Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression

PLASMID

20, 17-22 ( 1988)

Construction of Shuttle Cloning Vectors for Bacteroides fragilis and Use in Assaying Foreign Tetracycline Resistance Gene Expression DONALDG.GUINEY,KATHRYN BOUIC,PATRICIA HASEGAWA, ANDBARBARAMATTHEWS Department of Medicine HNlF,

VCSD Medical Center, 225 Dickinson, San Diego, California

92103

Received February 22, 1988; revised May 26, 1988 Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDKl and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOAl0 is a cosmid vector containing the replication region of pCP1 necessaryfor maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistancegene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments.pDK3 was usedto test the expression in B. fragilis of five foreign tetracycline resistance(TcR)genes.The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a Tea determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene,originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes. 0 1988Academic PI=, ITIC.

Genetic analysis of Bacteroides specieshas been greatly facilitated by the recent development of conjugation systems capable of transferring plasmids between Escherichia coli and Bacteroidesjiragilis (Guiney et al., 1984~; Shoemaker et al., 1985). Despite the great evolutionary distance between Bacteroides and the Enterobacteriaceae, the broad host range IncP plasmids are capable of mediating conjugal transfer of DNA from E. coli donors to Bacteroides recipients. Using these plasmid transfer systems,basic differences in plasmid maintenance and gene expression between E. coli and Bacteroides have been discovered. The IncP plasmids are not maintained in Bacteroides despite their broad host range replication properties in other gram-negative bacteria. The Bacteroides plasmids tested to date do not replicate in E. coli. In addition, no antibiotic resistance marker has been found that is expressed in both E. coli and B. fragilis.

Ampicillin (TEM /3-lactamase)and trimethoprim resistancefrom facultative bacteria were not expressedin Bacteroidesdespite successful introduction of the genes. The only Bacteroides resistancemarker characterized to date, clindamycin resistance (CIR,’ or erm), is not expressedin E. coli. In addition, an unusual tetracycline resistance determinant has been detected on the Bacteroides R plasmids pBF4 and pCP 1 (Guiney et al., 1984a). This marker is expressedin E. coli only under aerobic conditions and thus is cryptic in Bacteroides. These genetic studies have defined the requirements for shuttle cloning vectors that can be transferred from E. coli to Bacteroides. Vector plasmids must contain (1) an E. coli replicon and drug resistance marker, (2) a Bacteroides replicon and the CIRgene,and (3) an oriT region capable of being mobilized at high frequency by the IncP conjugation system. The initial shuttle vector, pDP1, consisted of a hybrid plasmid containing pDG5 (pBR322 oriV, AmR, RK2 oriT) and the Bac’ Abbreviations used:CIR,clindamycin resistance;MIC, minimal inhibitory concentration; TcR,tetracycline resis- teroides CIR plasmid, pCP1 (Guiney et al., 1984b, 1984c).We now describe the constructance; Ama, ampicillin resistance; BCH, brain-heart infusion containing cysteine and hemin. tion of pDP1 derivatives with specialized 17

0147-619X/88 $3.00 Copyright Q 1988 by Academic Rss, Inc. Au rights of reproduction in any form merved.

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GUINEY

properties for useasE. ColilBacteroidesshuttle vectors. One of these plasmids, pDK3, was used to study the expression of a variety of tetracycline resistance genesin Bacteroides. MATERIALS

