PLASMID
21,242-245 (1992)
Molecular Analysis of a Gene from Bacteroides fragilis Involved in Metronidazole Resistance in Escherichia co/i GABI U. WEHNERT, VALERIE R. ABRATT, AND DAVID R. WOODS Department of Microbiology, University of Cape Town, P B Rondebosch 7700, South Africa Received June 11, 199I ; revised September 20, 1991 The region of Bacteroidesfragilis DNA on the recombinant plasmid pMT 100 responsible for conferring metronidazole resistancein Escherichia coli strains was characterized. An open reading frame (ORFl) of 195bp encoded a protein of 64 amino acids with a predicted M, of 7.3 kDa. Deletion analysis indicated that ORFl conferred the metronidazole resistance phenotype and encoded a protein with an apparent h4, of approximately 8-10 kDa. o 1992 Academic PWSS, IX.
Investigations have been carried out on DNA damage and repair in the obligate anaerobic bacterium Bacteroidesfragilis (Jones et al., 1980; Jones and Woods, 198 1; Slade et al., 1983a,b, Abratt et al., 1985, 1986; Goodman and Woods, 1990), and a B. fragilis gene affecting metronidazole resistance and ultraviolet survival in Escherichia coli has been cloned (Wehnert et al., 1990). The recombinant plasmid pMT 100 cloned in pEcoR25 1 (Zabeau and Stanley, 1982) conferred increased resistance to metronidazole under aerobic conditions in E. coli AB1157 and E. coli AB 1886 (uvrA). pMT 100 also conferred increased sensitivity to far-ultraviolet irradiation under aerobic conditions in these E. coli strains. The loci affecting metronidazole resistance and uv sensitivity were located on a 5kb DNA fragment which originated from the small cryptic plasmid pBFC 1 from B. fragilis Bf-2. The region of pMT100 encoding increased resistance to metronidazole in E. coli was located on a MluI-Hind111 restriction endonuclease fragment (Wehnert et al., 1990). The role of pMT 100 in B. fragilis BF2 is not known since it has not been possible to cure the strain of pBFC 1. The MIC of B. fragilis BF2 and the metronidazole-sensitive B. fragilis 638 strain (Breuil et al., 1989) to metronidazole has been determined and under test conditions the B. fragilis BF2 strain was resistant to approximately twice the concentra0147-619X/92 $5.00 Copyright 0 1992 by Academx Press, Inc. All rights of reproduction in any form reserved
tion of metronidazole as the B. fragilis 638 strain. To determine whether expression of the gene(s) encoding increased metronidazole resistance was controlled by the X rightward promoter of the cloning vector pEcoR25 1, the metronidazole resistance of E. coli AB1157 (pMT 100) in the presence and absence of the plasmid ~~1857 (Remaut et al., 1983), which carries the gene encoding the X repressor, was investigated. The presence of ~~1857 repressed the increased metronidazole resistance phenotype in E. coli AB 1157 (pMT100). It was therefore concluded that the gene(s) on pMT 100 was expressed from the X promoter of pEcoR251 in E. coli ABI 157 (pMT100) cells. To facilitate nucleotide sequencing the Ml&Hind111 fragment of pMT 100 was subcloned into the EcoRV-Hind111 sites of Bluescript plasmid SK (Stratagene, San Diego, CA). The subclone, pMT 115, was shortened from both the 5’ and the 3’ ends of the insert using exonuclease III (Henikoff, 1984) and a range of nested deletions spanning the entire insert was chosen (Fig. 1). The nucleotide sequence of both strands of the pMT 115 insert DNA was determined using the dideoxynucleotide triphosphate chain termination method of Sanger et al. (1977), according to the protocol of Tabor and Richardson (1987) using the Sequenase DNA Sequenc-
242
243
SHORT COMMUNICATIONS
.
. .
.
7
pMTll6
.,.
.
1822
0 ~ 182
pMTll~3 pMTll@
532
.._..._._._._,_.
