plasmidtransfer in pediococcus intergeneric intrageneric of · 37°c) at a final concentration of...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1983, P. 81-890099-2240/83/070081-09$02.00/0Copyright 0 1983, American Society for Microbiology

Vol. 46, No. 1

Plasmid Transfer in Pediococcus spp.: Intergeneric andIntrageneric Transfer of pIP5O1CARLOS F. GONZALEZt* AND BLAIR S. KUNKA

Microlife Genetics, Sarasota, Florida 33578

Received 20 January 1983/Accepted 21 April 1983

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcusfaecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurredbetween cells immobilized on nitrocellulose filters in the presence of DNase.Expression of the pIP501-linked erythromycin and chloramphenicol resistancedeterminants was observed in transconjugants. Intrageneric transfer of pIP501from a P. pentosaceus donor to various pediococcal recipients occurred atfrequencies of 10-4 to 10-7 transconjugants per input donor cell. Intergenerictransfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcussanguis (Challis), and Streptococcus lactis was observed. Similar mating experi-ments showed no evidence for the transfer of the broad-host-range R-plasmidpAMP1 to Pediococcus spp. recipients.

The pediococci are a group of the homofer-mentative lactic acid bacteria that are ecologi-cally, morphologically, and physiologically simi-lar to the lactic streptococci. However, thepediococci can be differentiated from the lacticstreptococci by their failure to react with groupN streptococcal antiserum and by examinationof their mode of division. The pediococci formtetrads upon division, whereas the streptococciform chains (30). Members of the pediococci aregenerally found on plant material (20), ferment-ed vegetables (23), and in beer (24). They com-prise a group of economically important bacteriain the fermented food industry. These lactic acidbacteria are utilized for the fermentation ofvegetables (23) and meats (5).Although many taxonomic and physiological

studies have been conducted on the genus Pedi-ococcus (3, 10, 21, 30), no evidence for theexistence of genetic transfer systems, such astransduction, transformation, or conjugation,has been reported. However, reports of conjuga-tion have been described for other gram-positivebacteria such as the streptococcal strains be-longing to Lancefield groups A, B, D, F, H, andN (1, 7, 13, 14, 17, 19, 27), Lactobacillus casei(8), Staphylococcus aureus (25), and Bacillussubtilis (1Sa).

In an initial attempt to establish a genetictransfer system in Pediococcus spp., we reporthere the DNase-resistant transfer of the strepto-coccal macrolide-lincosamide-streptogramin B(MLS) resistance plasmid pIP501 (20 megadal-

t Address inquiries to: Microlife Genetics, P.O. Box 2339,Sarasota, FL 33578.

tons [Mdal]) from Streptococcusfaecalis to Ped-iococcus pentosaceus and Pediococcus acidilac-tici. Additionally, we show the intragenerictransfer of plasmid pIP501 by a conjugation-likemechanism from Pediococcus spp. donors toPediococcus spp. and intergeneric transfer toStreptococcus lactis, S. faecalis, and Strepto-coccus sanguis (Challis) recipients. The pres-ence of a resident plasmid(s) ranging in size from4.7 to 30 Mdal in Pediococcus spp. was ob-served during the course of this study.

MATERIALS AND METHODSBacterial strains and media. The bacteria and plas-

mids used in this study are listed in Table 1. S. faecalisand S. sanguis (Challis) were routinely maintained onbrain heart infusion (BHI; Difco Laboratories, De-troit, Mich.). Pediococcus spp. and S. lactis isolateswere routinely carried on APT (Difco). As necessary,the appropriate antibiotic(s) was added to sterile mediato maintain selection for resistance markers. Cultureswere grown at 32°C unless otherwise indicated.A modification of Elliker broth (6) not containing

lactose or sucrose, but only glucose, was used asgrowth medium and designated as BMG broth. Agar(Difco) was added to give a final concentration of 2.0%when a solid medium was desired. Carbohydrate fer-mentation was carried out in medium BMG withoutglucose (BM) containing bromocresol purple at a finalconcentration of 0.08% and the desired filter-sterilizedcarbohydrate at a final concentration of 0.5%.

Selective antibiotic concentrations were as follows:erythromycin (Sigma Chemical Co., St. Louis, Mo.)for S. faecalis and S. sanguis (Challis), 10 ,ug/ml;erythromycin for Pediococcus species and S. lactis, 2,ug/ml; streptomycin sulfate (Sigma), 1,000 pLg/ml; fu-sidic acid, sodium salt (Fus) (Leo Pharmaceutical,Denmark; gift of W. 0. Godtfredsen), 20 ,ug/ml; rifam-

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82 GONZALEZ AND KUNKA

pin (Sigma), 400 jig/ml; chloramphenicol (Calbiochem,La Jolla, Calif.), 20 ,ug/ml.Mating conditions. Donor and recipient strains were

grown overnight on APT agar containing the appropri-ate selective antibiotic, unless otherwise specified.After overnight growth at 32°C, a turbid suspensionwas prepared from the culture and inoculated intoBMG broth, unless otherwise indicated. The cellswere incubated statically at 32°C and allowed to growto approximately 108 CFU/ml. Mating conditions wereessentially as described by Smith and Guild (26). Thedonor and recipient cells were mixed at ratios of 1:1and 0.1:1 and filtered onto a 0.45-ji.m nitrocellulosefilter (Millipore Corp., Bedford, Mass.). Mating mix-tures, suspended in broth, were diluted and plated onBMG agar medium (unless otherwise specified) con-taining the selective antibiotics. Plates were incubatedfor 48 h at 32°C and scored for CFUs. Controlsconsisting of donor and recipient cells alone were

treated similarly to determine frequency of spontane-ously appearing, drug-resistant mutants in the popula-tion. Transfer frequency was expressed as the numberof resistant colonies per number of donor CFU at timezero. Transconjugants were purified by serial colonyisolation on medium containing selective antibiotics.Transconjugants were also subjected to analysis bytransfer of individual colonies to medium containingantibiotic(s) to which the recipients were resistant andwhich had not been used in the mating selection.Additionally, the ability to ferment selected carbohy-drates was used for such analysis. For convenience,the following characteristics were used to differentiatestrains: S. faecalis strain JH2-2 fermented xylose andmannitol, but not arabinose; P. pentosaceus and P.acidilactici fermented arabinose, but not mannitol; S.lactis strain SLA1.3 was arabinose and xylose nega-tive.