AND METHODS

Bacterial strains and plasmids. The E. coli strains JA22 1 and HB 101, used for plasmid constructions, B. fragilis 638, the helper RK2 derivative pRK23 1, and pDP1 A4 have been described previously (Guiney et al., 1984~; Matthews and Guiney, 1986). pTJS245 (tetA) was obtained from D. Helinski (Department of Biology, UCSD, San Diego, CA). pBT 107 (tetB) was from L. Smith (Research Institute of Scripps Clinic, La Jolla, CA). pJI3, from V. Burdett, and pJIR39, from J. Rood, have been described(Burdett, 1980;Abraham and Rood, 1985). Plasmid constructions. Restriction enzymes, Klenow fragment, and T4 DNA ligase were from BRL and were used according to the supplier’s specifications. Specific restriction fragments were purified from agarosegels by electroelution in an Elutrap (Schleicher & Schuell) apparatus. Ligated DNA was transformed into either E. coli JA22 1 or HBlOl. Bacterial matings. Plasmids were transferred into B. fragilis 638 using a triparental mating procedure with pRK23 1 as the helper plasmid as describedpreviously (Guiney et al., 1984~;Matthews and Guiney, 1986).Plasmids were purified from B. fragilis 638 recipients by CsCl/ethidium bromide density gradient centrifugation (Guiney et al., 1983). Minimal inhibitory concentration (MIC) determinations. The B. fragilis 638 transconjugants containing TcR derivatives of pDK3 were tested for tetracycline sensitivity as follows: (1) overnight cultures were grown in BCH (Guiney et al., 1983) containing 0.01 clg! ml tetracycline as an inducer of TcR expression, (2) 0.0 1 ml of the culture (about 10’ cells) was inoculated into 2 ml of BCH containing serial increasing concentrations of tetracycline, and (3) the tubes were incubated anaerobically for 48 h. The MIC was recorded as the lowest concentration that prevented visible growth. In each determination, the plasmid-containing

ET AL.

strain was compared with the isogenic plasmid-free parent. RESULTS

Derivatives of pDP1 A4 containing deletions of the CIRgene. The parent plasmid for all the vectors is pDP 1A4, shown in Fig. 1. This plasmid was constructed by a BglII deletion of pDP 1, our original shuttle plasmid formed by ligating pDG5 to pCP 1. The CIR,aerobic TcR, and replication region of pCP1 were mapped previously on pDP1 A4 by Tn5 mutagenesis (Matthews and Guiney, 1986). Becauseof the relatively large size of pDP 1A4 and the lack of convenient cloning sites, our overall ap preach was to reduce the size of the plasmid and introduce regions containing appropriate restruction enzyme sites. The first derivative, pDP1 A5, was constructed by a partial TaqI digest of pDP1 A4, followed by ligation and transformation of E. coli JA22 1. The transformants were pooled and mated into B. fragilis 638 using the helper plasmid pRK231. Plasmid DNA from the B. fragilis CIR transconjugants wasextracted and transformed into E. coli JA22 1. Analysis of one of these transformants yielded pDP 1A5 which had lost 4 kb of DNA around the BglII site (Fig. 1). Passage through B. fragilis ensured that the replication region of pCP1 was still intact. The CIRand aerobic TcR geneswere deleted from pDP1 A5 by EcoRI digestion and religation of the plasmid to form pDPlA6 (Fig. 1). This plasmid contains a single EcoRI site suitable for cloning selectable markers in Bacteroides. To increase the versatility of pDP1 A6, a HaeII fragment from pUC9, containing the multiple cloning site and 1acZ complementation region (Vieira and Messing, 1982), was inserted in two different locations to yield pDK 1 and pDK2.1. A cosmid vector based on pDP1 A5 was constructed by replacing the 4-kb EcoRI fragment with a 3-kb EcoRI/PvuII piece from pHC79 containing the cos site and TcR gene (Hohn and Collins, 1980). The resulting plasmid, pOA 10 (Fig. 1), has a single BamHl site within the TcR gene to facilitate cloning large fragments generated by SadA or Mb01 digests.

CLONING VECTORS FOR Bucteroides fragilis

19

FIG. 1. Map of pDP1 A4, and the construction of pDPlA5, pOA 10, and pDP 1A6. Seethe text for details. The individual plasmids are not drawn to the same scale. The heavy line in pDPl A4 representsthe DNA derived from pDG5, while the heavy line in pOA 10 representspHC79 DNA. The arrows represent the 1.2kb direct repeats, and the locations of the clindamycin resistance (CIR) and aerobic tetracycline resistance (Tp) genes are indicated by lines. AmR is the TEM fl-lactamase gene of pBR322. oriV pBR322 is the vegetative origin of replication of pBR322; oriT is the transfer origin of RK2, and rep B. jkzgifis refers to a region of pCP1 required for maintenance in Bucteroides.

