recombinants,0.02 ml of 1 M CaC12 was added to the suspension at

TABLE 1. Staphylococcal strainsStrain Relevant genetic traits Comments Reference or source

UT0002 ET A', ET B+, Bac Rl+, cad Contains a 27- and a 2.4-Mdal Rogolsky et al. (6, 7, 9)plasmid

UT0007 ET B+, Bac Rl+ Contains a 27-Mdal plasmid Rogolsky et al. (6, 7, 9)UT0100 rif Substrain of UT0007 that has been R. W. Warren

cured of the 27-Mdal plasmid forBac R, and ET B synthesis andmade rifampin resistant

UT0017-T2 cad Obtained after transduction of the Rogolsky et al. (9)2.4-Mdal plasmid for cadmiumresistance from strain UT0002 tothe plasmidless strain UT0017

VR0822 nov-9 Obtained after transformation of the This papernov-9 marker from strain MR103(4) to strain UT0007.

MR101 ery-J Substrain of the plasmidless UT0017 Martin et al. (4)strain that was made erythromycinresistant.

SM0005 rif Obtained after protoplast fusion of This paperstrains UT0100 (rif) and UT0002

502A An indicator strain for Bac R, B. F. Anthonyactivity

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this point and gently mixed. From this suspension, 0.1-ml samples were plated onto regeneration medium.Dilutions were made to determine the number ofregenerated protoplasts per milliliter. The regenera-tion medium consisted of 163.8 g of sucrose, 18 g ofheart infusion broth, 1.8 ml of 1 N NaOH, 15 g ofBacto-Agar (Difco), 300 mg of sodium citrate *2H2O,and 475 ml of distilled water. In DNase control experi-ments, 0.05 ml of DNase I (10 mg/ml) was spread oneach regeneration medium plate immediately beforeuse.

Protoplast suspensions were grown for 4 days at37°C on regeneration medium and were then harvestedin 0.85% saline (5 ml per plate) and sonicated for 1 minat a sufficient output to dispense cell clumps. The cellsfrom 10 regeneration medium plates were then pooled,and dilutions of these pools were plated on bothselective medium and HIA. Selective medium wasHIA supplemented with the proper concentration ofselective agents. The final concentration of agentsadded to HIA for the selection of specific fusants orfor the scoring of specific unselected markers was 80Fg of CdNO3, 40 ,ug of rifampin, 10 ,ug of erythromy-cin, and 2 ,ug of novobiocin per ml. The cells wereincubated on selective medium at 37°C for 48 h.Fusants were streaked twice on selective mediumbefore being scored for unselected markers. Modifica-tions of the fusion procedure were made so that thenecessary controls could be observed. Cells of theparent strains were not lysostaphin treated (cell con-trol). Protoplasts of the parent strains were treatedwith SMM instead of PEG (protoplast control). PEGsolution was used to treat a double portion of proto-plasts of one parental strain (reversion control); thiswas done for each parent strain. When CaCl2 wasused, it was added in the same way to each of thesecontrols, and another control experiment, which wasrun without CaCl2 (experimental control), was alsodone.

