Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

PLASMID

23,

Cloning

155-158 (1990)

of Bacteroides fragilis Piasmid Genes Affecting Metronidazole Resistance and Ultraviolet Survival in Escherichia co/i GABIU.

WEHNERT,VALERIER. ABRATT,HEIDEJ.K. AND DAVID R. WOODS’

Department of Microbiology,

GOODMAN,

University of Cape Town, Rondebosch 7700, South Africa

Received October 3 1, 1989; revised February 2, 1990 Since reduced metronidazole causesDNA damage, resistanceto metronidasole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia cob’. Genes from B.frag&s Bf-2 were cloned on a recombinant plasmid pMTlO0 which made E. co/i AB 1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistanceand uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fiagiils Bf-2 cells. o two Academic

We have been investigating the mechanisms of the repair of DNA damage in Bacteroides fragilis (Jones et al., 1980; Jones and Woods, 1981; Slade et al., 1983a,b; Abratt et al., 1985, 1986). To extend these studies to the molecular level we screened a B. fragilis gene bank in an attempt to isolate B. fiagilis DNA repair genes. DNA repair deficient mutants of Escherichia coli (uvrA,B, C and recA) were shown by Yeung et al. (1984) to be more sensitive to metronidazole than the wild-type strain. We therefore screened the B. fragilis gene bank in an E. coli uvrA mutant for increased metronidazole resistance. Metronidazole is a nitroimidazole which on reduction forms a reactive intermediate compound able to interact with DNA and cause strand breakage (Ings et al., 1974; Muller, 1983; Walker, 1984). The B. fragiili Bf-2 strain (Mossie et al., 1979) contained a cryptic plasmid pBFC1 of approximately 6 kb. A library of B. fragilis Bf2 DNA was established in E. coli DKl by insertional inactivation of the EcoRI gene of the positive selection vector pEcoR25 1 (Zappe et a/., 1986). Plasmid DNA prepared from pools of clones (about 10,000) containing B. fragih DNA was used to transform the E. cob ’ To whom correspondence should be addressed.

ABl886 uvrA mutant which was unable to grow on LB agar containing 500 @g/ml of metronidazole. An E. coli AB1886 transformant was isolated on LB agar containing 500 &ml of metronidazole. The transformant contained a recombinant pEcoR25 1 plasmid which on retransformation conferred ampicillin resistance and increased resistance to metronidazole. The recombinant plasmid was designated pMT 100. The ability of pMT 100 to confer increased metronidazole resistance in E. co/i AB 1157 (uvr+) and the uvr mutants, AB1884 (uvrc), AB1885 (uvrB), and AB1886 (uvrA) (HowardFlanders et al., 1966), was determined under aerobic conditions on LB agar plates. The MIC2 for metronidazole of E. coli AB 1157 was increased from 500 to 800 pg/ml metronidazole in cells containing pMT 100. The MIC for metronidazole of each of the uvrA, B, or C mutant was increased from 300 to 500 Kg/ml metronidazole in cells containing pMT 100. Transformation with a control recombinant pEcoR25 1 plasmid (pMTA104) (Fig. 1) did not affect their resistance to metronidazole. Although these increases in metronidazole re* Abbreviation used: MIC, minimal inhibitory concentration.

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0147-619X/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduftion in any form resewed.

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FIG. 1. Restriction maps of pMT100, pBFC1 (the B. fragilis Bf.2 plasmid from which pMTlO0 was derived), and the pMTlO0 deletion plasmids, pMTAlO1, pMTAlO2, pMTA103, and pMTA104. The bold bar in pMT 100 representsthe insert from pBFC1 and the thin bar representsthe cloning vector pEcoR25 1. The arrow indicates the location and direction of transcription of the X promoter on pEcoR25 1. The hatched area represents an area of spontaneous rearrangements which occurred during cloning.