Control matings with nonviable donor cells were as

TABLE 1. Bacterial strains

Strain [Chromosomal Plasmid Plasmid I Plasmid phenotype' Source or derivationphenotype' size (Mdal)jNoneNoneEmr, Tra+Emr, Cmr, Tra+Emr, Cmr, Tra+

Lac'None

None

NoneEmr, Cmr, Tra+

UnkNone

UnkNoneNoneUnk/Emr, Cmr,Tra+

Emr, Cmr, Tra+UnkNoneUnk/Emr, Cmr,Tra+

UnkUnkUnk

Unk/UnkUnk

Unk/Emr, Cmr,Tra+

D. ClewellbD. ClewellbD. ClewellbD. ClewellbThis study

W. Sandine'Lac-, LM0231 (15), cured

of pLM2103, this studySmr, Fusr, LM0230, this

study

F. MacrinadF. Macrinad

NRRL B-11465PPE1.0 cured of pSRQ1,

this studySmr, PPE1.0, this studySmr, PPE1.2, this studyFusr, PPE1.4, this studyPPE1.3(pIP501), this study

PPE1.4(pIP501), this studyRifr, PPE1.0, this studyRifr, PPE1.2, this studyPPE1.10(pIP501), this

studyATCC 25745Smr, PPE3.0, this studyFusr, PPE3.3, this study

ATCC 33316Smr, PPE4.0 cured ofpSRQ8, this study

PPE4.2(pIP501), this study

Streptococcus faecalisJH2-2JH2SSJH2-2JH2-2JH2SS

S. lactisLM0231LM0230

SLA1.3

S. sanguis (Challis)V879V683

Pediococcus pentosaceusPPE1.0PPE1.2

PPE1.3PPE1 .4PPE1.5PPE1.8

PPE1.9PPE1 .10PPE1.11PPE1.12

PPE3.0PPE3.3PPE3.4PPE4.0

PPE4.2

PPE4.4

Rifr, FusrSmr, Sp'Rifr, FusrRifr, FusrSmr, Sp,

NoneNone

Smr, Fusr

SmrNone

NoneNone

SmrsmrSmrSmr, FusrSmr

SmrRifrRifrRifr

NoneSmrSmr, FusrNone

Smr

Smr

NoneNonepAMI1pIP501pIP501

pLM2103None

None

NonepIP501

pSRQ1None

pSRQ1NoneNonepSRQ1/

pIP501pIP501pSRQ1NonepSRQ1/

pIP501pSRQ7pSRQ7pSRQ7pSRQ8/pSRQ9

pSRQ9

pSRQ9/pIP501

172020

20 to 22

30

30

30, 20

2030

30, 20

121212

17, 6.76.7

6.7, 20

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CONJUGAL TRANSFER IN PEDIOCOCCUS SPP. 83

TABLE 1-Continued

Strain Chromosomal Plasmid Plasmid Plasmid phenotypea Source or derivationphenotype' size (Mdal)

P. acidilacticiPAC1.0 None pSRQ10/ 23, 4.7 Unk/Unk NRRL B-5627

pSRQ11PAC1.1 Smr pSRQ10/ 23, 4.7 Unk/Unk Smr, PAC1.0, this study

pSRQ11PAC1.2 Smr, Fusr pSRQ10/ 23, 4.7 Unk/Unk Fusr, PAC1.1, this study

pSRQ11PAC1.3 Smr pSRQ10/ 23, 4.7, Unk/Unk/Emr, PAC1.1(pIP501), this study

pSRQ11/ 20 Cmr, Tra+pIP501

PAC1.5 Smr, Fusr pSRQ10/ 23, 4.7, Unk/Unk/Emr, PAC1.2(pIP501), this studypSRQ11/ 20 Cmr, Tra+pIP501

PAC2.0 None None None ATCC 33314PAC2.1 Smr None None Smr, PAC2.0, this studyPAC2.3 Smr, Fusr None None Fusr, PAC2.1, this studyPAC2.4 Smr, Fusr pIP501 20 Em', Cm', Tra+ PAC2.3(pIP501), this studyPAC2.5 SmT pIP501 20 Emr, Cmr, Tra+ PAC2.1(pIP501), this study

a Abbreviations: Rif, rifampin; Fus, fusidic acid; Sm, streptomycin; Sp, spectinomycin; Em, erythromycin;Cm, chloramphenicol; r, resistant; Tra+, transfer proficient; Unk, unknown; Lac+, lactose positive; Lac-,lactose negative.

b University of Michigan.Oregon State University.

d Virginia Commonwealth University, Richmond.

described by Hershfield (13). Filter matings in thepresence of DNase were done as above, except thatpancreatic DNase I (Sigma; type 1) was added to cellmixtures, and the medium was as described by Smithet al. (27). For broth matings, the donor and recipientwere grown as described above. The donor and recipi-ent were mixed at the same ratios as above andincubated statically for 3 to 4 h. Mixtures were mixedin a Vortex mixer, diluted, and plated on appropriateselective medium. To rule out the possibility of trans-duction, the donor and recipients were grown asabove, the donor culture was centrifuged, and thesupernatant was filtered through a 0.22->±m filter (Mil-lipore). The donor filtrate and recipient cells werecombined at ratios of 1:1 and 0.1:1. The mixtures werethen treated by two methods: (i) filtration through a0.45-,um filter (Millipore) and treatment as above; (ii)incubation of 0.2 ml of the mixture on a BMG agarplate for 3 h and then treatment as above.