Agarose gel electrophoresis. Plasmids were analyzedby agarose gel electrophoresis after in-well lysis ofcells. Cells were grown for 6 to 8 h on HIA at 37°C,and then ca. 1 mm3 of cells was taken up on aninoculating needle and suspended into 200 ,u of stabili-zation buffer. This buffer consisted of 5 ml of glycerol,1 ml of 10-fold concentrated standard electrophoresisbuffer, 4 ml of distilled H20, and 10 ,ug of RNase perml (Sigma). The standard electrophoresis buffer con-sisted of a final concentration of 89 mM Tris (hydroxy-methyl) aminomethane, 2.5 mM disodium EDTA and8.9 mM boric acid (Fisher Scientific Co., Pittsburgh,Pa.). Lysostaphin was added to the stabilization bufferto a final concentration of 200 pLg/ml; lysozyme (Sig-ma) was also added to a final concentration of 2 mg/ml.A 25- to 30-1I portion of this solution was then addedto the wells of a 0.75% agarose (Agarose type II;Sigma) gel and incubated at 37°C for 30 min. Sodiumdodecyl sulfate lysing solution was then added to eachwell and mixed. The sodium dodecyl sulfate lysingsolution consisted of 2% sodium dodecyl sulfate, 10%Ficoll 400,000 (Sigma), and 0.025% bromophenol bluein standard electrophoresis buffer. The gel was thenelectrophoresed for 18 h at 50 V in a Bio-Rad model1415 electrophoresis cell. The gel was removed andstained for 15 min with 1 ,g of ethidium bromide perml. The gel was then washed with distilled H20 andvisualized with a Chromato-Vue Transilluminator (Ul-

tra-Violet Products, Inc., San Gabriel, Calif.). Thestained gel was then photographed with Kodak Tri-Xfilm in a Pentax Spotmatic camera fitted with a Macro-Takumar lens and Wratten Number 2 and Number 23Afilters.

Assay for ET production. ET was assayed by amodification of the procedure previously designed byRogolsky et al. (9). Cultures were grown overnight in aCO2 atmosphere on 5% sheep blood agar. Cells werethen suspended in 0.85% saline, and 0.05 ml of thesuspension was injected into the scapular region of aneonatal mouse under 5 days old. Exfoliation or apositive Nikolsky sign which occurred at the site ofinjection before 24 h was indicative of ET production.Bac R, resistance assay. A crude Bac R1 solution

from strain UT0002 was prepared as described previ-ously (8). The (NH4)2SO4-precipitated crude Bac R1was dissolved 1:3 (wt/vol) in 0.02 M phosphate buffer(pH 7.2). This solution was centrifuged at 20,000 x gfor 20 min and then filter sterilized. One milliliter ofthis solution was spread on HIA and allowed to dry.Strains were streaked onto the plate, and the presenceof growth was indicative of resistance to Bac RI.Resistance to Bac RI was also determined by stabbinga Bac R1+ strain into a lawn of fusant cells on HIA.After overnight growth at 37°C in a CO2 atmosphere,Bac Rl-resistant bacterial lawns contained no clearzones.Bac RI production assay. The assay for Bac R,

production was performed in two steps. Indicatorstrains of S. aureus (UT0017 and 502A) were grown intryptic soy broth (Difco) and then allowed to sit at 4°Cfor 1 week. Lawns of strain UT0017 were then pre-pared on HIA, and 6- to 12-h old cultures of the teststrains from HIA plates were stabbed into the lawns.Agar plates were then incubated in a CO2 atmospherefor 20 h. Colonies surrounded by a clear zone ofinhibition were scored Bac R1l. The same procedurewas repeated on indicator strain 502A, and only thosecolonies producing a clear zone in lawns of strains502A and UT0017 were designated as Bac R1l. Thismethod appeared to minimize the scoring of false-positive Bac R1 producers.

Preparation of ET extracts. Staphylococcal wild-type and fusant strains were grown for 48 h in heartinfusion broth at 37°C in a shaking water bath with agas hood. The cultures were flushed three times dailywith 90%o CO2 throughout the growth period. Theywere then centrifuged at 10,000 x g, and the superna-tant fluids were collected. To precipitate the crudetoxin, saturated (NH4)2SO4 was added to a final con-centration of 80%. The extracts were allowed to standovernight at 4 to 6°C. The precipitated crude ET wascollected by centrifugation at 10,000 x g and taken upin 200 ,ul of phosphate-buffered saline (pH 7.4). Theprecipitates were allowed to stand overnight at 4 to6°C to dissolve and were centrifuged at 10,000 x g forclarification before use in immunodiffusion tests.Immunodiffusion tests. A micromethod for immuno-

diffusion was carried out on microscope slides. Staph-ylococcal ET extracts (10 pu) were added to sixperipheral wells. A central well received 10 p.l of ETantiserum, which was either undiluted or diluted 1:2.The agar medium consisted of 1% agarose and 1%PEG 6000 in phosphate-buffered saline (pH 7.4). ETwas purified after isoelectricfocusing as described byWiley and Rogolsky (14). Specific antiserum for ET A

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or ET B was prepared in rabbits by injection ofpurified ET by a procedure previously designed byWiley and Rogolsky (14).