sistance conferred by pMT 100 were relatively small, they were highly reproducible and identical MICs were obtained for each strain in 10 different experiments. Since the plasmid pMT 100 increased the metronidazole resistance of DNA repair deficient mutants, the ability of pMT 100 to confer resistance to far uv irradiation was investigated. The deletion plasmid pMT104 was used as a control. The uv survival of E. coli strain ABl157 uvr+ was decreased following transformation with pMT100. Survival of E. coli ABl157(pMT104) and ABl157(pMTlOO) at 60 J/m* was 13.7 and 0.270%, respectively. The DNA repair deficient E. coli mutants AB1885 uvrB and AB1886 uvrA were also much more sensitive to far uv irradiation after transformation with pMT 100. The survival of both mutants at 6 J/m* was reduced approximately 200-fold by pMT 100. E. coli AB 1884

uvrC (pMT 100) was lessaffected,and survival at 6 J/m2 was reduced approximately 5-fold. All these differences in uv survival were highly reproducible. The DNA regions determining the increased metronidazole resistance and uv sensitivity phenotypes were determined by the isolation of pMT100 deletion plasmids pMTA101, pMTA102, and pMTAl03 (Fig. 1). Plasmid pMTA 101 conferred increased metronidazole resistance but did not confer uv sensitivity which indicated that the locus conferring sensitivity waslocated on the pVuI/ClaI restriction endonuclease fragment. Deletion of the Ck.zI/ M/u1 restriction endonuclease fragment in pMTA 102 resulted in increasedmetronidazole resistance and partial uv sensitivity. Deletion of a MluI/HindIII restriction endonuclease fragment in pMTAl03 eliminated both the uv sensitive and the increased metronidazole re-

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sistancephenotypes. It was concluded that the locus conferring increased metronidazole resistance was located on the MluI/HindIII restriction endonuclease fragment. Although pMTA103 contained the pVuII/ClaI restriction endonuclease fragment which contained the uv sensitivity locus, it was unable to confer the uv sensitivity phenotype on E. coli cells. It is suggestedthat in constructing pMTA103 a distal regulatory region may have been deleted. The origin of the 5-kb DNA insert in pMT 100wasinvestigated by Southern blotting and DNA hybridization between B. fragilis total DNA and 32P-labeledpMT100 and between pMTlO0 and 32P-labeledpBFC1 (Fig. 2). pMTlO0 hybridized to itself and to the B. jkzgilis total DNA (Fig. 2), but did not hybridize to E. coli total DNA (results not shown). Digestion of pMT100 with PstI endonuclease resulted in an internal insert DNA fragment of 3.3-kb (Fig. 1) and as expected this internal fragment hybridized with the equivalent DNA fragment when the B. fragilis DNA was digested with the P.stIendonuclease (Fig. 2). pMTlO0 hybridized strongly to a discrete band of uncut B. fragilis DNA lying below the bulk chromosomal DNA (Fig. 2). It was concluded that this band was the DNA of the cryptic plasmid pBFC1 present in the B. jkzgilis Bf-2 strain. These hybridization results suggested that the insert DNA on pMTlO0 hybridized with pBFC1 and not with the B. fragilis chromosomal DNA. To determine whether the cloned fragment originated from pBFC 1, the plasmid from B. fragilis was isolated, labeled with 32P,and hybridized against pMTlO0 digested with PstI and Sty1 endonucleases(Fig. 2). Labeled pBFC 1 hybridized to the 3.3-kb PstI and to the 3.0- and 0.65-kb Sty1 internal restriction endonuclease fragments of pMT 100. As expected the restriction endonuclease maps of the insert DNA in pMTlO0 and pBFC1 (J. A. Southern, Ph.D. thesis, University of Cape Town, Cape Town, 1986) were similar (Fig. 1). Plasmid pBFC1 is interesting in that it appeared to be cryptic in B. fragilis Bf-2 cells, but when it was cloned on a recombinant