Derivatives of pDPlA4 that retain CF. In order to clone nonselectable DNA segments in Bacteroides, it is necessaryto retain the CIR gene on the shuttle plasmid to select for transfer into B. jiragilis. Beginning with pDPlA5, a 2.6-kb region around the ClaI site wasdeleted by partial TaqI digestion as described previously. The resulting plasmid, pDK3 (Fig. 2), was recovered after transfer to B. fragilis to ensure that both the CIR gene and the pCPl replication region remained intact. pDK3 contains a single EcoRI site which has been used for cloning TcR genesfor testing in Bacteroides (seebelow). To extend the versatility of pDK3, the EcoRI site was removed by the filling-in reaction catalyzed by Klenow fragment DNA polymerase (Maniatis et al., 1982)

followed by ligation, to form pDK3.2. The HaeII fragment from pUC9 containing the multiple cloning region and IacZ complementation was inserted to give pDK4.1 and pDK4.2 (Fig. 2). Introduction and expression of foreign tetracycline resistance genes in B. fragilis. Although studies to date have indicated that foreign drug resistance genes are not expressed in Bacteroides, relatively few markers have been examined. In order to test systematically for expression of heterologous TcR genes in Bacteroides, five different markers, chosen to represent genes from gram-negative, grampositive, and anaerobic bacteria, were cloned into the EcoRI site of pDK3, as listed in Table 1. The tetA, -B, and -C determinants are widely

20

ET AL.

distributed on drug resistanceplasmids in facultative gram-negative bacteria (Mendez et al., 1980). tetA is found on broad host range IncP plasmids such asRK2/RP4; tetB is from Tn 10; and tetC was originally from pSC101. The tetM gene was first characterized in streptococci (Burdett, 1980; Burdett et al., 1982), but recently has been reported in both gram-negative and gram-positive bacteria as well as Mycoplasma and Ureaplasma (Morse et al., 1986; Roberts et al., 1985,1986a, 1986b).The TcR marker on pJIR39 was cloned from the Clostridium perfringens plasmid pCW3. Except for pOA 13, the TcR plasmids were constructed by blunt-end ligation of the gel-purified restriction fragment into the EcoRI site of pDK3 after the ends of both fragment and vector had been filled in with Klenow polymerase.Since the TcR marker from pJIR39 is located on two EcoRI fragments, a partial EcoRI digest of pJIR39 was ligated to pDK3 to form pOA13. All five plasmid constructs expressedTcR in E. coli with MIC of greater than 20 pg/ml. Each plasmid was transferred into B. frugilis 638 using the RK2 helper plasmid pRK23 1 and selecting for CIR in the Bucteroides recipients. Individual CIR transconjugants were picked and tested for expression of the TcR marker by tube dilution MIC as listed in Table 1. No increase in the tetracycline MIC was seen with tetA, tetB, tetC, or the C. perfringens determinant. The tetM marker on pPH4 gave a small but reproducible increment in tetracycline resistance.The MIC of cells containing pPH4 was fivefold higher than parent cells without the plasmid although the absolute value of TcR remained low. To rule out deletions or mutations in the TcR genes,each of the plasmid constructs was extracted from B. frugilis and transformed back into E. coli: all of these plasmids expressed normal levels of TcR in E. coli. DISCUSSION

The ability to transfer cloned genesfrom E. co/i to Bucteroides specieswill greatly facilitate the genetic analysis of theseanaerobic bacteria. In this paper, we describe the construction of

CLONING

VECTORS

21

FOR Bucteroides fiagih

TABLE

I

TETRACYCLINE RESISTANCEGENE EXPREWON IN B. fiagilis Tc MIC of B. jiiagiiris 638 Plasmid construct

