Vol. 164, No. 1JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 294-3010021-9193/85/100294-08$02.00/0Copyright © 1985, American Society for Microbiology

Development and Use of Cloning Systems for Bacteroides fragilis:Cloning of a Plasmid-Encoded Clindamycin Resistance Determinant

C. JEFFREY SMITH

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer ResearchFacility, Frederick, Maryland 21701

Received 25 April 1985/Accepted 19 July 1985

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were

constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmidpBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection ofthe plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing theclindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4; Ccr was not expressed in E. coli.The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme

recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependentupon the source of the DNA; generally 105 transformants ug-1 of DNA were recovered when plasmid purifiedfrom B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the numberof transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used inan analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr waslocated on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing thegene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids couldtransform B. fragilis to clindamycin resistance. These results together with previous structural data show thatthe gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-likestructure.

The Bacteroidesfragilis group of organisms is the predom-inant gram-negative species indigenous to the human gastro-intestinal tract (6, 16). These strictly anaerobic bacteria arealso significant opportunistic pathogens, being the anaerobesmost frequently isolated from clinical specimens (5). Geneticapproaches to the problems of the virulence of B. fragilis andtheir role as part of the normal flora have been hampered bya lack of genetic exchange systems suitable for manipulationof these organisms. Most genetic analyses have centered oncharacterization of the R plasmids associated with this group(9, 18, 20, 21, 25, 27, 28). Each of the R plasmids studied todate is a distinct replicon, but they do have a number ofcommon features: they are transmissible, they encode forresistance to clindamycin (Ccr), and the Ccr determinants areapparently homologous and bounded by the same directrepeat sequences. Although these studies have resulted in abetter understanding of genetic transfer in B. fragilis, theyhave had to rely heavily upon physical and structural anal-yses of the plasmids and on the use of spontaneous deletionderivatives. Further progress in understanding the geneticorganization of these R plasmids has been limited by prob-lems associated with expression ofB. fragilis genes cloned inEscherichia coli or the apparent differential expression ofphenotypes in E. coli and B. fragilis (8, 10).The problems encountered with expression can be

avoided by development of gene cloning systems and vec-tors suitable for use in Bacteroides spp. The first step in thisdevelopment has been the description of a method forgenetic transformation of B. fragilis with plasmid DNA (22;C. J. Smith, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985,H119, p. 128). In the present work, this transformationsystem has been applied in the construction of cloningvectors capable of replicating in B. fragilis, and shuttleplasmid vectors which replicate in both E. coli and B.fragilis. These plasmids ranged in size from 5.3 to 7.3

kilobase pairs (kb) and contained single restriction endonu-clease recognition sites for at least 10 different enzymes.Two of these shuttle vectors were used here in an analysis ofthe Ccr determinant located within a transposon-like struc-ture on the Bacteroides ovatus plasmid pBI136 (21, 23). Theresults demonstrated that a 0.85-kb fragment inserted intothe unique ClaI site of the vectors was sufficient for expres-sion of Ccr and that this fragment lies directly adjacent to acopy of the repeated sequence which is associated with thetransposon-like structure. This work and the successfulconstruction of the plasmid vectors have proven that it ispossible to apply routine recombinant DNA techniques tostudies of B. fragilis, and they also have shown the utility ofthe cloning systems developed here.

MATERIALS AND METHODSBacterial strains, media, and growth. The E. coli and

Bacteroides spp. strains used in this study are described inTable 1. E. coli strains were propagated in a complex mediumcontaining the following (per liter): tryptone, 10 g; yeastextract, 5 g; NaCl, 5 g; agar, 15 g (when required). Ampicillinand 5-bromo-4-chloro-3-indoyl-,-D-galactoside (X-gal) wereused at 40 ,ug ml-' where appropriate. Bacteroides spp.strains were grown in brain heart infusion broth supple-mented with the following (per liter): L-cysteine, 1 g; hemin,5 mg; menadione, 1 mg; NaHCO3, 2 g. Both broth culturesand agar plates of this medium were incubated at 37°C in ananaerobic chamber containing an atmosphere of80% N2-10%C02-10% H2. Clindamycin was routinely used at aconcentration of 5 ,ug ml-' in this medium. Antibioticsensitivities (MICs) were determined by an agar dilutionmethod (13). For strains containing unstable plasmids, MICswere determined with cultures that had been grown in thepresence of 1 ,ug of clindamycin ml-' to ensure maintenanceof the plasmids. To determine the stability of plasmids,

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TABLE 1. Relevant characteristics of bacterial strains andplasmids