1271
pMT121
1528
B ECORI “l”dlll sty, pMT100
sty,
PatI , SlYl
Sfml
M’“’
’ met resistant
7 pMT106 pMTlO7 pMT108
+
pMT100 pMT110 pMT111
+
I-
1 440 ORFl
FIG. 1. (A) Plasmid pMTl15 contained the 1.622-kb metronidazole resistance-encoding B. fiugih fragment from pMTlO0 subcloned into Bluescript SK. The exonuclease IIIderived shortenings are shown as arrows above the restriction map of pMTll5. The arrows indicate the direction and extent of sequence derived from the different nested deletions. Plasmids pMTll6, pMT 118, pMT I 19, and pMT 121 contain exonuclease III-shortened DNA fragments of pMT 115. The lengths of the B. frugilis inserts in nuc&otide basesare indicated. The location and direction of transcription of the T7 promoter are indicated (T7 ). (B) Plasmids pMT106 to pMTll1 were derived by subcloning Bluescript SK exonuclease III-shortsed insert DNA Fragments(pMTll6 to pMT12 1) into pEcoR25 1 adjacent to the X rightward promoter (P ). At the top the figure the &&I-~/u1 DNA fragment of pMTlO0 is shown for comparison. The metronidazole resistance phenotypes conferred in E. coli AB 1I57 by pMT106 to pMTll1 are indicated: +, metronidazole resistance; -, parental level of metronidazole sensitivity. The 440-bp region containing ORFl (0) is shown. Bluescript SK DNA (. . .); pEcoR25 1 DNA (-); B. frudis DNA (I).
ing kit (US Biochemical Corp., Cleveland, OH) (Fig. 2). The 1.6-kb nucleotide sequencecontained several small open reading frames (ORF) and therefore a smaller DNA region encoding metronidazole resistance had to be defined. The exonuclease III-shortened insert DNA fragments (pMTl16 to pMT121) were subcloned back into pEcoR251 to make use of the X promoter for expression studies (Fig. 1). The resulting plasmids (pMT106 to
pMTll1) were transformed into E. co/i ABl157 and the transformants were plated onto different concentrations of metronidazole to determine their minimum inhibitory concentration under aerobic conditions. As controls E. coli ABl157 transformed with pMT 100 and the deletion plasmid pMT 104, where the entire insert was deleted (Wehnert et al., 1990), were used. Only E. coli AB 1157 (pMT108) and ABl157 (pMT111) showed increased resistance to metronidazole similar
244
SHORT COMMUNICATIONS
fiG. 2. Nucleotide sequence of the 440-bp DNA region of pMT 115 containing ORFl _The deduced amino acid sequence for ORFI is given in single-letter code below the nucleotide sequence.
to that of AB 1157(pMT 100), whereasE. coli AB1157 transformed with the other subclones showed parental levels of metronidazole resistance(Fig. 1). This indicated that metronidazole resistance was encoded by gene(s)contained within a 440-bp DNA region. This region contained only one complete open reading frame (ORFI) of 195 bp which encoded a protein of 64 amino acids (Fig. 2). ORFl was initiated by an ATG start codon and terminated by a TAG stop codon. A consensusribosome binding site (Shine and Dalgamo, 1976)upstreamof the start codon was not detected, but an ATGA sequence was located 10 bp upstream of the ATG start. The nucleotide sequenceof the pMTl15 insert DNA was analyzed on a VAX 6000330 computer using the University of Wisconsin GeneticsComputer Group suite of sequence analysis programs (Devereux et al., 1984).The programsTESTCODE, which detects possible protein coding sequencesby plotting a measureof the nonrandomnessof the composition at everythird base,and CODONFREQUENCY, which recognizespossible protein coding sequencesby comparing the codon preferenceof the query sequence to a codon frequency table, were used to ob-
