molecular cloning of an erythromycin resistance determinant in

JOURNAL OF BACTERIOLOGY, Nov. 1980, p. 806-8130021-9193/80/11-0806/08$02.00/0

Vol. 144, No. 2

Molecular Cloning of an Erythromycin ResistanceDeterminant in Streptococci

DETLEV BEHNKEt AND JOSEPH J. FERRETTI*Department ofMicrobiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma

City, Oklahoma 73190

The erythromycin resistance determinant of plasmid pDB102, a derivative ofplasmid pSM19035, was cloned into the single HindIII site of the 3.6-megadaltoncryptic Streptococcus mutans plasmid pVA318 and introduced into Streptococcussanguis strain Challis by transformation. Plasmid pDB201, which was isolatedfrom one of the transformants, consisted of the vector plasmid and the 1.15-megadalton HindIII fragment D of pSM19035. HindIlI fragment D containedwithin it one of the two unique "spacer" sequences between the unusually largeinverted repeat sequences of pSM19035. Electron micrographs of self-annealedmolecules of the recombinant plasmid revealed classical stem-loop structures, andthe resistance determinant of pSM19035 appeared as a transposon-like structure.No differences were observed in either the type or the level of erythromycinresistance by pSM19035 or pDB201. The availability of a cloned erythromycinresistance determinant should be useful for future comparative studies of ma-crolide, lincosamide, and streptogramin B resistance plasmids in streptococci.

The streptococci as a group of organisms areresponsible for a wide variety of diseases and areaccordingly of considerable medical importance.The emphasis of research concerning these or-ganisms has been of a biochemical and immu-nological nature, and this work has resulted inthe availability of relatively easy detection andscreening methods for many of the clinicallyimportant streptococcal characteristics. In con-trast, the genetics of these organisms has beenpoorly characterized, in part because gene trans-fer systems have been only recently described,but primarily because of the fastidious nature ofthese organisms and a lack of selective markers.The ability to utilize recombinant DNA tech-nology in streptococci would provide a new ap-proach for an improved genetic analysis of theseorganisms and would open new avenues of in-vestigation for the study of gene expression,regulation, and plasmid evolution. The possibil-ity of performing recombinant DNA experi-ments in streptococci became more of a realitywith the description of potential streptococcalvector plasmids such as pVA318 (16) andpDB1O1 (1). In this report, we describe the mo-lecular cloning of the erythromycin resistancedeterminant of streptococcal plasmid pDB102and the first step towards expanding the possi-bilities of genetic analysis in streptococcci.Among the various types of antibiotic resist-

t Present address: Zentralinstitut fur Mikrobiologie undExperimentelle Therapie, DDR-69 Jena, German DemocraticRepublic.

ance described in the genus Streptococcus, re-sistance to erythromycin is of the greatest inter-est since this antibiotic is the drug of choice forpatients who are sensitive to penicillin. Plasmid-mediated resistance to erythromycin has beenreported in group A (5), B (10), and D (6) strep-tococci, and transfer is promiscuous not onlyamong these strains (11, 14, 17, 18), but alsoamong group F and H streptococci, oral strep-tococci (15), and certain strains of lactobacilli(8). The incidence of erythromycin-resistantstreptococci appears to be increasing (13, 19, 23),and of great interest is the recent description ofan erythromycin resistance transposon in Strep-tococcus faecalis which exhibits enhanced trans-position in the presence of erythromycin (24).Many of the erythromycin resistance plasmidsstudied to date are in the same molecular weightrange, and some show similarities in restrictionendonuclease digestion patterns (la, 9) or shareextensive nucleotide sequence homologies (25,27), or both. One of these plasmids, pDB1l1 (2),is a derivative of the MLS resistance plasmidpSM19035 originally obtained from a group Astreptococcal clinical isolate (7). A detailed phys-ical map of this plasmid has been constructed(1), and electron microscopy results indicate thepresence of unusually large inverted repeat se-quences situated between two "spacer" seg-ments of unique nucleotide sequences (3). Wepresent the results of the first molecular cloningexperiments in streptococci which have allowedthe identification of the erythromycin resistance

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MOLECULAR CLONING IN STREPTOCOCCI 807

determinant as a transposon-like structure onone of the unique spacer segments of plasmidpSM19035 or its derivatives pDB1l1 andpDB102.