RESULTSGenetic transfer after PEG-induced protoplast

fusion of strains UT0002 and UT0100. StrainUT0002 contains a 27-Mdal plasmid (pRWO02)with genes for ET B and Bac R1 synthesis (12).It also contains the chromosomal locus for ET Asynthesis and a 2.4-Mdal plasmid (pRWO10) forcadmium resistance (cad). The ET-, Bac Rl-strain UT0100 contains no plasmids and has achromosomal locus for rifampin resistance (riJ).After PEG-induced protoplast fusion of thesetwo strains, regenerated fusants were selectedfor the rifand cad markers. Genetic transfer wasnot affected by DNase, which indicated thattransfer did not occur by PEG-induced transfor-mation of protoplasts (2). The frequency of Rifr,Cadr fusants among regenerated protoplasts wasca. 5 x 10-4. This frequency was in closeagreement with that found by Gotz et al. (1) afterprotoplast fusion of phage group III strainswhere selection was made for at least one plas-mid-associated trait. At the concentration ofantibiotics used, controls routinely showed nogrowth, which indicated that the frequency ofreversion to antibiotic resistance was less than 1X lo-9.Table 2 summarizes the phenotypes of the

progeny obtained after protoplast fusion ofstrains UT0002 and UT0100. The largest class offusants was Bac Ri+, Bac R, , ET B. Thisclass constituted ca. 40% of total number offusants. Of the 115 isolated fusants, 18 were BacRl-, Bac R1s, ET B-. All strains within thisclass lacked the 27-Mdal pRWO02 plasmid afteranalyses by agarose gel electrophoresis (Fig. 1,

TABLE 2. Fusant phenotypes resulting from thetransfer of plasmid pRWO02 from strain UT0002 to

strain UT0100 by protoplast fusiona

Phenotypeb No. of fusants

Bac RI', Bac R1l, ET B.............. 45Bac Rl-, Bac Rlr, ETB ............. 24Bac R1-, Bac Ril, ETB.............. 18Bac Rl-, Bac Rlr, ET B+.............. 17Bac Rj+, Bac RI , ET B .............. 11

a Strain UT0002 has a 27-Mdal plasmid (pRWO02)with genetic determinants for Bac R, production (BacRj+), Bac R, resistance (Bac RIr), and ET B produc-tion (ET B+). Strain UT0002 also contains a 2.4-Mdalplasmid that encodes for cadmium resistance (cad) anda chromosomal determinant for ET A synthesis. StrainUT0100 is Bac Rl-, Bac Rl, ET B-, ET A-, Cads,contains no plasmids, and has a chromosomal determi-nant for rifampin resistance (rij). Fusants were select-ed for rif and cad.

b None of the fusants were ET A+.

FIG. 1. Agarose gel electrophoresis of DNA fromfusants obtained after transfer of the pRWO02 plasmidfrom strain UT0002 to strain UT0100 by protoplastfusion. DNA from strain (lane 1) UT0100 (Bac Rl-,Bac Rls, ET B-); (lane 2) SM0104 (Bac R1l, Bac Rl ,ET B-); (lane 3) SM0103 (Bac Rl+, Bac Rl , ET B);(lane 4) SM0102 (Bac Rl, Bac Rl(, ET B+); (lane 5)SM0101 (Bac RI-, Bac Rl, ET B-); (lane 6) SM0100(Bac R1l, Bac Rl, ET B-); (lane 7) SM0099 (Bac R1l,Bac Ri', ET B-); (lane 8) SM0098 (Bac R1l, Bac RI',ET B-); (lane 9) SM0097 (Bac RJ-, Bac Rlr, ET B+);(lane 10) UT0002 (Bac R1l, Bac RI', ET B+). SeeTable 2 for information about the properties ofpRWO02 and pRWO10 and about how the fusants wereobtained.