Plasmid isolation and purification. Survey lysis ofcells to determine the acquisition or loss of plasmidDNA was done by the procedure of LeBlanc and Lee(16), with only the modification of growth medium. S.lactis for lysis was grown in the lysis broth of Klaen-hammer et al. (15); Pediococcus spp. isolates weregrown in APT broth; and S. faecalis and S. sanguis(Challis) were grown in BHI broth. Samples of etha-nol-precipitated DNA were subjected to electrophore-sis on vertical 0.7% agarose slab gels as previouslydescribed (11). Photography of gels was as previouslydescribed (9).

Large quantities of plasmid DNA were obtainedfrom 1-liter broth cultures grown to the mid-exponen-

tial phase (4 h at 32°C) in the appropriate medium asdescribed above. Cells were concentrated by centrifu-gation and washed in 1/10 volume of TE buffer (4).Washed cells were suspended in 1/40 the originalvolume and lysed by a scaled-up modification of theLeBlanc and Lee (16) procedure. The cells wereexposed to pronase (Sigma) (predigested for 90 min at37°C) at a final concentration of 0.5 mg/ml afterlysozyme treatment. Centrifugation of CsCI-ethidiumbromide gradients was carried out at 18°C in a SorvallTV865 vertical rotor for 18 h at 55,000 rpm with aSorvall OTD65B ultracentrifuge. Plasmid DNA wasextracted with CsCI-saturated isopropanol to removeethidium bromide and dialyzed against 1 mM EDTA(Sigma)-10 mM Tris (Sigma) buffer (pH 8.0) at 4°C.

Reference DNA. Plasmid DNA for use in agarosegels was prepared from sodium dodecyl sulfate-lysedcells (28) and purified by cesium chloride-ethidiumbromide centrifugation as described above. Bacterialstrains and plasmid sizes were as follows: Escherichiacoli V517, 1.4, 1.8, 2.0, 2.6, 3.4, 3.7, 4.8, and 35.8Mdal (18); Pseudomonas aeruginosa PAO2(pRO161),24.5 Mdal (12); and Salmonella typhimurium cryptic,60 Mdal (12).

Restriction enzyme analysis. Plasmid DNA was di-gested with AvaIl, BstEII, HaeIII, HindIII, HpaI,Sall, and SphI restriction enzymes (Bethesda Re-search Laboratories, Inc., Gaithersburg, Md.) accord-ing to manufacturer's instructions. Analysis of frag-ments was by electrophoresis on 0.7 or 1.0% agaroseslab gels.MIC. Cultures to be tested were grown on APT agar

containing the appropriate selective antibiotic. After

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overnight growth at 32°C, a turbid suspension wasprepared in BMG broth and diluted so that approxi-mately 100 CFU was plated onto agar plates. Theminimal inhibitory concentration (MIC) was deter-mined on BMG agar containing doubling concentra-tions of the antibiotic, starting from 0.015 ug/Iml. TheMIC was recorded as the lowest concentration ofantibiotic preventing colony formation after 24 h ofincubation at 32°C.

Induction of erythromycin resistance. The regulation(inducible or constitutive) of erythromycin resistancewas determined essentially by the method of Hersh-field (13). These assays were conducted with BMGbroth. Challenge concentrations were 20 or 40 ,ug/ml.Growth of the cultures was followed with a Spec-tronic-20 (Bausch & Lomb, Inc., Rochester, N.Y.) at600 nm.

Curing procedure. The stability of plasmid-encodedtraits was evaluated by two methods: growth of cells atvarious temperatures and exposure of cells to a plas-mid curing agent. Strains were grown overnight onAPT agar containing the appropriate selective antibiot-ic. After overnight incubation at 32°C, a suspension inbroth containing 108 CFU/ml was prepared from theplate culture. The cells (50 ,u) were inoculated into 10ml of BMG broth and incubated in water baths equili-brated at 32, 35, 42, 45, and 48°C for 18 h. Temperatureexposed cultures were diluted and plated on BMGagar. For curing with acriflavin (Sigma), the cells wereinoculated similarly into BMG broth containing acri-flavin at final concentrations of 0, 5, 10, 12, 15, and 20p.g/ml, respectively, and grown in the dark for 18 h at32°C. Acriflavin exposed cultures growing at the high-est non-inhibitory concentration were diluted and plat-ed on BMG agar. Individual colonies from the abovetreatments were tested for susceptibility by streakingonto plates containing the appropriate antibiotic.

RESULTSMating experiments. To establish mating con-

ditions in our laboratory, S. faecalis strain JH2-2containing the broad-host-range plasmid pIP501(20 Mdal) or pAMP1 (17 Mdal) was used in filtermatings with S. faecalis strain JH2SS as arecipient. Transfer of these plasmids to theisogenic recipient occurred at the expected fre-quencies (Table 2). Transfer of plasmid pIP501from the S. faecalis donor to P. pentosaceus andP. acidilactici ranged from 10-5 to 10-8 trans-conjugants per donor (Table 2). P. pentosaceusstrain PPE3.3 showed no evidence as an effec-tive recipient in transfer experiments with the S.faecalis donor strains (Table 2). In filter matingsunder conditions that allowed the transfer ofplasmid pAMP1 to S. faecalis, no evidence forthe transfer of pAMp1 to Pediococcus spp.recipient strains was observed (Table 2). Selec-tive plating of donor-recipient mixtures after themating period showed that there was no reduc-tion in the viability of either.