MATERIALS AND METHODSBacteria and media. Streptococcus sanguis strain

Challis (pSM19035) contained the naturally occurringMLS resistance plasmid pSM19035. The origin ofChallis (pDB102), which harbors the deletion mutantpDB102 of plasmid pSM19035, has been reported else-where (2). The plasmid-free strain Challis-6 served asthe recipient in the transformation experiments.Streptococcus mutans V318, the host strain of thecryptic 3.6-megadalton (Mdal) plasmid pVA318 (16),was kindly provided by F. Macrina. Brain heart infu-sion broth (Difco) was used as a broth medium andwas solidified with agar throughout these experiments.The special media used for transformation of strainChallis were as described previously (14).

Materials. Restriction endonucleases EcoRI,BglII, HindII, HindIII, KpnI, and SalI, as well asbacteriophage T4 ligase, were prepared and generouslyprovided by M. Hartmann and F. Walter, Jena. En-zymes BamHI, HpaI, Sau3A, and TaqI were pur-chased from New England Biolabs, HaeII was fromBethesda Research Laboratories, and HpaII was fromBoehringer/Mannheim. The other reagents and theirsources were described previously (1).

Isolation of plasmid DNA. Growth in the pres-ence of D,L-threonine (4) and glycine (21) facilitatedthe lysis of S. sanguis and S. mutans, respectively.Plasmid DNA was isolated after dye-buoyant densitygradient centrifugation of deproteinized cleared ly-sates (1).

Restriction enzyme analysis and gel electro-phoresis. Plasmid DNA preparations (0.5 to 1 ,ug)maintained in 10 mM Tris-hydrochloride (pH 7.4)were subjected to cleavage with different restrictionendonucleases under conditions described previously(1) or according to the instructions of the manufac-turer of the enzyme. When double digestions wereperformed, the enzyme requiring the lower salt con-centrations was used first, and appropriate adjust-ments were made to the reaction buffer before theaddition of the second enzyme. Separation of restric-tion fragments was accomplished on vertical 1% aga-rose slab gels in Tris-acetate buffer (2) or on 3.5%polyacrylamide gels containing 25% glycerol (12, 20).

Ligation of the restriction fragments andtransformation. Plasmids pDB102 (5 jg) andpVA318 were mixed in a concentration ratio of 2:1 toensure that the fragment to be cloned was present inat least a twofold higher amount than that of thevector plasmid. After digestion with Hindlll the sam-ple was heated for 5 min at 65°C to inactivate theendonuclease, and the buffer conditions were subse-quently adjusted to 100 mM Tris-hydrochloride (pH7.6), 10mM MgCl2, 50mM NaCl, 1 mM dithiothreitol,and 1 mM adenosine 5'-triphosphate. The tempera-ture was allowed to equilibrate to 12.5°C, and T4 ligase(2 U) was added after an annealing time of 2 h.Ligation was stopped after 36 h by heating the sampleat 65°C for 5 min. The previously described protocol

(14) was followed in the subsequent transformation ofS. sanguis strain Challis. Before plating at selectiveantibiotic concentrations (10 ,ug/ml), the transforma-tion mixture was incubated for 1 h with 0.07 ,ug oferythromycin per ml to allow induction of the resist-ance.

Level and type of resistance. Minimal inhibitoryconcentrations of erythromycin and lincomycin weredetermined by growing the strains in serial dilutionsof the antibiotics in brain heart infusion broth inmicrotiter plates. Quantitative determinations ofgrowth rates in liquid medium cultures revealed thetype of resistance, i.e., inducible or constitutive (17).

Electron microscopy. The electron microscopytechniques used were as described previously (5, 22,26). 4X174 DNA served as a molecular weight stand-ard for both single- and double-stranded DNA.

Containment. The present experiments have beenclassified as self-cloning experiments by the Office ofRecombinant DNA Research and are exempt fromthe National Institutes of Health guidelines. All ma-nipulations were carried out in P2 facilities.