lane 5). Of the 115 fusants, only 11 were BacR1, Bac R1', ET B+.None of the fusants listed in Table 2 produced

ET A. This indicates that chromosomal recom-bination was not occurring after protoplast fu-sion. It appears that unidirectional transfer ofthe 27-Mdal pRWO02 and 2.4-Mdal pRWO10plasmids from the UT0002 strain to the UT0100strain after fusion was a common event. Ifgenetic interaction after protoplast fusion ofthese strains included mixing and genetic recom-bination of the parental chromosomes, some ofthe fusants would have been expected to be ETA'. However, this might have not been the caseif the strain UT0002 chromosome had multiplesites for ET A synthesis or if the frequency of

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chromosomal recombination was below the lim-its of detection with our procedure.

All fusants listed in Table 2 which were able toinduce exfoliation or a positive Nikolsky sign inneonatal mice were subsequently shown to pro-duce ET B and to contain the 27-Mdal pRWO02plasmid. None of the fusants which lackedpRWO02 gave a positive Nikolsky sign. Everyfusant that possessed pRWO02 was always resis-tant to Bac R1 but not necessarily ET B+ or BacR1 . This indicated that genetic transfer ofpRWO02 after PEG-induced protoplast fusioninduced molecular rearrangements, which led tomutation of the genetic determinants for ET Band Bac R1 synthesis. However, this did notappear to be the case for the genetic locus thatdetermines Bac R1 resistance.

Plasmid analyses by agarose gel electrophore-sis. All of the fusant progeny listed in Table 2were analyzed after electrophoresis on 0.75%agarose gels for their plasmid content. Some ofthese analyses are shown in Fig. 1. None of theET B-, Bac R1s, Bac Rl- strains had the 27-Mdal pRWO02 plasmid. The DNA from one ofthese strains is clearly missing the pRWO02plasmid at the top of the gel (Fig. 1, lane 5).Every fusant strain that was resistant to Bac R1had the 27-Mdal pRWO02 plasmid. The DNAfrom such strains are shown in lanes 2, 3, 4, 6, 7,8, 9, and 10 of Fig. 1. Strains used to obtain theDNA bands shown in lanes 2, 3, 6, 7, and 8 wereBac R1, Bac R1,, ET B, and the DNA in lane 4came from a Bac R1l, Bac R1', ET B+ strain.The strain used for lane 9 was Bac RJ-, Bac R1l,ET B+. All fusants contained the 2.4-MdalpRWO10 plasmid for cadmium resistance sincethey were selected for this resistance trait afterprotoplast fusion. This plasmid can be seen ineach lane of the gel shown in Fig. 1 with theexception of lane 1, which contains DNA fromthe plasmidless UT0100 strain. DefectivepRWO02 plasmids within a few of the fusantsappeared to have slightly faster migration ratesduring electrophoresis than the wild-typepRWO02 plasmid (Fig. 1, lane 10). This indicatedthat small deletions might have impaired thegenes for ET B or Bac R1 synthesis, but accu-rate detection of these deletions was beyond thesensitivity of the technique that was used.