Intrageneric transfer of plasmid pIP501 from aP. pentosaceus transconjugant, PPE1.12, to P.pentosaceus and P. acidilactici recipients varied

TABLE 2. Frequency of transfer of plasmids pIP501and pAM01 in S. faecalis and Pediococcus Spp.a

Transfer StrainPlasmid Recipient frequency designation

per donorb ofugantcnpIP501 S. faecalis 1.0 X 10-4 JH2SS

JH2SS (pIP501)P. pentosaceusPPE1.3 1.0 x 10-6 PPE1.8PPE1.4 1.3 x 10-6 PPE1.9PPE3.3 <10-8PPE4.2 1.0 x 10-6 PPE4.4

P. acidilacticiPAC1.1 4.3 x 10-5 PAC1.3PAC2.1 3.0 x 10-8 PAC2.5

pAMP1 S. faecalis 1.9 x 10-4 JH2SSJH2SS (pAM1)

P. pentosaceusPPE1.3 <10-8PPE1.4 <10-8PPE4.2 <10-8

P. acidilacticiPAC1.1 <10-8PAC2.1 <10-8

a The donor was S. faecalis JH2-2.b Transfer frequency is expressed as the number of

resistant colonies per donor CFU. Donor CFU weredetermined before mating. Transconjugants were se-lected for the chromosomal resistance of the recipientand the donor plasmid. Frequency is the average ofresults obtained in three independent experiments.The number <10-8 represents the lower limit of detec-tion.

from 10-4 to 10-7, with no transfer detected toP. pentosaceus strain PPE3.4 (Table 3). OtherP. pentosaceus and P. acidilactici donors ofpIP501 showed transfer frequencies of 10-4 to0-5 to a P. pentosaceus recipient (Table 3).Intergeneric transfer of plasmid pIP501 from a

P. pentosaceus donor to S. faecalis, S. sanguis(Challis), and S. lactis was lower than thatobserved in intrageneric matings (Table 3). Fre-quencies of 7.5 x 10-5 and 1 x 10-2 transconju-gants per donor were obtained when S. sanguis(Challis) containing pIP501 was mated with P.pentosaceus and S. lactis, respectively (data notshown).The transconjugants obtained in each mating

were analyzed for selected and unselected mark-ers. Erythromycin-resistant Pediococcus spp.recipient mutants were not detected in any of theexperiments. Streptomycin-resistant S. faecalisdonors were easily distinguished by carbohy-drate fermentation characteristics routinely usedin the unselective marker analysis of transconju-gants. Likewise, streptomycin-resistant Pedio-coccus spp. donors could be easily distinguished



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TABLE 3. Intergeneric and intrageneric transfer ofplasmid pIP501

TransferDonor Plasmid Recipient frequency

per donor'

P. pentosaceus pIP501 P. pentosaceusPPE1.12b PPE1.5 4.8 x 1o-5

PPE3.4 <10-8PPE4.2 1.3 x 1o-4

P. acidilacticiPAC1.2 2.4 x 1O-4PAC2.3 3.8 x 10-7

S. faecalis 9.0 x 10-7JH2SS

S. sanguis 5.0 x 10-7(Challis) V879C

S. lactis SLA1.3 2.7 x 10'6P. pentosaceus P. pentosaceusPPE4.4 PPE1.11 5.4 x 10-5

P. acidilactici P. pentosaceusPAC1.5 PPE1.11 8.3 x 10-4PAC2.4 PPE1.11 1.5 x 10-4

a Transconjugants were selected for the resistancesof the recipient chromosome and the donor plasmid.Donor CFUs were determined before mating. Fre-quency is the average of results obtained in threeindependent experiments.

b A transconjugant of a JH2SS(pIP501) x PPE1.10mating.

c Donor and recipient were grown on APT broth formating with S. sanguis (Challis); selection for trans-conjugants was on BHI agar containing streptomycin(1,000 pLg/ml) and erythromycin (10 p.g/ml).

from recipients by unselective marker analysiswith fusidic acid and carbohydrate fermentationcharacteristics.The filter matings described in Tables 2 and 3

using the S. faecalis and P. pentosaceus donorstrains containing plasmid pIP501 were also con-ducted in the presence of 70 ,ug of DNase I perml. In two independently performed experi-ments, no reduction of transfer frequencies wasobserved when matings were performed in thepresence of DNase I. No transconjugants weredetected in mating studies with sodium hypo-chlorite-treated donor cells, chloroform-treateddonor cells, or cell-free supernatants as donorswhen mixed with recipients. Furthermore, brothmatings of donor and recipient combinationsdescribed in Tables 2 and 3 did not show evi-dence for transfer of plasmid pIP501.

Plasmid analysis. The presence or absence ofcovalently closed circular (CCC) DNA in eachof the Pediococcus spp. strains used in the studywas confirmed by subjecting lysates to cesiumchloride-ethidium bromide density gradient cen-trifugation. For accurate sizing of the residentplasmids in the Pediococcus spp. strains, theCCC DNA obtained from gradients was subject-

ed to agarose gel electrophoresis in parallel withplasmid DNA ranging in size from 1.4 to 60Mdal. P. pentosaceus strain PPE1.1 contained asingle plasmid of 30 Mdal designated pSRQ1(Fig. 1, lane D). P. pentosaceus PPE3.0, showedthe presence of a single 12-Mdal plasmid desig-nated pSRQ7 (Table 1). Strain PPE4.0 showedtwo plasmids of 17 and 6.7 Mdal designatedpSRQ8 and pSRQ9, respectively (Table 1). Ex-amination of P. acidilactici strain PACO. re-vealed the presence of two plasmids of 23 and4.7 Mdal, designated pSRQ10 and pSRQ11, re-spectively (Fig. 1, lane I). Strain PAC2.0 con-tained no detectable plasmid DNA (Table 1).