RESULTS

Construction of the recombinant plas-mids. Plasmid pDB102, which served as thesource of the restriction fragment to be cloned,was cleaved by endonuclease HindIII into 10fragments, with molecular sizes between 0.45and 1.9 Mdal. The actual number of differentHindIII fragments, however, was only six, sincefour of the fragments were present as identicalpairs (2). The cryptic S. mutans plasmidpVA318, which had been shown to be cleavedonly once by HindIII (16), wvas selected as thecloning vehicle. A mixture of both plasmids di-gested with HindIII was subjected to ligation asdescribed above. The disappearance of the nor-mal HindIII fragment pattern of pDB102 on 1%agarose tube gels indicated successful ligation.S. sanguis strain Challis served as the recipientin the subsequent transformation procedure. Atotal of 52 transformants were obtained, corre-sponding to a transformation frequency of 1.4X 10-7 per recipient colony-forming unit. Inagreement with earlier observations (2), a com-parable concentration of native plasmid DNA(pDB102) yielded a transfornation frequencythat was one order of magnitude higher. Severaltransformants were isolated and purified by sin-gle-colony isolation.Restriction enzyme analysis of recombi-

nant plasmids. Plasmid DNA isolated fromtransformants was subjected to analysis withHindIII. The HindIII fragment patterns for twoof the recombinant plasmids, pDB201 andpDB202, are shown in Fig. 1. Plasmid pDB202yielded four fragments upon digestion withHindIII, the largest of which migrated to thesame position as the linear vector plasmid

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808 BEHNKE AND FERRETTI

pVA318. The three other fragments of pDB202were identified as HindIII fragments B, C, andD of plasmid pDB102 according to their positionon the gel (Fig. 1, lane 3). Digestion of a secondrecombinant plasmid, pDB201, with HindIIIgave rise to only two fragments, one of whichagain corresponded to the linear vector plasmidpVA318 (Fig. 1, lanes 1 and 5). The otherHindIII fragment migrated to the same positionas HindIII fragment D of plasmid pDB102.Proof that this fragment was HindIII fragmentD ofpDB102 was derived from analysis with theenzyme HindII. HindIII fragment D of pDB102is known to be cleaved twice by HindII, resultingin 0.25-, 0.3-, and 0.58-Mdal subfragments. The0.58-Mdal subfragment is flanked on either sideby the two smaller subfragments (1).Upon digestion with HindII, six fragments

were observed for pDB201 compared with fourfragments for pVA318 (Fig. 2). One of the newHindII fragments of pDB201 was 0.58 Mdal insize and comigrated with the respective HindIIfragment of pDB102 (Fig. 2). The resistancedeterminant of pSM19035 and its derivative

1 2 3

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plasmids, including pDB102, was therefore lo-cated on HindIII fragment D, which had beenshown to be the small unique sequence spacingthe unusually large inverted repeat sequences ofthese plasmids (1, 2).Restriction enzyme map of pVA318/

pDB201. Cleavage of plasmid pVA318 with theenzyme HindII generated four fragments (Fig. 2,lane 3). Their arrangement on the pVA318 mol-ecule was concluded from partial digestion withHindII. Of several partial digestion productsthree were observed that were smaller in sizethan HindII fragment A, but larger than frag-ment B by differences that were equivalent tothe molecular weights of either fragment C or Dor both fragments together (Table 1). HindIIfragments C and D, therefore, flanked the largerfragments A and B on either side. Enzyme HpaIcleaved pVA318 into two fragments (2.35 and1.25 Mdal), the smaller of which was identical insize to HindII fragment B. Since HpaI cleavessequences that are also recognized by HindII,these two fragments were identical. Conse-quently, the larger HpaI fragment covered

4 5 6

B B

CC'

F F

H H

x

FIG. 1. HindIII restriction fragment patterns of recombinant and parental plasmids. Separation of thefragments was achieved on vertical 1% agarose slab gels at 60 Vfor 4 h. Lanes 1 and 5, pDB201; lanes 2 and4, pDB102; lane 3, pDB202; lane 6, pVA318.


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1 2 3

0.58-mda I

FIG. 2. HindII restriction fragment patterns afterelectrophoresis on 3.5% polyacrylamide gels at 85 Vfor 13 h. Lane 1, pDB102; lane 2, pDB201; lane 3,pVA318.

TABLE 1. Molecular sizes ofHindII fragments andpartial HindII digestion products ofpVA318

HindII fragments MolecularHindufragments ~size (Mdal)

A ................................ 1.85B ................................ 1.25C ................................ 0.3D ................................ 0.20

Partial digestion products0B, C, D ................. ......... 1.75B, C ........................... 1.55B, D ............................ 1.45

a HindII fragments covered.