Strain or plasmid Phenotype or genotypea Reference

BacteriaE. coli RR1 hsdS20 (HsdR- HsdM-) supE44 2

ara-14 galK20 lacYl proA2rspL20 xyl-5 mtl-l

E. coli TB1 ara A(pro-lac) rspL thi-J 4)80 dlacZAM15 hsdRJ7 (HsdR7 HsdM+)

B. fragilis 638 Rif' Tets Ccs 18B. ovatus Tetr Rif' clinical strain, NIH-cc/cp S. Gersch

IB138 #2642-3B. fragilis Tetr Rif' transconjugant of IB138 x This study

IB143 638

PlasmidspBF4 Ccr, transmissible, 41 kb 27pBFTM10 Ccr, transmissible, 14.6 kb 25pBI136 Ccr, transmissible, 80 kb 24pBI143 Cryptic, 2.7 kb, from strain IB143 This studypUC19 Apr, lacP (polylinker) lacZ', 2.7 kb 17pFD139 Apr, pUC19:: pBI136 EcoRI-C 21

chimeric plasmid, 9.9 kbpFD158 Apr, pUC19:: pBF4 EcoRI-D 23

chimeric plasmid, 6.6 kb

a Drug resistance phenotypes: Rif, rifampicin; Tet, tetracycline; Cc, clin-damycin, Ap, ampicillin.

I TB1 is an HsdR- HsdM I derivative of JM83 (26) and was obtained fromBethesda Research Laboratories, Inc.

cultures were diluted to 107 cells ml-1, grown overnight in theabsence of antibiotics, diluted to 106 cells ml-', and thengrown to late logarithmic phase (a total of about 10generations). These cells then were grown on a nonselectivemedium; after growth, individual colonies were replicated tomedia with and without the appropriate antibiotic.

Preparation and analysis of DNA. Plasmid DNAs werepurified from crude lysates by ultracentrifugation (19). Crudelysates of E. coli were prepared by the method of Guerry etal. (7), and crude lysates of B. fragilis were prepared by aslightly modified Currier and Nester technique (4, 21). Rou-tine screening of both E. coli and B. fragilis transformantswas performed by the rapid alkaline extraction procedure (1)except that before precipitation of DNA, lysates were ex-tracted with phenol and chloroform. DNA samples wereanalyzed by agarose gel electrophoresis with horizontal slabgels in Tris-acetate or Tris-borate buffers containing 0.5 ,ugof ethidium bromide ml-' (15). Restriction endonucleasedigestions were performed as described in the specificationsof manufacturers. Individual restriction fragments were pu-rified from agarose gels by electroelution followed by puri-fication and concentration with the Elutip-d system(Schleicher & Schuell, Inc., Keene, N.H.).

Cloning methodologies. All of the plasmid vectors andclones described in this work were constructed by using theplasmids shown in Table 1. In addition, most of the recom-binant plasmids made here were produced by in vitro ligationof. blunt-ended DNA fragments. Linearized DNAs witheither 3' or 5' extensions were made blunt ended with DNApolymerase I large fragment by incubation for 1 h at ambienttemperature in a solution containing 50 mM Tris (pH 7.2), 10mM MgSO4, 1 mM dithiothreitol, 50 ,ug of bovine serumalbumin ml-', and 80 ,uM each of the four nucleosidetriphosphates. Where indicated, the blunt-ended plasmidvectors were treated with calf intestine alkaline phosphatase(Boehringer GmbH, Mannheim, Federal Republic of Ger-many). For ligation reactions, linearized DNAs were mixed

at a molar ratio of 3:1 (insert to vector) and ligated with 2 to5 U of T4 DNA ligase in a 20-,u volume with the buffersdescribed by the supplier (Bethesda Research Laboratories,Inc., Gaithersburg, Md.). T4 RNA ligase (5 U) was alsoadded to some of the blunt-end ligations. Frozen competentcells of E. coli were transformed with plasmid DNA orligation mixtures by the method of Hanahan (11). Transfor-mation of B. fragilis 638 with plasmid DNA was facilitatedwith polyethelene glycol by a recently described method(22). The term transformation frequency as used in thispaper refers to the number of antibiotic-resistant transform-ants obtained per microgram of DNA.