Confirmation of the presence of plasmidpIP501 was accomplished by examination oflysates of donor, recipients, and transconjugantsfrom matings shown in Tables 2 and 3. Cesiumchloride-ethidium bromide gradient-purifiedplasmid DNA from lysates of select isolates wassubjected to agarose gel electrophoresis. Atleast two to three forms of pIP501 were found

A

35.cc,cI-LIN

4.8 f

1A-

FIG. 1. Agarose gel electrophoresis of CsCl-ethidi-um bromide-purified plasmid DNA from donor S.faecalis and recipient pediococcal strains. Electropho-resis of DNA was in 0.7% agarose at 100 V for 2 h.Bands are identified from top to bottom. (A) S. typhi-murium LT2 cryptic; 60-Mdal CCC DNA and frag-mented chromosomal DNA. (B) V517 35.8-Mdal CCCDNA; chromosomal DNA; 4.8-, 3.7-, 3.4-, 2.6-, 2.0-,1.8-, and 1.4-Mdal CCC DNA. (C) JH2-2(pIP501); 20-Mdal CCC DNA band and linear (LIN) DNA form ofpIP501 (marked to left of the figure). (D) PPE1.10; 30-Mdal CCC DNA pSRQ1 and chromosomal DNA. (E)PPE1.12; CCC pSRQ1; small amount of CCC pIP501;chromosomal DNA and linear pIP501 (F) PPE1.9;faint CCC pIP501 and linear pIP501. (G)PPE4.2; open circular pSRQ9 and CCC pSRQ9, 6.7Mdal. (H) PPE4.4; CCC pIP501; linear pIP501; opencircular pSRQ9 and CCC pSRQ9. (I) PAC1.1; opencircular pSRQ10; CCC pSRQ10, 23 Mdal; faint chro-mosomal DNA; open circular PSRQ11 and CCCPSRQ11, 4.7 Mdal. (J) PAC1.3; CCC pSRQ10; chro-mosomal DNA; linear pIP501; open circular pSRQ11and CCC pSRQ11. The molecular size of standardDNA is noted to the left of the figure.

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upon dialysis of gradient samples. The CCCform of plasmid pIP501 was identified from itselectrophoretic migration relative to CCC DNAfrom E. coli V517. The linear form was identifiedby two methods: its characteristic migrationbelow linear A DNA and digestion of the CCCform with HaeIII, which cleaves pIP501 in onlyone site. Examination of gel patterns revealedthe presence of resident plasmid(s) in recipientsand the addition of plasmid DNA identical inmigration with that of the S. faecalis donorstrain (Fig. 1). Parental strains are shown in Fig.1, lanes D, G, and I, whereas their correspond-ing transconjugants containing pIP501 areshown in lanes E, H, and J. The phenotypesexpressed by these transconjugants were thoseexpected, that is, erythromycin and chloram-phenicol resistance (see below). Additionally,the transconjugants used as donors in subse-quent mating experiments were able to transferplasmid pIP501 in intergeneric and intragenericmatings (see above).

Restriction enzyme analysis of plasmid DNAfrom Pediococcus spp. transconjugants. Furtherphysical evidence for the identity of plasmidpIP501 among the pediococcal transconjugantswas obtained by restriction enzyme analysis ofplasmid DNA from these strains. Plasmid DNAfrom the transconjugant strain PPE1.9 (Fig. 1,lane F), which contained only pIP501, was com-pared by restriction enzyme analysis to DNAfrom the donor strain JH2-2(pIP501). The analysiswith HaeIII, HpaI, BstEII, AvaIl, and HindIllshowed that pIP501 DNA isolated from the JH2-2donor strain and strain PPE1.9 had identical frag-ment patterns (Table 4). The identity of pIP501 instrain PPE1.12 (Fig. 1, lane E) was further con-firmed by SphI digestion of its plasmid DNA.Digestion of plasmid pIP501 with SphI resulted intwo fragments measuring 11.6 and 9.3 Mdal (Fig.21, lane B). SphI digestion of plasmid pSRQ1resulted in fragments of 11.6, 5.4, 4, 3.5, 2.3, 1.5,1.4, and 0.9 Mdal (Fig. 21, lane C). An SphI digestofPPE1.12 plasmid DNA revealed the presence ofthe 11.6- and 9.3-Mdal fragments from pIP501 inthe pediococcal transconjugant (Fig. 2I, lane D).Similarly, conclusive evidence for the presence ofundeleted pIP501 was confirmed in strain PPE4.4(Fig. 1, lane H). HpaI digestion of plasmid pIP501yielded fragments that were 15.3, 4.0, and 1.2Mdal in size (Table 4; Fig. 2II, lane B). The HpaIdigestion of plasmid pSRQ9 revealed two frag-ments of 4.0 and 2.7 Mdal (Fig. 2II, lane C).Digestion of plasmid DNA from the transconju-gant PPE4.4 showed the addition of the 15.3- and1.2-Mdal fragments of pIP501 in the transconju-gant (Fig. 21I, lane D). The 4.0-Mdal fragments ofpSRQ9 and pIP501 co-electrophoresed and aretherefore indistinguishable.MIC testing and resistance expression. The

TABLE 4. Restriction enzyme analysis of plasmidpIP501 from S. faecalis and P. pentosaceus host