HindII fragments A, C, and D. Double digestionof pVA318 with HindII/HindIII yielded 0.05-and 0.27-Mdal subfragments of Hindll fragmentC. The 0.27-Mdal subfragment was linked to

HindII fragment B as determined by HindIII/HpaI double digestions. A 0.27-Mdal piece wascleaved from the large HpaI fragment in suchdouble digestions. The physical map of plasmidpVA318 for the enzymes HinduI, HindIII, andHpaI is presented in Fig. 3. The determinationof the map positions of the HindII and HaeIIrestriction sites on the cloned HindIII fragmentD of pDB102 has been described (1).The molecular size of pDB201 was determined

by contour length measurement to be 4.77 +0.38 Mdal. The recombinant plasmid, therefore,contained only one copy of HindlIl fragment D.Several other restriction endonucleases weretested to find single cutting enzymes for pDB201.The endonucleases EcoRI, Bgl II, HpaII, KpnI,and SalI all failed to cleave pDB201. Also, nodigestion was observed with BamHI. SinceBamHI was reported to cleave pVA318 once(16), the pVA318 plasmid DNA was mixed withbacteriophage A DNA and treated with BamHI.Although the normal BamHI fragment patternfor A was obtained, no cleavage of pVA318 wasdetectable. The enzymes Sau3A and TaqIyielded six and eight fragments, respectively,with pVA318. Their positions were not deter-mined.Level and type of resistance. The level of

resistance to either erythromycin or lincomycinwas identical for the wild-type plasmidpSM19035 and the two recombinant plasmidspDB201 and pDB202 (Table 2). Both plasmidsalso mediated a resistance that was inducible bylow concentrations of erythromycin, but not oflincomycin, as was observed with the wild-typeplasmid pSM19035 (Fig. 4). However, differ-ences were observed in the response of eitherinduced or uninduced cultures to challenge withlower concentrations of antibiotic. Strain Chal-lis(pDB201) responded in essentially the samemanner as Challis(pSM19035) (Fig. 4). Thecloned HindIII fragment D of pSM19035, there-fore, codes for all necessary information regard-ing both the type and the level of antibioticresistance. The induction of the resistance ap-peared to be somehow impaired inChallis(pDB202). Challenge of uninduced cul-tures of this strain with 20 pg of either antibioticper ml resulted in drastic growth inhibition fora prolonged time (Fig. 4). Also, after inductionwith low concentrations of erythromycin therewas still a short lag phase after challenge withhigher erythromycin concentrations (Fig. 4).The reason for this different behavior of cellsharboring pDB201 remains unclear, sincepDB202 had also acquired HindIII fragment Dof pDB102. A different orientation of fragmentD on the recombinant plasmid pDB202 seems

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Hpa IHIn HINDnHIND a

Hpa IHIno NINOm

I I .I I I I IV%

0 0.5 1.0 1.5 2.0 2.5 3.1 34'If\

Cs to %a

HAEU HINDI NAEU

1

FIG. 3. Linear restriction map ofplasmidpVA318 and the cloned HindIII fragment D ofplasmidpDB102.Molecular sizes are given in megadaltons.

TABLE 2. Minimal inhibitory concentrations oferythromycin and lincomycina

Minimal inhibitory concn (ug/ml)Strain

Erythromycin Lincomycin

Challis 0.5 0.5Challis(pSM19035) 1,000 250Challis(pDB201) 1,000 250Challis(pDB202) 1,000 250Challis(pDB210) 250 8

a Determined after incubation at 37°C for 48 h.

to be a possible explanation. A third transform-ant, tentatively designated Challis(pDB210), ex-

pressed a dramatically reduced level of resist-ance to both erythromycin and lincomycin com-pared with Challis(pSM19035) (Table 3). Fur-thermore, Challis(pDB210) expressed constitu-tive resistance to both antibiotics as opposed tothe inducible type of pSM19035 and the otherrecombinant plasmids (Fig. 4). Attempts to dem-onstrate extrachromosomal DNA by dye-buoy-ant density gradient centrifugation of total DNAisolates of Challis(pDB210) repeatedly failed.The occurrence of a spontaneous chromosomalmutation of Challis to antibiotic resistance ap-peared to be unlikely since resistant Challis col-onies have never been observed with controlcultures not treated with DNA. Either integra-tion of the resistance determinant of pSM19035into the Challis chromosome or a small unstableplasmid generated from ligation of only pDB102fragments could account for the observed effects.In the latter case posttransformational rear-

rangement (2) of the in vitro constructed plas-mid may have affected regions responsible forthe regulation of the antibiotic resistance mech-anism.Electron microscopy of self-annealed

pDB201 molecules. Restriction enzyme map-ping of plasmid pDB1l1, a derivative plasmid of

pSM19035, revealed that the large inverted re-

peat sequences extended into HindIII fragmentD (2). This was confirmed by electron micros-copy of denatured and self-annealed pDB201molecules. The typical stem-loop structure thatis formed when inverted repeats are present wasobserved (Fig. 5). The short double-strandedstem was approximately 240 base pairs, and thesmaller of the two single-stranded loops was