RESULTS

Shuttle plasmids for B. fragilis and E. coli. The crypticBacteroides plasmid pBI143 was chosen as the replicon forthese plasmid vectors because of its small size (2.7 kb) andapparent high copy number. One disadvantage with thischoice was a paucity of restriction endonuclease sites suit-able for cloning (Fig. 1). pBI143 was originally observed aspart of the plasmid complement of a tetracycline-resistant(Tetr) B. ovatus clinical isolate, strain IB138. During studieson the conjugal transfer of tetracycline resistance, pBI143was mobilized from IB138 to the recipient strain 638. Theresulting Tetr transconjugant, IB143, contained only pBI143and no other detectable extrachromosomal elements. Sub-sequent studies have shown that pB3I143 did not play a rolein either tetracycline resistance or its transfer (unpublishedobservations). The source of pBI143 for all plasmid con-structions was the transconjugant IB143. The E. colireplicon used for the plasmid vectors was pUC19 (17, 26).This is a small pBR322-derived plasmid containing a modi-fied E. coli lac operon with numerous restriction recognitionsites located in a 57-base-pair multiple cloning sequence(MCS).The shuttle plasmids were constructed by performing all

cloning in the E. coli host. To maintain the integrity of theMCS, pUC19 was digested with either AatII or NdeI. Theseenzymes have recognition sites in nonessential regions of themolecule outside of the ampicillin resistance (Apr) determi-nant and the lac region. The linearized plasmids were madeblunt ended, treated with alkaline phosphatase, and thenligated with XbaI- or HaeII-linearized (and blunt-ended)pBI143. After transformation of E. coli RR1, Apr transform-ants were screened for inserts, and two of the four possiblecombinations were observed; these were designated pFD160and pFD165. pFD160 was formed by the ligation of HaeII-digested pBI143 atd NdeI-cleaved pUC19, whereas pFD165was a product of ligation between XbaI-digested pBI143 andAatII-digested ptJC19 (Fig. 1). None of the endonucleaserecognition sites used in these blunt-end ligations wereregenerated in the recombinant plasmids. To test whetherthese plasmids could replicate in B. fragilis, the purifiedpBF4 EcoRI D fragment containing a Bacteroides Ccr deter-minant (28) was cloned into the EcoRI site (within the MCS)of each plasmid. The source of this fragment was thechimeric plasmid pFD158 (Table 1 and Fig. 1). Previouswork has shown that this fragment also carries a cryptic Tetrdeterminant which is expressed in E. coli but not in B.fragilis (8). Thus, putative E. coli clones with this insert wereeasily selected as Apr Tetr white colonies on X-gal plates,Plasmid DNAs obtained from representative colonies trans-formed B. fragilis 638 to Ccr at a high frequency (ca. 5 x 102ug'1). Examination of these Ccr transformants revealed thepresence of a plasmid of the expected size in all cases tested.

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pUC19 pBI 143

Aat II CIa I

~Nds I 1..

2i a1J Ava IEndonuclease l-w DigestionXbIBlunt-ended Has II

Ligation

Xba IHas II Aat II Nd. I

MCS

5.4/0

ClaaI pFD165Xba XAat123

AvaaI1Xba I

Aat IINd. I

Has II

__ ba I

5.410

Ava I

4 pFD160

MCS-.

Cla I

Nde IHaI II

Nde I Ava I + Hae IIBlunt-ended Blunt-ended

/(MCS) 'EcoR I

Hae 11 Nde IAva I

7 7.3/0CaI 6 1 Hind III

pFD173 1

Ava 1-Hae II

4 3 Nde I~~~~MCS Aat IIBlunt-ended

MCS

FIG. 1. Construction of B. fragilis-E. coli shuttle plasmids. All of the shuttle plasmids shown here were constructed by ligation ofblunt-ended fragments followed by transformation and selection in E. coli. The fragments were made blunt ended as described in the Materialsand Methods section. For construction of pFD165, XbaI-cleaved pBI143 was ligated to AatII-cleaved pUC19; and for pFD160, HaeII-cleavedpBI143 was ligated to NdeI-digested pUC19. These two recombinant plasmids were then digested with the enzyme indicated and ligated tothe purified, blunt-ended, 1.9-kb Aval-Haell fragment of pFD158 containing the Ccr determinant from pBF4. The restriction endonucleasecleavage site maps of each plasmid were drawn to scale, and the numbers around the inside of each map are the size references in kb. Solidregions on the maps represent pUC19-derived sequences, and the stippled regions were derived from pBI143. The hatch-marked regions wereoriginally derived from the pBF4 EcoRI D fragment and have been implicated in resistance to clindamycin (28); the dashed lines on the insideof the maps indicate the approximate location of the Ccr determinant based on physical analyses (23) and on the cloning data described in thetext. Small gaps in the maps were used to indicate the sites and orientations of the blunt-end ligations; none of the restriction sites used inthese blunt-end ligations were preserved or regenerated. MCS refers to that region of pUC19 containing the MCS with recognition sites for13 restriction enzymes (17). For the sake of clarity, the three HaeII sites of pUC19 are not shown in these maps; for their locations seereference 26.