Restric- No. of Calculated size of

enzyme fragments fragment (Mdal)a

S. faecalis HaeIII 1 20JH2-2(pIP501) HpaI 3 15.3, 4.0, 1.2

BstEII 4 8.5, 6.5, 3.6, 1.3AvaIl 3 10.7, 5.1, 4.8HindIlI 13 4.2, 3.2, 2.3, 1.4,

1.2, 1, 0.9,0.8, 0.7, 0.6,0.5, 0.4, 0.1b

P. pentosaceus HaeIII 1 20PPE1.9 HpaI 3 15.3, 4.0, 1.2

BstEII 4 8.5, 6.5, 3.6, 1.3AvaIl 3 10.7, 5.1, 4.8HindIlI 13 4.2, 3.2, 2.3, 1.4,

1.2, 1, 0.9,0.8, 0.7, 0.6,0.5, 0.4, 0.1

a The sizes offragments were calculated by compar-ison with A DNA fragments produced by Sall andHindlIl digestion. The size determination was basedon at least two independently performed digests.

b Calculation for fragments of 0.5, 0.4, and 0.1 Mdalwere taken from Evans and Macrina (6a).

level of resistance to erythromycin and chloram-phenicol was investigated in parental and trans-conjugant strains of Pediococcus spp. that hadacquired the plasmid pIP501 (Table 5). Theresistance to erythromycin expressed by the P.pentosaceus and P. acidilactici transconjugantswas equivalent to those observed in S. faecalis(>4,000 ,ug/ml). The parental P. pentosaceusand P. acidilactici strains showed sensitivity toerythromycin at 0.24 and 0.12 jxg/ml, respective-ly. The chloramphenicol resistance level for P.pentosaceus isolates containing pIP501 was 64pug/ml, whereas P. acidilactici showed a resist-ance level of 32 ,ug/mI.We examined the regulation of erythromycin

resistance to determine whether the expressionwas inducible in a P. pentosaceus transconju-gant containing plasmid pIP501. The growth ofstrain PPE1.12 was temporarily inhibited by achallenge dose of 20 or 40 ,ug of erythromycinper ml when not previously treated for 4 h at aninducing subinhibitory concentration (0.1,Ig/ml). Induced cultures showed no lag ingrowth (data not shown).

Stability of pIP501 in Pediococcus spp. Thestability of the antibiotic traits encoded for byplasmid pIP501 was evaluated in P. pentosaceusand P. acidilactici. Growth of P. pentosaceusPPE1.12 at temperatures of 32, 35, or 42°Cresulted in the detection of erythromycin-sensi-



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FIG. 2. Restriction enzyme analysis of CsCl-ethidi-um bromide-purified plasmid DNA from Pediococcusspp. recipients and transconjugants. Agarose gels(0.7%) were electrophoresed at 100 V for 2 h. (I)Lanes: A, Hindlll digest of XDNA (size in Mdal notedto left of figure); B, SphI digest of plasmid pIP501 fromstrain JH2-2; C, SphI digest of plasmid pSRQ1 fromstrain PPE1.10 (Fig. 1, lane D); D, SphI digest ofplasmid DNA from PPE1.12 (Fig. 1, lane E), a trans-conjugant of PPE1.10 containing pIP501. (II) Lanes:A, Hindlll digest of XDNA; B, HpaI digest of pIP501;C, HpaI digest of plasmid pSRQ9 from strain PPE4.2(Fig. 1, lane G); D, HpaI digest of plasmid DNA fromPPE4.4 (Fig. 1, lane H), a transconjugant of PPE4.2containing pIP501.

tive and chloramphenicol-sensitive segregants ata frequency of 0.25%. At 45°C, erythromycin-and chloramphenicol-sensitive segregants wereobserved at a frequency of 22% (98 of 450).Exposure of the same strain to 5 ,ug of acriflavinper ml at 32°C resulted in erythromycin- andchloramphenicol-sensitive segregants at a fre-quency of 0.25%. Similar studies with P. acidi-lactici strain PAC1.3 resulted in erythromycin-and chloramphenicol-sensitive segregants at afrequency of 0.75% at 32, 35, or 42°C. Frequen-

cies of 4 and 47% (212 of 450) were observed at45 and 48°C, respectively. Acriflavin exposure

of strain PAC1.3 yielded antibiotic-sensitive seg-regants at a frequency of 1%. Erythromycin- andchloramphenicol-sensitive derivatives of strainsPPE1.12 and PAC1.3 showed sensitivity to bothantibiotics at a concentration equivalent to thoseobserved in the wild-type parental strains. Ex-amination of lysates by slab agarose gel electro-phoresis revealed the erythromycin- and chlor-amphenicol-sensitive isolates to be devoid ofplasmid pIP501 DNA.

DISCUSSIONThe data presented here provide evidence for

a genetic transfer system in Pediococcus spp.

The intra- and intergeneric transfer of plasmidDNA establishes Pediococcus as an exchangerof genetic information with other gram-positivegenera.

Mating experiments suggested that the modeof transfer of plasmid DNA was by conjugation.This premise was supported by the followinglines of evidence: (i) the addition of DNase I tomating mixtures did not affect the mating fre-quencies observed; (ii) no plasmid transfer wasobserved from nonviable donor cells; and (iij)when filtrates from either Pediococcus spp. orS. faecalis donor strains were mixed with recipi-ent cells, no erythromycin-resistant colonieswere detected. It is therefore unlikely that trans-formation or transduction was involved in thetransfer process observed.