1,500 base pairs in length. The total molecularsize of this structure (1.3 ± 0.14 Mdal) was inagreement with the molecular size of HindIIIfragment D of pSM19035. The larger single-stranded loop was 3.52 + 0.16 Mdal (12 mole-cules measured) in size, a value that was in goodagreement with the molecular size of 3.6 Mdalfor pVA318 (16). When the vector plasmidpVA318 was subjected to denaturation and self-annealing no stem-loop structures were ob-served, indicating that the cloned HindIII frag-ment D was responsible for the respective struc-ture in pDB201. The size of the small single-stranded loop observed for pDB201 was identicalto the small spacer sequence between the twoinverted repeats of pSM19035 and its derivativeplasmids (D. Behnke et al., manuscript in prep-

aration). From these results it seems clear thatthe resistance determinant of pSM19035 repre-sents physically a transposon-like structure, al-though actual transposition remains to be dem-onstrated.

DISCUSSIONIn the present communication, we have de-

scribed the first molecular cloning experimentsin streptococci. The gene(s) specifying inducibleerythromycin resistance ofpDB102 (a derivativeof pSM19035) has been cloned into the singleHindIII site of the cryptic S. mutans plasmidpVA318. The recombinant plasmids were sub-sequently introduced into S. sanguis strain

I

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A- r

A ,C

2 * * * u s 2 4 6 S US@ 2 4 S S U

CHALLIS (S0S202)

wih2-goflnoyi per ml ,ncalne

*~~

9*2 * * *FIG. ~4. Grwhrtso ulue:A nuedwth .07yferyhoyi pe m; , ndce wth0.5u

oflncmcierm;C,uinued ymos * halng it 0ygo eyhrmci e m-O,calegwih0oflicoyinpe m;A, nocalne

811


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4~~~~~~~~~~~~

I'/

Is</;tP '4 ,ss sX/ r{A~~~~~

FIG. 5. Electron micrograph of self-annealedpDB201.

Challis by transformation. The insertion of fheerythromycin resistance determinant intopVA318 neither impaired essential plasmid func-tion nor affected the inducibility or level ofantibiotic resistance.The cloned fragment was identified as the

HindIII fragment D of pSM19035. The exactlocation of this 1.15-Mdal fragment is betweenthe two large inverted repeat sequences of themolecule. Electron micrographs of self-annealedmolecules of the recombinant plasmid pDB201demonstrated the classical stem-loop structurefound in most known transposons. Although thephysical evidence of a transposon-like structuresuggests that the erythromycin resistance deter-minant of pSM19035 is a transposon, actualtransposition remains to be demonstrated. It isof interest that an inducible erythromycin re-sistance transposon has been described recentlyin S. faecalis (24). This transposon, Tn917, is 3.3Mdal in size with a 290-base-pair inverted repeatsequence on either side. Preliminary experi-ments have shown that HindII digestion ofpAMal::Tn9l7 results in a 0.58-Mdal fragmentidentical in size to a HindII fragment foundwithin the cloned erythromycin resistance de-terminant of pSM19035. This 0.58-Mdal frag-ment was part of Tn917 since it was absent in acomparative digestion of pAMal alone. A moredetailed comparison of Tn917 and the erythro-mycin resistance determinant of pSM19035 willbe necessary to reveal the relationship betweenthese two structures.The 1.15-Mdal size of the HindIII fragment

containing the erythromycin resistance deter-minant and its 0.58-Mdal HindII component

may be of importance in comparative studieswith other MLS resistance plasmids. In supportof this possibility, antibiotic-sensitive mutantsof the MLS resistance plasmid pAM,B1 werefound to have suffered a 1-Mdal deletion (V.Burdett, personal communication). On the otherhand, the erythromycin resistance determinantof Staphylococcus aureus plasmid pI258 hasbeen shown to be located on a 2.8-Mdal HindIIIfragment, and although different in size from theHindIII fragments containing erythromycin re-sistance genes in streptococci, the actual deter-minants may be identical (25). It would thereforebe of interest to determine whether a 0.58-MdalHindII fragment, also found in Tn917, is a com-mon fragment present in the MLS plasmids ofother gram-positive organisms.A major advantage of having cloned the 1.15-