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pFD158 pBlI143

Hae II Ha II

Hae

III

EcoR I(MCS)

Ava I Hind III

FIG. 2. Restriction endonuclease cleavage site map of the Bac-teroides vector pBI191. Construction of pBI191 was performed by invitro ligatlon of HaeII-digested pBI143 and p]FD158 with selection inB. fragilis for resistance to clindamycin. The HaeII fragment frompFD158 contains the Ccr determinant from pBF4 plus a smallsegment of the pUC19 lac region containing the MCS. On the map

of pFD158 (Fig. 1), this fragment extends from the Haell site at kbcoordinate 2.2 to an HaeII site at kb coordinate 4.8 which is not

shown on the pFD158 map. All of the symbols used here have beendescribed in the legend to Fig. 1.

This result indicated that pFD160 and pFD165 could repli-cate in both the E. coli and B. fragilis hosts.The final step in construction of the shuttle vectors was to

clone a selective marker for Bacteroides spp. into a sitewhich would not affect replicatioh ih either host or interferewith the lac region of pUC19. Based on the differencesbetween pFD160 and pFD165, it was clear that their AatIIand Ndel sites, respectively, could be used for this purpose

(Fig. 1). It also was apparent that the pBF4 Ccr determinantwould be a suitable selective marker. Previous DNA-DNAhybridization analyses suggested that this determinant was

located between the AvaI and HaeII sites of the pBF4 EcoRID fragment (23). Therefore, the pFD158 chimeric plasmidwas digested with AvaI plus HaeII, and a 1.9-kb fragmentwas purified, made blunt ended, and ligated into the appro-

priate sites (also nmade blunt ended) on pF160 or pFD165(Fig. 1). These ligations were used to transform E. coli RR1,and the Apr transformants were screened for plastnid con-

tent. Clones containing the desired inserts were identified,their plasmids were mapped for restriction sites, and two ofthese, pFD173 and pFD176, were retained for further study.Neither of these plasmids had preserved nor regenerated theoriginal restriction sites used in its construction (Fig.1), andin transformation studies with B. fragilis, both were able to

transform strain 638 to clindamycin resistance.Construction of pBI191. Successful construction of the

shuttle plasmids suggested several possible strategies for thedevelopment of a Bacteroides cloning vector. As mentionedabove, a major problem in the use of pBI143 was its lack ofrestriction recognition sites. The problem was overcome bythe use of pFD158 as a source for both the Ccr selectable

marker and restriction enzyme cleavage sites. This waspossible because the Ccr determinant could be excised on a2.6-kb HaeII fragment. This fragment extended from anHaeII site which is shown on the pFD158 map in Fig. 1 at kbcoordinate 2.2 to an HaeII site near the MCS at kb coordi-nate 4.8 (not shown on map), and it included 250 to 300 basepairs of the pUC19 lac region containing the MCS. Construc-tion of pBI191 was accomplished by a straightforward liga-tion of HaeII-digested pBI143 and pFD158 mixed in a ratioof 1:1 (Fig. 2). Transformation of B. fragilis 638 with 0.5 ,ugof this ligation mixture and selection on clindamycin resultedin a total of 52 transformants. All Ccr colonies tested con-tained a plasmnid about 5.3 kb in size; one of these strains,IB191, was retained for further study.The results from restriction endonuclease digestions ver-

ified the structure of pBI191 and that it was indeed the resultof a ligation between two HaeII fragments of nearly equalsize (Fig. 2). To demonstrate the presence of the completeMCS, pBI191 was digested with several representative en-zymes. EcoRI, BamHI, PstI, and Sall each produced asingle linear fragment of pBI191 (Fig. 3). HindIII producedtwo fragments because there is a HindIll site within theMCS and the Ccr detertminant. Results from AvaI digestionwere consistent with sites shown on the map in Fig. 2 plus acleavage site within the MCS (i.e., the SmaI site is also anAvaI site). These data show that the entire MCS, which hassome 12 restriction recognition sites bounded by an EcoRIand a Hindlll site, was present on pBI191.