Analysis of plasmid pIP501 DNA from lysatesof transconjugants showed no difference in themobility when compared with that from the S.faecalis donor. Restriction digests of plasmidpIP501 DNA from S. faecalis and Pediococcusspp. transconjugants showed identical fragmentpatterns (Fig. 2, Table 4) and agreed with frag-ment patterns reported by Hershfield (13) 4ndfragment sizes reported by Evans and Macrina(6a). No deletion mutants of plasmid pIP501

TABLE 5. MIC of erythromycin and chloramphenicol for S. faecalis donor and Pediococcus spp. donor andrecipient strains

MIC t>g/mI)Strain Plasmids

Erythromycin Chloramphenicol

S. faecalis JH2-2 pIP501 >4,000 64

P. pentosaceusPPE1.10 pSRQ1 0.24 2PPE1.12 pSRQ1/pIP501 >4,000 64PPE1.9 pIP501 >4,000 64

P. acidilacticiPAC1.2 pSRQ10/pSRQ11 0.12 1PAC1.3 pSRQ10/pSRQ11/pIP501 >4,000 32

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were observed in any of the Pediococcus spp.transconjugants surveyed.Pediococcus spp. transconjugants from pri-

mary matings were used as donors for furtherexperiments (Table 3), and the plasmid andresistance characteristics observed in secondarytransconjugants were as expected. P. pentosa-ceus and P. acidilactici transconjugants utilizedas donors to transfer pIP501 into other Pedio-coccus recipients were found to transfer theplasmid at a higher frequency than the S. faecal-is donor (JH2-2) to the same recipients. Transferof pIP501 from P. pentosaceus to S. faecalisshowed a reduction in transfer frequency, per-haps due to host restriction mechanisms. Stud-ies with plasmid pIP501 in S. pneumoniae haveshown its ability to mobilize a nonconjugativeplasmid (27). A survey of transconjugants fromthe Pediococcus spp. x Pediococcus spp. mat-ings revealed no evidence for mobilization ofany resident Pediococcus plasmid(s) by plasmidpIP501. The inability to obtain any transconju-gants of isolates P. pentosaceus PPE3.3 orPPE3.4 with either an S. faecalis or a P. pento-saceus donor, respectively, may have been dueto host-controlled restriction mechanisms in therecipients or the inability to form a mating pairwith the donor.

Conjugal transfer of plasmid pAMP1 has beendemonstrated in nine different species of Strep-tococcus (1), as well as L. casei (8), S. aureus(25), and B. subtilis (15a). It would therefore beanticipated that transfer of pAMP1 to Pediocor-cus spp. would have occurred; however, noevidence for transfer was observed. A report byClewell et al. (2) has shown that, in S. faecalis,the transferability of pAMP1 was inhibited dra-matically when a highly transmissable plasmidsuch as pAMyl was present in the donor. Thiswas not the case with the JH2-2 donor used inour experiments, which contained only pAMP1.Failure to transfer into Pediococcus spp. maythen be related to the inability of pAMp1 toinitiate replication, lack of expression of itsMLS determinant, or deleterious deletionscaused during or after transfer into Pediococcusspp. LeBlanc et al. (17) have suggested thatstreptococcal plasmids often undergo deletionsor molecular rearrangemnnts during or aftertransfer to new host species by either transfor-mation or conjugation. Such rearrangements ordeletions could have resulted in the loss oftransmissability or failure to mediate resistanceto MLS antibiotics. Evidence from DNA anneal-ing studies with plasmids containing genes thatcode for MLS resistance suggests that there maybe substantial sequence diversity among theMLS resistance determinants, which may affectexpression in different hosts (22). Alternatively,Hershfield (13) has suggested that 17 to 20-Mdal

MLS plasmids, which include pIP501 andpAM,1, probably represent a class of sex fac-tors analogous to an incompatibility group in E.coli and that the MLS plasmids form a cohesive,yet evolving, family of plasmids. This evolvingpattern was inferred by the similarities of pat-terns generated by restriction digests of MLSplasmids. However, one basic difference doesexist in the control of expression of the MLSresistance determinant in pAMP1 and pIP501,the former having constitutive expression (29)and the latter an inducible type (13). In any case,the host range of pAMP1 does not extend intoPediococcus spp., as might be expected of abroad-host-range plasmid.The role which the resident plasmid(s) ob-

served in the examined Pediococcus spp. mayplay in the survival or properties expressed bythis group of microorganisms is unknown. Theestablishment of a conjugal transfer system inPediococcus spp. will serve as a useful tool forpossible transfer or mobilization and identifica-tion of plasmid phenotype(s) in Pediococcusspp. Increased knowledge of this group ofmicroorganisms will aid in the development ofimproved strains for food fermentation.

LITERATURE CITED

1. Clewell, D. B. 1981. Plasmids, drug resistance and genetransfer in the genus Streptococcus. Microbiol. Rev.45:409-436.

2. Clewell, D. B., Y. Yagi, Y. Ike, R. A. Craig, B. L. Brown,and F. An. 1982. Sex pheromones in Streptococcusfaeca-lis: multiple pheromone systems in strain DS5, similaritiesof pAD1 and pAMyl, and mutants of pAD1 altered inconjugative properties, p. 97-100. In D. Schlessinger(ed.), Microbiology-1982. American Society for Microbi-ology, Washington, D.C.

3. Coster, E., and H. R. White. 1964. Further studies on thegenus Pediococcus. J. Gen. Microbiol. 37:15-31.

4. Currier, T. C., and E. W. Nester. 1976. Isolation of cova-lently closed circular DNA of high molecular weight frombacteria. Anal. Biochem. 76:431-441.

5. Diebel, R. H., G. D. Wilson, and C. F. Niven, Jr. 1961.Microbiology of meat curing. IV. Lyophilized Pediococ-cus cervisiae starter culture for fermented sausage. AppI.Microbiol. 9:239-243.