TcR gene/plasmid source

pPH 1 pPH3 pPH5 pPH4 POA13

tetClpBR322 tetAJpTJS245 tetBlpBT107 tetMlpJI3 pJIR39

Size/restriction

shuttle vectors based on pDP1, our original hybrid plasmid containing pDG5 and pCP1, the most extensively studied Bacteroidesplasmid. pCP1 (probably identical to pBFTM10, described independently: Tally et al., 1982) is 14 kb in size and contains the CIR gene and aerobic TcR marker on a transposable DNA segment,designatedTn4400, bounded by 1.2kb direct repeats (Robillard et al., 1985). In order to construct stable vectors, we sought to remove portions of one or both of the direct repeats while still retaining the desired drug resistance and replication properties. In addition, we needed to reduce the size of the plasmid and introduce new restriction sites for cloning. Deletions around the Bg/II sites of pDP 1, yielding pDP 1A4 and pDP 1A5, removed 8 kb of DNA, including all of the EcoRI C fragment of pCP 1 and part of the A fragment. The only known trait mapping in this region is a mobilization function that allows pCP1 to be transferred by other conjugation systems (Shoemaker et al., 1986). This function is dispensable since all of our derivatives contain the RK2 oriT sequence.Deletion of the EcoRI B fragment in pDP1 A6 and pOAl0 removes the CIR gene, aerobic TcR, and one direct repeat. The vectors pDK 1, pDK2.1, and pOA 10 retain the pCP 1 replication region and should prove useful in cloning selectable markers in Bacteroides, such as the non-plasmid-mediated CIR and TcR genes. In order to introduce cloned genesthat do not have a selectablephenotype in Bacteroides, we constructed plasmid vectors that retain the

fragment

1.4 kb/EcoRI-AvaI 4 kb/EcoRI-Hind111 2.2 kb/HindIII-BstEII 5 kb/HincII 4 kb/EcoRI-EcoRI

- plasmid

+ plasmid

0.2 0.2 0.1 0.1 0.1

0.2 0.2 0.1 0.5 0.1

CIR gene. These plasmids are based on pDK3 and have lost 2.6 kb of DNA around the C/a1 site, inactivating the aerobic TcR gene and removing the adjacent EcoRI site. pDK4.1 and 4.2, aswell as pDK3, allow cloned DNA to be transferred into B. fragilis and maintained as a plasmid by clindamycin selection. The vector pDK3 was usedto introduce and test the expression of different tetracycline resistancegenesin B. fragilis. This study (Table 1) showed that only the tetM gene from strep tococci expresseddetectable tetracycline resistance in Bacteroides.The molecular basis for the lack of tetracycline resistance expression by the other markers could be at the level of transcription, translation, or function of the gene products. The tetA, -B, and -C determinants encodean energy-dependenttetracycline efflux system (McMurry et al., 1980), and the tetA marker on RK2/RP4 is expressed in a wide variety of gram-negative bacteria (Thomas and Smith, 1987). The tetM gene encodes tetracycline resistance by a different mechanism and appears to act at the level of the ribosome (Burdett, 1986). This biochemical difference may explain the detectable expression of tetM in Bacteroides.The tetM marker appears quite versatile and has been found in both gram-positives and gram-negatives, as well as Mycoplasma, Ureaplasma, and the anaerobe, C. di@cile (Burdett et al., 1982; Hachler et al., 1987; Morse et al., 1986; Roberts et al., 1986a, 1986b, 1985). Finally, we tested a tetracycline resistance gene from the anaerobe C. perji+ngens(Abraham and Rood, 1985). Although the mechanism of ac-

22

GUINEY ET AL.

resistance, tetracycline resistance, and a replication tion of this gene is not known, it is expressed function on the Bacteroides R plasmid pCP 1. J. Bacin E. co/i but not in B. fragilis. This work teriol. 167, 5 1l-52 1. shows that the only antibiotic resistance gene MCMURRY, L., PETRUCCI,R. E., AND LEVY, S. B. ( 1980). expressedwell in B. fragilis is the CIR deterActive efflux of tetracycline encoded by four genetically minant cloned from Bacteroidesplasmids. different resistance determinants in Escherichia coli.