Genetic characteristics of the chimeric plasmids. B. fragilis638 is extremely sensitive to clindamycin, with an MIC of 0.5,ug ml-'. Selection of transformants containing the plasmidvectors was routinely accomplished on media with 5 ,ug ofclindamycin ml-' with no background growth of the recipi-ent. The MICs of clindamycin for the B. fragilis strainscontaining pFD173, pFD176, and pBI191 were 100 pug ml-'.The MIC of ampicillin for strain 638 was 20 ,ug ml-', andnone of the shuttle plasmids conferred an increased resist-ance to anmpicillin; thus, it seems that the pUC19 Aprdeterminant was not expressed in B. fragilis. Likewise, therewas no expression of Ccr or erythromycin resistance in anyof the plasmid-containing E. coli strains when they weretested by the method of Malke and Holm (14). In addition,these plasmids did not mediate Tetr in the E. coli host. Thissuggests that the cryptic Tetr determinant associated withthe pBF4 EcoRI D fragment (8) was either not present or notintact on the AvaI-HaeII subfragment used to construct theshuttle plasmids.

Stabilities of the plasmid vectors were determined after 10generations of growth in the absence of antibiotics. Theseresults (Table 2) indicate that there were significant differ-ences in the ways pFD173 and pFD176 were maintained. InB. fragilis, pFD173 was extremely unstable, with a fre-quency of loss greater than 97%. In E. coli, however, thisplasmid was stably maintained with no measurable loss.Conversely, pFD176 was not spontaneously lost from the B.fragilis host, but curing frequencies approached 60% in E.coli RR1. pBIl91, which was constructed by insertion intothe HaeII site of pBI143 similar to that of pFD176, appearedto be very stable in B. fragilis 638, with no detectable lossafter more than 10 generations of growth (Table 2).

Transformations with plasmid vectors. The ability to trans-form B. fragilis 638 to Ccr with the chimeric plasmids wasdependent upon the source of plasmid DNA. When pFD173or pFD176 was purified from the 638 strain background,frequencies were greater than 105 Ccr transformants ,ug ofplasmid DNA-' (Table 3). These frequencies were more

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than 25-fold higher than the typical results obtained with thecontrol, R plasmid pBFTM10. Even when considered on amolar basis, pBFTM10, which is twice the size of pFD173and pFD176, yielded 10-fold fewer transformants. The plas-mid p1I191 transformed B. fragilis with the highest frequen-cies, approaching 5 x 105 transformants ,ug of DNA-1.When B. fragilis strain 638 was transformed with either ofthe shuttle plasmids purified from an E. coli host, the numberof Ccr transformants recovered decreased by nearly 1,000-fold (Table 3). Complementary studies were performed bytransformation of the restriction-negative RR1 strain of E.coli. These results showed that the source of transformingDNA had no significant effect on the number of Apr trans-formants recovered in experiments with either pFD173 orpFD176. There was, however, a 10-fold decrease in trans-formations with pFD173 or pFD when compared with trans-formations with pUC19 (Table 3), but this difference may bedue to the smaller size of pUC19.

Cloning of the pBI136 gene encoding Ccr. The plasmidcloning systems described above were used in a molecularand genetic analysis of the transposon-like structure encod-ing Ccr in pBI136. In these studies, plasmids pFD160 andpFD165 were used for the cloning since they did not containthe pBF4 Ccr determinant (Fig. 1). Based on hybridizationstudies comparing the pBI136 EcoRI C and pBF4 EcoRI Dfragments, the Ccr determinant was tentatively located on a0.85-kb EcoRI-HaeII subfragment of pBI136 EcoRI-C (23)(Fig. 4). Therefore, this small fragment was purified from thechimera pFD139, made blunt ended, and ligated to ClaI-digested, blunt-ended vector DNA. This ligation mixturewas then used to transform E. coli RR1 to ampicillin resist-ance, and two of the resulting transformants were retainedfor further study. These were designated pFD167 andpFD174 and were the result of ligation between the pBI136fragment and pFD160 and pFD165, respectively. Restrictionanalyses of the recombinant plasmids revealed that theblunt-end cloning strategy used for their construction regen-erated the EcoRI site of the pBI136 fragment. Results frommapping this EcoRI site demonstrated that the fragment wasin opposite orientations in pFD167 and pFD174 (Fig. 4).Plasmid DNA (1 ,ug) from each of these clones was used to

TABLE 2. Stability of chimeric plasmids in B. fragilis andE. colia

Total no. of No. of Ccr or %Plasmid Strain colonies tested Apr coloniesb Resistant

pFD173 B. fragilis 638 200 7 3.5150 2 1.3

E. coli RR1 150 150 100100 100 100

pFD176 B. fragilis 638 200 200 100150 150 100

E. coli RR1 150 80 53100 38 38

pBI191 B. fragilis 638 150 150 100100 100 100

a Stability was tested after 10 generati9ns of growth in nonselective media.Cells were then plated on nonselective media, and after growth overnight,colonies were replicated onto antibiotic-containing selective media or nonse-lective media (see Materials and Methods).