6. Elliker, P. R., A. Anderson, and G. H. Hannessen. 1956.An agar culture medium for lactic streptococci and lacto-bacilli. J. Dairy Sci. 39:1611-1612.

6a.Evans, R. P., Jr., and F. L. Macrina. 1983. StreptococcalR plasmid pIP501: endonuclease site map, resistancedeterminant location, and construction of novel deriva-tives. J. Bacteriol. 154:1347-1355.

7. Gasson, M. J., and F. L. Davies. 1980. Conjugal transferof the drug resistance plasmid pAMP in lactic streptococ-ci. FEMS Microbiol. Lett. 7:51-53.

8. Gibson, E. M., N. M. Chace, S. B. London, and J. Lon-don. 1979. Transfer of plasmid-mediated antibiotic resist-ance from streptococci to lactobacilli. J. Bacteriol.137:614-619.

9. Gonzalez, C. F., and A. K. Vidaver. 1979. Bacteriocin,plasmid and pectolytic diversity in Pseudomonas cepaciaof clinical and plant origin. J. Gen. Microbiol. 110:161-170.

10. Gunther, H. L., and H. L. White. 1961. The cultural andphysiological characters of the pediococci. J. Gen. Micro-biol. 26:185-197.



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11. Hansen, J. B., Y. Abiko, and R. C. Curtiss III. 1981.Characterization of the Streptococcus mutans plasmidpVA318 cloned into Escherichia coli. Infect. Immun.31:1034-1043.

12. Hansen, J. B., 4nd R. H. Olsen. 1978. Isolation of largebacterial plasmids and characterization of the P2 incom-patibility group plasmids pMG1 and pMG5. J. Bacteriol.135:227-238.

13. Hershfield, V. 1979. Plasmids mediating multiple drugresistance in group B streptococcus: transferability andmolecular properties. Plasmid 2:137-149.

14. Horodniceanu, T., D. H. Bouanchaud, G. Bieth, and Y. A.Chabbert. 1976. R plasmids in Streptococcus agalactiae(Group B). Antimicrob. Agents Chemother. 10:795-801.

15. Klaenhammer, T. R., L. L. McKay, and K. A. Baldwin.1978. Improved lysis of group N streptococci for isolationand rapid characterization of plasmid deoxyribonucleicacid. Appl. Environ. Microbiol. 35:592-600.

15a.Landman, 0. E. D., D. J. Bodkin, C. W. Finn, Jr., andR. A. Pepin. 1981. Conjugal transfer of pAMB1 fromStreptococcus anginosis to Bacillus subtilis and plasmid-mobilized transfer of chromosomal markers between B.subtilis strains, p. 219-228. In M. Polsinelli and G. Mazza(ed.), Transformation-1980.

16. LeBlanc, D. J., and L. N. Lee. 1979. Rapid screeningprocedure for detection of plasmids in streptococci. J.Bacteriol. 140:1112-1115.

17. LeBlanc, D. J., L. N. Lee, J. A. Donkersloot, and R. J.Harr. 1982. Plasmid transfer in streptococci (an over-view), p. 82-87. In D. Schlessinger (ed.), Microbiology-1982. American Society for Microbiology, Washington,D.C.

18. Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers,and S. M. McCowen. 1978. A multiple plasmid containingEscherichia coli strain: convenient source of size refer-ence plasmid molecules. Plasmid 1:417-420.

19. McKay, L. L., K. A. Baldwin, and P. M. Walsh. 1980.

Conjugal transfer of genetic information in group N strep-tococci. Appl. Environ. Microbiol. 40:84-91.

20. Mundt, J. O., W. G. Begttle, and F. R. Wieland. 1969.Pediococci residing on plants. J. Bacteriol. 98:938-942.

21. Nakagawa, A., and K. Kitahara. 1959. Taxonomic studieson the genus Pediococcus. J. Gen. Appl. Microbiol. 5:95-126.

22. Ounissi, H., and P. Courvalin. 1982. Heterogenicity ofMacrolide-lincosamide-streptogramin B type antibioticresistance determinants, p. 167-169. In D. Schlessinger(ed.), Microbiology-1982. American Society for Microbi-ology, Washington, D.C.

23. Pederson, C. S. 1949. The genus Pediococcus. Bacteriol.Rev. 13:225-232.

24. Pederson, C. S., M. N. Albury, and R. S. Breed. 1954.Pediococcus cerevisiae the beer sarcina. Wallerstein Lb.Commun. 17:7-17.

25. Schaberg, D. R., D. B. Cleweil, and L. Glatzer. 1982.Conjugative transfer of R- plasmids from Streptococcusfaecalis to Staphylococcus aureus. Antimicrob. AgentsChemother. 3:315-324.

26. Smith, M. D., and W. R. Guild. 1980. Improved methodfor conjugative transfer by filter mating of Streptococcuspneumoniae. J. Bacteriol. 144:457-459.

27. Smith, M. D., N. B. Shoemaker, V. Burdett, and W. R.Guild. 1980. Transfer of plasmids by conjugation in Strep-tococcus pneumoniae. Plasmid 3:70-79.

28. Vandenbergh, P. A., S. A. Syed, C. F. Gonzalez, W. J.Loesche, and R. H. Olsen. 1982. Plasmid content of somemicroorganisms isolated from subgingival plaque. J. Dent.Res. 61:497-501.

29. Weisblum, B., S. B. Holder, and S. M. Halling. 1979.Deoxyribonucleic acid sequence common to staphylococ-cal and streptococcal plasmids which specify erythromy-cin resistance. J. Bacteriol. 138:990-998.

30. Whittenbury, R. 1965. A study of some pediococci andtheir relationship to Aerococcus viridans and the entero-cocci. J. Gen. Microbiol. 40:97-106.

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