Serological testing of ET' fusants. Immunodif-fusion assays between ET extracts from theET' fusants listed in Table 2 and either ET A orET B antiserum showed that every ET+ strainproduced ET B but not ET A. These agar doublediffusion analyses proved to be even more inter-esting and significant when they showed that 4of the 28 ET B+ strains were cross-reactingmutants. Figure 2 shows the immunodiffusionpatterns of ET B antiserum and ET B from threeof these cross-reacting mutants. The spurs pro-

FIG. 2. Immunodiffusion analysis of ET B pro-duced by fusants obtained after the transfer of thepRWO02 plasmid from strain UT0002 to strain UT0100by protoplast fusion. The center well contains anti-ETB serum. Peripheral wells 1, 2, and 3 contain extractsof ET B from fusant strains SM0050, SM0108, andSM0109, respectively. Alternating peripheral wellscontain purified preparations of ET B.

duced by the ET B protein from strains SM0050(well 1), SM0108 (well 2), and SM0109 (well 3)indicate that these toxin proteins are serological-ly related to, but not identical to, the wild-typeET B protein in the altdrnating peripheral wellsof the gel shown in Fig. 2. The cross-reactingmutants had reduced ET activity in comparisonwith the other ET B+ fusants as determined byneonatal mouse test assays. The isolation ofthese cross-reacting mutants provides furtherevidence that genetic transfer of the pRWO02plasmid by PEG-induced protoplast fusion Canresult in mutation of the genetic determinant forET B synthesis.Chromosomal recombination after protoplast

fusion. In the genetic transfer experiment afterprotoplast fusion of strains UT0002 and UT0100,evidence was provided to indicate that recombi-nation between parental chromosomes did notoccur. To be more certain about this observa-tion, we performed further genetic transfer ex-periments by protoplast fusion (Table 3). In thegenetic crosses shown in experiments 1, 5, and 7of Table 3, it can be seen that few to no fusantswere recovered when a chromosomal markerfrom each parent strain was selected. However,in experimental crosses 3, 4, 5, and 6, it can beseen that when a chromosomal marker from oneparent strain and an extrachromosomal markerfrom the second parent were selected, fusants

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TABLE 3. Genetic transfer of chromosomal and plasmid genetic traits after protoplast fusion of phage groupII staphylococcal strainsa

Expt Parent strains fusion mixture Selected traits Frequencyb

1 VR0822 (nov-9, ET B+, Bac RI+) and UT0100 (rif) nov rif 9 x 10-82 VR0822 (nov-9, ET B+, Bac RI+) and UT0100 (rif) CaCl2 nov rif 8 x 10-63 UT0002 (cad, ET A', ET B+, Bac RI+) and UT0100 (rif) cadc rif 5.8 x 10-4d4 VR0822 (nov-9, ET B+, Bac RI+) and UT0017-T2 (cad) nov cadc 3.9 x 10-55 VR0822 (nov-9, ET B+, Bac RI+) and SM0005 (cad, rif) nov cad' 6.9 x 10-6e

nov rif 8.1 x 10-96 VR0822 (nov-9, ET B+, Bac RI') and SM0005 (cad, rif) CaCl2 nov cad' 1.6 x 10-5

nov rif 1.4 x 0-77 VR0822 (nov-9, ET B+, Bac RI+) and MR101 (ery-1) nov ery 0a nov, Novobiocin resistance; cad, cadmium resistance; rif, rifampin resistance; ery, erythromycin resistance.b Frequency was defined as the number of fusants on selective medium divided by the total number of

regenerated CFU per milliliter, which was in the range of 109 to 1010 CFU per ml.c The cad marker for cadmium resistance is located on plasmid pRW010.d None of the fusants were ET A'.e None of the fusants had the rif trait. Addition of DNase I did not reduce the number of fusants.