The selective medium used for B. fragilis strains contained 5 ,ug ofclindamycin ml-', and for E. coli strains the medium contained 40 ,ug ofampicillin ml-'.

1 2 3 4 5 6 7 8 9

FIG. 3. Electrophoretic analysis of restriction endonuclease di-gested pBI191. The restriction enzymes used for these analyseswere chosen in order to demonstrate the presence of the wholeMCS. Purified pBI191 was cleaved with the restriction enzymeindicated and then electrophoresed on a 0.8% agarose slab gel inTris-borate buffer containing 0.5 p.g of ethidium bromide ml-'.Lanes 1 and 9 are the 1-kb ladder size reference markers (BethesdaResearch Laboratories, Inc.); the sizes from bottom to top are 0.5,1.0, 1.6, 2.0, 3.0, 4.1, 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.2, and 12.2 kb.Lanes 2 through 8 contain pBl191 digested with the followingenzymes: lane 2, SmaI; lane 3, BamHI; lane 4, PstI; lane 5, SaIl;lane 6, EcoRI; lane 7, Hindlll; lane 8, AvaI.

transform B. fragilis with selection for resistance to clinda-mycin. Several hundred Ccr colonies were obtained fromtransformations with each plasmid, and screening of theseCcr strains for plasmid content demonstrated the presence ofplasmids indistinguishable from pFD167 and pFD174 in allstrains tested. The level of Ccr displayed by clones contain-ing each of the plasmids was tested, and the MIC ofclindamycin for each was 100 ,ug ml-'. This result indicatesthat the orientation of the EcoRI-HaeII fragment within theplasmid vector did not significantly affect expression of theCcr determinant.

During the course of this work, shuttle plasmids contain-ing the pBI136 EcoRI-HaeII fragment ligated into a numberof restriction sites other than the ClaI site were constructedin E. coli RR1. However, none of these recombinant plas-mids was able to express Ccr when transformed into B.fragilis strain 638 (data not shown). The reasons for this lackof expression are not known, but this is presently underinvestigation.

DISCUSSION

The results presented in this communication have for thefirst time demonstrated a useful system for cloning in the B.fragilis group of gram-negative anaerobic bacteria. Theplasmid vectors described here were designed around theBacteroides replicon pBI143. This small plasmid appears tobelong to the class I group of cryptic plasmids widelydisseminated among the intestinal Bacteroides spp. (3). Thebroad host range of this class suggests that the cloningvectors derived from pBI143 may be able to replicate inmany of the Bacteroides spp. In this regard, pBI191 has been

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TABLE 3. Transformation of B. fragilis or E. coli withplasmid DNA

Plasmid Transformants ,ug of DNA-1 inb:source,plasmida B. fragilis 638 E. coli RR1

B. fragilispFD173 1.6 x 105 8.6 x 105pFD176 3 x 105 3.1 x 106pBI191 4.5 x 105pBFTM10 6 x 103

E. coli RR1pFD173 2 x 102 1.4 x 106pFD176 4.2 x 102 2.2 x 106pUC19 1.1 x 107a Plasmid DNA was purified as described in the text and added to each

transformation in a final volume of 20 p.1. In transformations, 1.0 ,ug of DNAwas used for B. fragilis, and 0.2 ,ug of DNA was used for E. coli.bThe number of transformants obtained per microgram of DNA was the

average of at least three independent experiments. B. fragilis transformantswere recovered on media containing 5 ,ug of clindamycin ml-', and E. colitransformants were selected on 40 ,ug of ampicillin ml-'.-, Not done.

successfully introduced into a second B. fragilis strain and aBacteroides uniformis strain (unpublished data). The choiceof pUC19 as the E. coli replicon in the shuttle plasmids wasimportant because the large number of restriction sitespresent in the MCS was necessary to complement the lack ofsuitable cloning sites in pBI143. In addition, the pUC19-modified ,-galactosidase gene used in conjunction withselection on ampicillin plus X-gal medium is a powerful toolfor one-step detection of cloned inserts in E. coli (17, 26).The selective marker for these plasmids in B. fragilis wasprovided by cloning a Ccr determinant from the R plasmidpBF4. Clindamycin has proven very effective in selectionsince the rate of spontaneous mutation to Ccr is extremelylow. The levels of Ccr conferred by the plasmid vectors weresomewhat lower than previously reported for pBF4 (27); thisresult was surprising in that pBF4 is present only in a singlecopy in B. fragilis V479-1 (28) whereas the pBI143-derivedplasmids seem to be in a much higher copy number. TheMIC values were, however, similar to those found forpBI136 when it is present in the 638 strain background (24).Two schemes were used to construct the shuttle plasmids,