Mdal HindIll fragment specifying erythromycinresistance is that the fragment can serve as asource of DNA and a sensitive probe for futurecomparative studies ofMLS resistance plasmids.Heteroduplex analysis, restriction cleavage pat-terns, and DNA sequencing of determinantswould provide further information about theevolution of this type of resistance. Moreover,transfer of pDB201 to Bacillus subtilis followedby the generation of minicells would facilitatethe isolation and study of the proteins actuallyinvolved in resistance.

S. mutans plasmid pVA318 is a convenientvehicle for cloning genes which have a selectablephenotype such as demonstrated in this study;however, its usefulness would be greatly en-hanced by the presence of a selective marker.Plasmid pDB201 has the potential of serving asan ideal cloning vehicle since it has an antibioticselective marker as well as a low molecularweight and high copy number (-30; unpublisheddata); however, it has two HindIII restrictionenzyme cleavage sites. Modification of pDB201to a derivative form with a single HindIII sitecould be achieved, and the modified pDB201would then have all of the desirable featuresessential for a streptococcal cloning vehicle. An-other streptococcal plasmid recently described,pDBlO1, offers the advantage of three differentsingle restriction sites (1). The availability ofthese streptococcal cloning vehicles should offernew avenues of approach for an improved ge-netic analysis of the streptococci.Plasmid pVA318 is representative of a cryptic

plasmid found in approximately 5% of naturallyoccurring strains of S. mutans (16). Attempts torelate these plasmids to cariogenicity havefailed, primarily because phenotypic propertiesassociated with the plasmid were not identifiedbut also because of the lack of a selectablemarker for the construction of isogenic pairs of

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strains differing only in the plasmid. The avail-ability of pDB201, which possesses a selectablemarker, should allow new approaches to betaken in experiments relating S. mutans plas-mids to cariogenicity.

ACKNOWLEDGMENTS

We thank D. B. Clewell and P. Tomich for assistance withthe electron microscopy techniques used in this study. Wealso thank M. Hartmann and F. Walter for the generous

supply of restriction endonucleases used in this work.This investigation was supported by a grant from the Roger

McCormick Foundation, Chicago, Ill.

ADDENDUM IN PROOF

A preliminary account of this work was reported in Plasmid(3:234, 1980). pDB201 can serve as a useful streptococcalcloning vehicle with single restriction sites, since J. B. Hansenand Y. Abiko (Abstr. Annu. Meet. Am. Soc. Microbiol., H47,p. 116, 1980) have reported that pVA318 has single cleavagesites for PstI and HaeIII restriction endonucleases.

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1. Behnke, D., and J. J. Ferritti. 1980. Physical mappingof plasmid pDB1l1: a potential vector plasmid for mo-lecular cloning in streptococci. Plasmid 4:130-138.

la.Behnke, D., V. I. Golubkov, H. Malke, A. S. Boitsov,and A. A. Totolian. 1979. Restriction endonucleaseanalysis of group A streptococcal plasmids determiningresistance to macrolides, lincosamides, and streptogra-min-B antibiotics. FEMS Microbiol. Lett. 6:5-9.

2. Behnke, D., H. Malke, M. Hartmann, and F. Walter.1979. Post-transformational rearrangement of an in vi-tro reconstucted group A streptococcal erythromycinresistance plasmid. Plasmid 2:605-616.

3. Boitsov, A. S., V. I. Golubkov, I. M. Iontova, E. N.Zaitsev, H. Malke, and A. A. Totolian. 1979. Invertedrepeats on plasmids determining resistance to MLSantibiotics in group A streptococci. FEMS Microbiol.Lett. 6:11-14.

4. Chassy, B. M. 1976. A gentle method for the lysis of oralstreptococci. Biochem. Biophys. Res. Commun. 68:603-608.

5. Clewell, D. B., and A. E. Franke. 1974. Characterizationof a plasmid determining resistance to erythromycin,lincomycin, and vernamycin B in a strain of Streptococ-cus pyogenes. Antimicrob. Agents Chemother. 5:534-537.