were recovered at high frequencies. It appearsthat chromosomal recombination between phagegroup II strains either does not occur or occursat low frequencies after the PEG-induced proto-plast fusion procedure used by our laboratory.The latter possibility is probably the general rulesince chromosomal recombination in the ab-sence of CaCl2 was sometimes observed (Table3, crosses 1 and 5) but at extremely low frequen-cies. Experimental crosses 2 and 6 show thatchromosomal recombinants are recovered at fre-quencies of 8 x 10-6 and 1.4 x 10-7, respective-ly, if a final concentration of 10 mM CaCl2 isadded to the PEG-protoplast suspension. Con-centrations of CaCl2 higher than 10 mM wereextremely toxic and lethal to the protoplasts.The frequency of chromosomal recombinationin the presence of 10 mM CaC12 was ca. 50 timeslower than the frequency of plasmid transfer.CaC12 did not affect the frequency of plasmidtransfer. Gotz et al. (1) reported that treatmentof protoplasts with 90 mM CaC12 was requiredfor recombination of phage group III strain 8325chromosomal genes when their protoplast fusionprocedure was used. Stahl and Pattee (10) ob-served high frequencies of recombination ofstrain 8325 chromosomal genes after protoplastfusion in the absence of CaC12. However, chro-mosomal recombination between the phagegroup II strains shown in Table 3 could not bedetected after utilizing the fusion procedure ofStahl and Pattee (10).

DISCUSSION

S. aureus provides an ideal model for thestudy of microbial genetics. It possesses manydiverse and unique plasmids which can housemany different types of genes for antibiotic and

metal ion resistance, genes for bacteriocin syn-thesis and for toxin production. One such plas-mid, pRW002, which contains genes for Bac R1and ET B synthesis, was the subject for thestudies reported here. A decade ago, only trans-duction was available to investigate the transferof staphylococcal chromosomal and extrachro-mosomal genes. Fortunately, this is no longerthe case. In addition to transduction, DNA-mediated transformation (3, 4, 11), a conjugal-like mechanism (5), and PEG-induced protoplastfusion are also now available as tools to investi-gate staphylococcal genetics.

In past efforts, our laboratory was unable totransfer a large 27-Mdal plasmid for Bac R1 andET B synthesis by either transduction or trans-formation. Therefore, our success in finallytransferring this plasmid after PEG-induced pro-toplast fusion was a welcome and satisfyingaccomplishment. Protoplast fusion has thereforeproven itself to be a novel method to transferlarge staphylococcal plasmids that cannot betransformed and are too large to be carried bytransducing phage.Although unidirectional transfer of plasmids

occurred at high frequencies after fusion ofphage group II strains, effective transfer andrecombination of chromosomal genes was diffi-cult to achieve. Gotz et al. (1) observed thatchromosomal recombination in fused strain 8325protoplasts only occurred when Ca2+ was com-bined with PEG. It was suggested that PEG, byitself, led to incomplete fusion, which allowedfor the transfer of plasmids but not for theproper transfer, mixing, and pairing of homolo-gous chromosomes from the parent strains.These events were probably simulated in oursystem, since frequencies of chromosomal re-combination were greatly enhanced in the pres-

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ence of Ca2" ions. Ca2+ ions had no effect on thefrequency of plasmid transfer. Chromosomalrecombination was observed to occur at muchlower frequencies than plasmid transfer in bothfused 8325 strains (1) and fused phage group IIstrains.A significant and interesting observation was

that transfer of the pRWO02 plasmid by proto-plast fusion induced mutations within the genesfor ET B and Bac R1 synthesis. Similar muta-tions of staphylococcal plasmid genes were ob-served after transfer of gentamycin resistanceplasmids by a conjugal-like mechanism (5). Fu-sants carrying mutations within the genetic locusfor ET B synthesis were found to produce eitherno ET B or ET B with decreased biologicalactivity that was serologically related to, but notidentical to, the wild-type toxin. Since onlyplasmid and not chromosomal genes were ob-served to be affected after protoplast fusion inthe absence of Ca2+ ions, the above resultsfurther substantiate the theory that genetic de-terminants for ET B synthesis do indeed resideon the pRWO02 plasmid.