ACKNOWLEDGMENTS This work was supported by Public Health Service Grants AI16463 and DE07344 from the National Institutes of Health. B. Matthews was supported by a training grant from the National Institute ofAllergy and Infectious Diseases.We thank V. Burdett, T. Schmidhauser,L. Smith, and J. Rood for providing plasmids used in this study.

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BURDETT,V., INAMINE,J., AND RAJAGOPALAN, S. (1982). Heterogeneity of tetracycline resistancedeterminants in Streptococcus.J. Bacterial. 149, 995- 1004. GUINEY, D., HASEGAWA,P., AND DAVIS, C. (1984a). Expression in Escherichia coli of cryptic tetracycline resistancegenesfrom Bacferoides R plasmids. Plasmid l&248-252.

GUINEY,D., HASEGAWA,P., AND DAVIS,C. (1984b). Homology between clindamycin resistance plasmids in Bacteroides. Plasmid 11, 268-27 1. GUINEY, D., HASEGAWA,P., AND DAVIS, C. (1984c). Plasmid transfer from Escherichia coli to Bacteroides fragilis: Differential expression of antibiotic resistance phenotypes. Proc. Natl. Acad. Sci. USA 83,7203-7206. GUINEY, D., HASEGAWA,P., STALKER,D., AND DAVIS, C. (1983). Genetic analysis of clindamycin resistancein Bacteroides species.J. Infect. Dis. 147, 55 l-558. HACHLER,H., KAYSER,F. H., AND BERGER-BACHI,B. (1987). Homology of a transferable tetracycline resistance determinant of Clostridium dtjicile with Streptococcus(Enterococcus)faecalis transposon Tn9 16.Antimicrob. Agents Chemother. 31, 1033-1038. HOHN, B., AND COLLINS,J. (1980). A small cosmid for efficient cloning of large DNA fragments.Gene 11,29 l298.

MANIATIS, T., FRITSCH,E., AND DAMBROOK,J. (1982). “Molecular Cloning: A Laboratory Guide.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MATTHEWS,B., AND GUINEY, D. G. (1986). Characterization and mapping of regions encoding clindamycin

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ROBERTS,M. C., KOUTSKY, A., HOLMES, K. K., LEBLANE,D. J., AND KENNY, G. E. (1985). Tetracyclineresistant Mycoplasma hominis strains contain streptococcal tetM sequences.Antimicrob. Agents Chemother. 28,141-143. ROBILLARD,N., TALLY, F., AND MALAMY, M. (1985). Tn4400, A compound transposon isolated from Bacteroidesfragilis functions in E. coli. J. Bacterial. 164, 1248-1255. SHOEMAKER,N. B., GETTY, C., GUTHERIE,E. P., AND SALYERS,A. A. (1986). Two Bacteroides plasmids, pBFTM 10 and pB8-5 1, contain transfer regions that are recognized by broad host range IncP plasmids and by a conjugative Bacteroides tetracycline resistanceelement. J. Bacterial. 166, 959-965. SHOEMAKER,N. B., GUTHERIE,E. P., SALYERS,A. A., AND GARDENER,J. F. (1985). Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposableelement. J Bacterial. 162,626-632.

TALLY, F., SNYDMAN,D., SHIMELL,M., AND MALAMY, M. (1982). Characterization of pBFTM 10, a clindamycin-erythromycin resistance transfer factor from Bacteroidesfragilis. J. Bacterial. 151, 686-689. THOMAS,C. M., AND SMITH,C. A. (1987).Incompatibility group P plasmids:Genetics,evolution, and usein genetic manipulation. Annu. Rev. Microbial. 41, 77-101. VIEIRA, J., AND MESSING,J. (1982). The pUC plasmids, an M 13 mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19,259-268.

Communicated by Francis L. Macrina