6. Courvalin, P. M., C. Carlier, and Y. A. Chabbert.1972. Plasmid-linked tetracycline and erythromycin re-sistance in group D Streptococcus. Ann. Inst. PasteurParis 123:755-759.

7. Dixon, J. M. S., and A. E. Lipinski. 1972. Resistance ofgroup A beta-hemolytic streptococci to lincomycin anderythromycin. Antimicrob. Agents Chemother. 1:333-339.

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

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

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12. Jones, B. B., and W. S. Reznikoff. 1977. Tryptophan-transducing bacteriophages: in vitro studies with re-striction endonucleases HindII+III and Escherichiacoli ribonucleic acid polymerase. J. Bacteriol. 132:270-281.

13. Jones, W. F., Jr., and M. Finland. 1957. Susceptibilityof enterococcus to eleven antibiotics in vitro. Am. J.Clin. Pathol. 27:467-481.

14. LeBlanc, D. J., and F. P. Hassel. 1976. Transformationof Streptococcus sanguis Challis by plasmid deoxyri-bonucleic acid from Streptococcus faecalis. J. Bacteriol.128:347-355.

15. LeBlanc, D. J., R. J. Hawley, L. N. Lee, and E J. St.Martin. 1978. "Conjugal" transfer of plasmid DNAamong oral streptococci. Proc. Natl. Acad. Sci. U.S.A.75:3484-3487.

16. Macrina, F. L., and C. L. Scott. 1978. Evidence for adisseminated plasmid in Streptococcus mutans. Infect.Immun. 20:296-302.

17. Malke, H. 1974. Genetics of resistance to macrolide anti-biotics and lincomycin in natural isolates of Streptococ-cus pyogenes. Mol. Gen. Genet. 135:349-367.

18. Malke, H. 1979. Conjugal transfer of plasmids determin-ing resistance to macrolides, lincosomides and strepto-gramin-B type antibiotics among group A, B, D and Hstreptococci. FEMS Microbiol. Lett. 5:335-338.

19. Nakae, M., T. Murai, Y. Kaneko, and S. Mitsuhashi.1977. Drug resistance in Streptococcus pyogenes iso-lated in Japan (1974-1975). Antimicrob. Agents Chem-other. 12:427-428.

20. Postle, K., and W. S. Reznikoff. 1978. Hindll andHindIII restriction maps of the atto8O-ton B-trp regionof the Escherichia coli genome, and location of thetonB gene. J. Bacteriol. 136:1165-1173.

21. Reider, J. L., and F. L. Macrina. 1976. Plasmid DNAisolation in Streptococcus mutans: glycine-enhancedcell lysis. Proc. Microb. Aspects Dent. Caries Spec.Suppl. Microbiol. 3:725-736.

22. Sharp, P. A., M. Hsu, E. Ohtsubo, and N. Davison.1972. Electron microscope heteroduplex studies of se-quence relations among plasmids of Escherichia coli.J. Mol. Biol. 71:471-497.

23. Toala, P., A. McDonald, C. Wilcox, and M. Finland.1969. Susceptibility of group D streptococcus (entero-coccus) to 21 antibiotics in vitro, with special referenceto species differences. Am. J. Med. Sci. 258:416-430.

24. Tomich, P. K., F. An, and D. B. Clewell. 1978. Atransposon (Tn917) in Streptococcus faecalis that ex-hibits enhanced transposition during induction of drugresistance. Cold Spring Harbor Symp. Quant. Biol. 43:1217-1221.

25. Weisblum, B., S. B. Holder, and S. M. Halling. 1979.Deoxyribonucleic acid sequence common to staphylo-coccal and streptococcal plasmids which specify eryth-romycin resistance. J. Bacteriol. 138:990-998.

26. Yagi, Y., and D. B. Clewell. 1976. Plasmid-determinedtetracycline resistance in Streptococcus faecalis: tan-demly repeated resistance determinants in amplifiedforms of pAMal DNA. J. Mol. Biol. 102:583-600.

27. Yagi, Y., A. E. Franke, and D. B. Clewell. Plasmid-determined resistance to erythromycin: comparison ofstrains of Streptococcus faecalis and Streptococcus py-ogenes with regard to plasmid homology and resistanceinducibility. Antimicrob. Agents Chemother. 7:871-873.

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