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FIG. 2. Southern blot analysis of pMTlO0 to B. frugilis total DNA (a-c) and of pBFC1 to pMTlO0 (d-g). The agarosegel is shown on the left (A) and the corresponding radiograph on the right (B). Molecular mass markers are indicated on the left. Digestion of pMT 100 with PstI resulted in an internal insert DNA fragment of 3.3 kb (a and b). pMTlO0 hybridized to a discrete band of uncut B. fragilis DNA below the bulk chromosomal DNA (c). Digestion of pMT 100 with PstI and Styl resultedin internal insert DNA fragments of 3.3 kb (PstI) (d and e) and 3.0 and 0.65 kb (StyI) (f and g). (A) Agarose gel. Lanes (a) pMTlO0 Pstl; (b) B. frugih total DNA PstI; (c) B.fiagilis total DNA uncut; (d) pMTlO0 PstI; (e) pBFC1 PstI; (f) pMTlO0 StyI; (g) pBFC1 StyI. (B) Autoradiograph of A.

plasmid, pEcoR25 1, in E. coli ABl157 and uv sensitive mutant strains it contained two loci which made the E. coli strains more resistant to metronidazole, but more sensitive to uv irradiation under aerobic conditions. The loci are being sequencedto enable an understanding of the mechanisms controlling these phenotypes. REFERENCES ABRATT, V. R., JONES, D. T., AND WOODS, D. R.

(1985). Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroides fragilis. J. Gen. Microbial. 131, 24792483. ABRATT, V. R., LINDSAY, G. L., AND WOODS, D. R.

(1986). Pyrimidine dimer excision repair of DNA in Bacteroides Jiragilis wild-type and Mitomycin C/uvsensitive mutants. J. Gen. Microbial. 132, 2577-2581. HOWARDFLANDERS, P., BOYCE, R. P., AND THERIOT, L. (1966). Three loci in Escherichia coli K-12 that control

the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53, 11191136.

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INGS, R. M. J., MCFADZEAN, J. A., AND ORMEROD, W. E. (1974). The mode of action of metronidazole in Trichomonas vaginalis and other microorganisms. Biochem. Pharmacol. 23,142 I- 1429, JONES,D. T., ROBB, F. T., AND WOODS,D. R. (1980). Effect of oxygen on Bacteroides fragilis survival after far-ultraviolet irradiation. J. Bacterial. 144(3), 11791181. JONES,D. T., AND WOODS,D. R. (198 1). Effect of oxygen on liquid holding recovery of Bacteroides fragilis. J. Bacteriof. 145(l), l-7. MOSSIE,K. G., JONES,D. T., ROBB,F. T., AND WOODS, D. R. (1979). Characterization and mode of action of a Bacteriocin produced by a Bacteroides fragilis strain. Antimicrob.

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MULLER, M. (1983). Mode of action of metronidazole on anaerobic bacteria and protozoa. Surgery 93, I65- 17I. SLADE,H. J. K., SCHUMANN,J. P., JONES,D. T., AND WOODS,D. R. (1983a). Peroxide inducible phage reac-

tivation in Bacteroides fragilis. FEMS Microbial. Lett. 20,401-405. SLADE,H. J. K., SCHUMANN,J. P., PARKER,J. R., JONES, D. T., AND WOODS,D. R. (1983b). Effect of oxygen on host cell reactivation in Bacteroides fragilis. J. Bacterial. 153, 1545-1547. WALKER, G. C. (1984). Mutagenesis and inducible responsesto DNA damage in Escherichia coli. Microbial. Rev. 48, 60-93.

YEUNG, T. C., BEAULIEU,B. B., MCLAFFERTY,M. A., AND GOLMAN,P. (1984). Interaction of metronidazole with DNA repair mutants of Escherichia coli. Antimicrab. Agents Chemother. 25, 65-70.

ZAPPE,H., JONES,D. T., AND WOODS,D. R. (1986). Cloning and expression of Clostridium acetobutylicum endoglucanase,cellobiase and amino acid biosynthesis genesin E.rcherichia coli. J. Gen. Microbial. 132, 13671372. Communicated by Francis L. Macrina