and these resulted in replicons with different properties ofmaintenance and stability. When the unique HaeII site ofpBI143 was used in construction, as in the case of pFD176and pBI191, the product was a plasmid with remarkablestability in the B. fragilis host (Table 2). However, plasmidconstructions with the single pBI143 XbaI site 150 base pairsaway from HaeII were lost from B. fragilis at a very highrate in the absence of selective pressure (e.g., pFD173;Table 2). In the presence of clindamycin, pFD173 wasmaintained in strain 638 without any obvious effect on thegrowth rate, but there appeared to be a significant decreasein the amount of plasmid DNA which could be recoveredfrom this strain (data not shown). As might be expected, thetwo clones expressing the pBI136 Ccr determinant, pFD167and pFD174 (Fig. 4), shared stability properties similar tothose of the related plasmids pFD176 and pFD173, respec-tively (data not shown). This result supports the idea that theinstability of pFD173 and pFD174 in B. fragilis is due to theinterruption of the XbaI site and not due to a position effectof the Ccr determinant. In addition, it is now clear that theClaI site of the plasmid vectors will be useful as a cloningsite since its interruption does not seem to interfere withplasmid replication or maintenance.

The differential stability of pFD173 and pFD176 in the E.coli host is difficult to understand (Table 2). Although thetwo plasmids were constructed differently, they both containforeign DNA inserted into the same two sites of pUC19. Thiswould suggest that the problem is not with the interruption ofan essential gene product.The rationale for development of the shuttle plasmids was

to facilitate genetic exchange between E. coli and B. fragilis.To be useful, DNA extracted from E. coli must be able totransform B. fragilis. Evidence presented in Table 3 suggeststhat there was a restriction of heterologous DNA by B.fragilis 638 during transformation. This amounted to a 1,000-fold decrease in the number of Ccr B. fragilis transformantsobtained when the source of plasmid was E. coli RR1 ratherthan B. fragilis 638. The modification system of E. coliexerted little or no influence on these frequencies as plasmidDNA isolated from the modification-proficient strain TB1also transformed 638 at a relatively low frequency (data notshown). However, even with the significant effect of restric-tion, more than 200 transformants ,ug of plasmid' wereobtained routinely when DNA from E. coli was used. Thislevel of sensitivity should be more than adequate to meetmost requirements.

Construction of pBI191 provided the first demonstrationof cloning directly in B. fragilis. Although the design ofpBI191 was not complex, the recovery of more than 50transformants was important in view of the facts that it wasnot previously known if ligated (nonsupercoiled) plasmidDNA would transform B. fragilis, and more than one-half ofthe recombinant molecule (i.e., fragment bearing the geneencoding Ccr) was composed of heterologous DNA from thechimera pFD158 purified from an E. coli host. This pBI191vector is stable in Bacteroides spp. and smaller than theshuttle plasmids; thus, it should be useful for subcloning offragments or cloning in B. fragilis of directly selectablemarkers. Recently, this plasmid has been used as a vectorfor shotgun cloning of chromosomal fragments from anotherB. fragilis strain. Results from these experiments are stillpreliminary, but there has been no indication of majorproblems with deletion formation or rearrangements; how-ever, the inserts obtained were generally only 2.0 to 4.0 kb insize.The plasmid vectors described here were developed pri-

marily as tools to study the genetic organization of Bacte-roides R plasmids. As part of this objective, two of theshuttle plasmids were used in an analysis of the geneencoding Ccr associated with an 8.4-kb transposon-likestructure found on the R plasmid pBI136. These experimentswere designed to take full advantage of the E. coli system forits ease of cloning and screening of recombinants, followedby use of the B. fragilis system to analyze expression of thecloned DNAs. By this strategy the pBI136 gene encoding Ccrwas cloned on a 0.85-kb EcoRI-HaeII fragment locatedwithin the transposon-like region (Fig. 4). This fragment wasthen shown to express Ccr in B. fragilis 638 at levelscomparable to those observed for the native pBI136 plasmidin the same strain background (24). In a previous report (23),we have tentatively identified the EcoRI-HaeII fragment ascontaining the Ccr determinant and showed that sequentialHindlIl, Avall, and DdeI sites within the fragment wereconserved in the three Bacteroides R plasmids pBF4,pBFTM10, and pBI136. The cloning results presented in thecurrent communication are in full agreement with the place-ment of the gene encoding the Ccr and with the idea that thethree sequential restriction sites are within the structuralgene.