If mutations of the genes for ET B or Bac R1synthesis were caused by deletions, insertions,or translocations, it would then be possible tomap these genes on the pRWO02 plasmid. Thiswould be accomplished by cleaving wild-typeand defective plasmids with different restrictionendonucleases and then attempting to correlatedefective plasmid fragments with the genes forBac R1 and ET B synthesis. The laboratory ofM.R. is presently initiating such studies.

ACKNOWLEDGMENTS

This investigation was supported in part by Faculty Re-search grant K2-10440 and Weldon Springs Endowment Fundgrant K3-40139 from the University of Missouri to M.R.

Gratitude is extended to Richard McDonnell for providingvaluable comments and suggestions on some aspects of thiswork.

LITERATURE CITED

1. Gotz, F., S. Ahrne, and M. Lindberg. 1981. Plasmidtransfer and genetic recombination by protoplast fusion instaphylococci. J. Bacteriol. 145:74-81.

2. Hopwood, D. A. 1981. Genetic studies with bacterialprotoplasts. Annu. Rev. Microbiol. 35:237-272.

3. Jackson, M. P., J. DeSena, J. Lednicky, B. McPherson, R.Haile, R. G. Garrison, and M. Rogolsky. 1983. Isolationand characterization of a bacteriophage factor that conferscompetence for genetic transformation to an exfoliativetoxin-producing strain of Staphylococcus aureus. Infect.Immun. 39:939-947.

4. Martin, S. M., S. Shoham, M. Alsup, and M. Rogolsky.1980. Genetic mapping in phage group 2 Staphylococcusaureus. Infect. Immun. 27:532-541.

5. McDonnell, R. W., H. M. Sweeney, and S. Cohen. 1983.Conjugational transfer of gentamicin resistance plasmidsintra- and interspecifically in Staphylococcus aureus andStaphylococcus epidermidis. Antimicrob. Agents Che-mother. 23:151-160.

6. Rogolsky, M. 1979. Nonenteric toxins of Staphylococcusaureus. Microbiol. Rev. 43:320-360.

7. Rogolsky, M., R. Warren, B. B. Wiley, H. T. Nakamura,and L. A. Glasgow. 1974. Nature of the genetic determi-nant controlling exfoliative toxin production in Staphylo-coccus aureus. J. Bacteriol. 117:157-165.

8. Rogolsky, M., and B. B. Wiley. 1977. Production andproperties of a staphylococcin genetically controlled bythe staphylococcal plasmid for exfoliative toxin synthesis.Infect. Immun. 15:726-732.

9. Rogolsky, M., B. B. Wiley, and L. A. Glasgow. 1976.Phage group II staphylococcal strains with chromosomaland extrachromosomal genes for exfoliative toxin produc-tion. Infect. Immun. 13:44-52.

10. Stahl, M. L., and P. A. Pattee. 1983. Computer-assistedchromosome mapping by protoplast fusion in Staphylo-coccus aureus. J. Bacteriol. 154:395-405.

11. Thompson, N. E., and P. A. Pattee. 1981. Genetic transfor-mation in Staphylococcus aureus: demonstration of acompetence-conferring factor of bacteriophage origin inbacteriophage 80a lysates. J. Bacteriol. 148:294-300.

12. Warren, R. L. 1980. Exfoliative toxin plasmids of bacte-riophage group 2 Staphylococcus aureus: sequence ho-mology. Infect. Immun. 30:601-606.

13. Warren, R., M. Rogolsky, B. B. Wiley, and L. A. Glasgow.1975. Isolation of extrachromosomal deoxyribonucleicacid for exfoliative toxin production from phage group IIStaphylococcus aureus. J. Bacteriol. 122:99-105.

14. Wiley, B. B., and M. Rogolsky. 1977. Molecular andserological differentiation of staphylococcal exfoliativetoxin synthesized under chromosomal and plasmid con-trol. Infect. Immun. 18:487-494.

VOL. 42, 1983

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