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300 SMITH

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0 1 2 3 4 5 6 7 8 9 kbFIG. 4. Cloning and expression of the pBI136 Ccr determinant. A gene encoding Ccr located within the transposon-like structure from

pBI136 was cloned in E. coli on a 0.85-kb EcoRI-HaeII fragment. A restriction enzyme cleavage site map of the Ccr region and the clonedfragment are shown at the top of the figure. The EcoRI-HaeII fragment was blunt ended and then cloned into the ClaI site (made blunt ended)of pFD160 and pFD165; the resulting recombinant plasmids were designated pFD167 and pFD174, respectively. Both plasmids weretransformed into B. fragilis and found to express resistance to clindamycin. The solid areas on the pFD167 map and the open areas on thepFD174 map are vector sequences, and the cloned fragment is indicated by the thin line. The striped areas on the map of the pBI136 regionencoding Ccr indicate the location of the direct repeat sequence except that the ends nearest the EcoRI sites have not yet been preciselymapped (21). A size reference marker in kb is given at the bottom of the figure.

The transposon-like structures encoding Ccr in the Bacte-roides R plasmids pBF4, pBFTM10, and pBI136 are com-posed of a somewhat variable region bounded by a homolo-gous 1.2-kb directly repeated sequence (9, 20, 21, 23). TheEcoRI site used in cloning the pBI136 Ccr determinant is partof one copy of this repeated sequence very near its terminus(21, 23) (Fig. 4); this would indicate that the pBI136 geneencoding Ccr lies directly adjacent to the repeated sequence.Furthermore, in construction of the plasmids pFD173 andpFD176, a 1.9-kb fragment originally from pBF4 was thesource of the Ccr determinant. This AvaI-HaeII fragmentcontains about 0.7 kb of the directly repeated sequenceextending from the AvaI site toward the HindIll site (23)(Fig. 1); this would place the pBF4 Ccr gene very near aterminus of a copy of the direct repeat sequence. The closeassociation of antibiotic resistance determinants with therepeated sequences of transposons is not uncommon, and inmany cases these sequences are involved in regulation ofadjacent genes (12). It will be interesting to determinewhether the directly repeated sequences from the Bacte-roides R plasmids play a role in expression of the geneencoding Ccr.

In summary, the work presented in this paper has added anew dimension to our ability to analyze the biologicalproperties of an important group of anaerobic bacteria. Thecloning systems described here have proved useful andshould allow a number of different genetic approaches to thestudy of Bacteroides R plasmids or of chromosomally en-coded properties. As with any newly developed system,

improvements can be made; this is especially apparent in thesole reliance on Ccr for selection of the cloning vectors in B.fragilis. But overall, the small size, number of uniquerestriction sites, and selectability of these plasmid vectorsare desirable traits which should ensure their utility.

ACKNOWLEDGMENTS

I thank Donald J. LeBlanc for his helpful comments and criticalreview of this manuscript and G. Harbaugh for help in preparation ofthe manuscript.

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pFD 174

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8. Guiney, D. G., P. Hasegawa, and C. E. Davis. 1984. Expressionin Escherichia coli of cryptic tetracycline resistance genes fromBacteroides R plasmids. Plasmid 11:248-252.

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11. Hanahan, D. 1983. Studies on transformation of Escherichia coliwith plasmids. J. Mol. Biol. 166:557-580.

12. Kleckner, N. 1981. Transposable elements in prokaryotes.Annu. Rev. Genet. 15:341-404.

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17. Norrander, J., T. Kempe, and J. Messing. 1983. Construction ofimproved M13 vectors using oligodeoxynucleotide-directed mu-tagenesis. Gene 26:101-106.

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22. Smith, C. J. 1985. Polyethylene glycol-facilitated transformationof Bacteroides fragilis with plasmid DNA. J. Bacteriol.164:466-469.

23. Smith, C. J., and M. A. Gonda. 1985. Comparison of thetransposon-like structures encoding clindamycin resistance inBacteroides R-plasmids. Plasmid 13:182-192.

24. Smith, C. J., and F. L. Macrina. 1984. Large transmissibleclindamycin resistance plasmid in Bacteroides ovatus. J. Bac-teriol. 158:739-741.

25. Tally, F. P., D. R. Snydman, M. J. Shimell, and M. H. Malamy.1982. Characterization of pBFTM10, a clindamycin-erythromy-cin resistance transfer factor from Bacteroides fragilis. J. Bac-teriol. 151:686-691.

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