tain further evidence for the presence of a functional ORFl . Both programspredicted a coding region in the area of ORFl (results not shown). The deduced amino acid sequence(64 aa) of ORFl was used to search the GenBank, EMBL, SWISS-Protein, and NBRF-Protein databasesfor related amino acid sequences using the FASTA and TFASTA computer programs. Although limited similarity was demonstratedto a number of unrelated gene products no specific regions of homology or similarity were apparent and the similar amino acids were distributed throughout the proteins. The DNA and amino acid sequenceshavebeendepositedin the GenBank data bank (AccessionNo. M7655 1). The synthesisof proteins from the insert in pMT 115 was investigatedusing the T7 promoter-directed expression system of Tabor and Richardson (1985), which is an in vivo system designed to express only plasmidborne genes.The following constructs were transformed into E. coli AB1157 and used for the T7 promoter-directed expressionsystem: Bluescript SK, pmTl16 and pmT 119, both deleted for ORFl ; and pMT 118 and pMT121, both containing ORFl (Fig. 1). Under aerobic conditions E. coli AB1157 (pMT118) and E. coli AB1157 (pMT121) produced a pronounced protein band with an apparent h& of approximately 8 kDa and a lessintenseprotein band with an apparent M, of approximateIy 10 kDa (Fig. 3). Ahhough the insert DNA of pMT 118 has the coding capacity only for ORF 1,it produced two proteins with apparent M, of 8 and 10 kDa. It is suggestedthat in E. coli AB1157 the 8-kDa protein is a processedor degradation product of the lo-kDa protein. The predicted M, of the protein produced by ORFl is 7.3 kDa. Although this is lessthan the 10-kDa protein observedon the SDS-PAGE gels,it is within the range of accuracy of the gel techniques. These proteins were not produced by E. coli AB1157 (pMT116) or E. coli AB1157 (pMTl19). These results indicated that ORF 1 encodeda protein in E. coli cells.This protein with an apparent A& of approxi-
SHORT COMMUNICATIONS
FIG. 3. T7 promoter-dire&d expression of the ORFI protein. Lanes A to Don the radiograph of the polyacrylamide gel contained cell extracts of the following: (A) E. coli ABI 157 (pMTl16), (B) E. coli AB1157 (pMTl18), (C) E. coli ABI 157 (pMTIl9), and (D) E. coli ABl157 (pMTl2 1). The positions of the proteins encoded by ORFI and ORF2 are indicated by arrows. Approximate M, values are indicated in kilodaltons.
mately 10 kDa appeared to be responsible for increased resistance to metronidazole in E. co/i AB1157. The results from the TESTCODE analysis predicted a second ORF downstream of ORFl (results not shown) which encoded a protein with a predicted &I, of 14.7 kDa. A protein with an approximate && of 16 kDa was produced in E. coli AB 1157 (pMT119) and E. coli AB1157 (pMT121), but not in E. coli AB1157 (pMTl16) or E. coli AB1157 (pMTl18) (Fig. 3). The presence or absence of ORF2 had no affect on the metronidazole phenotype of the cells. E. coli AB 1157 transformed with Bluescript SK did not produce either the 8 to 10 kDa or the 16-kDa proteins. REFERENCES ABRATT, V., JONES,D. T., AND WOODS,D. R. (1985). Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroidesfragilis. J. Gen. Microbial. 131,2479-2483. ABRATT, V. R., LINDSAY, G. L., AND WOODS,D. R. (1986). Pyrimidine dimer excision repair of DNA in Bacteroidesfragils wild-type and Mitomycin C-sensitive mutants. J. Gen. Microbial. 132,2577-258 1.
245
BREIJIL,J., DUBLAN~HET,A., TRUFFAUT,N., AND SEBALD, M. (1989). Transferable 5nitroimidaxole resistance in the Bacteroidesfragilis group. Plasmid 21, 151-154. DEVERBUX,J., HAEBERII, P., AND SMITH~S, 0. (1984). A comprehensive set of sequence analysis programs for the vax. Nucleic Acids Res. 12, 387-395. GOODMAN,H. J. K., AND WOOD$ D. R. (1990). Moleeular analysis of the Bacteroidesfragiiis recA gene. Gene 94,77-82. HENIKOFF,S. (1984). Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28,35 l-359. JONES,D. T., ROBB,F. T., AND WOODS,D. R. (1980). E&et of oxygen on Bacteroides fragilis survival after far-ultraviolet irradiation. J. Bacterial. 144(3), 11791181. JONES,D. T., ANJJWOODS,D. R. ( 1981). Effect of oxygen on liquid holding recovery of Bacteroidesfragilis. J. Bacterial. 145(l), l-7. REMAUT,E., TSAO,H., AND FIERS,W. (1983). Improved plasmid vectors with thermo-inducible expression and temperature regulated runaway replication. Gene 22, 103-I 13. SANGER,F., NICKLEN, S., AND COIJISON,A. R. ( 1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463. SHINE, J., AND DALGARNO,L. (1976). Determinant of cistron specificity in bacterial ribosomes. Nature 254, 34-38. SLADE,H. J. K., S~HIJMANN,J. P., JONES,D. T., AND WOODS,D. R. (1983a). Peroxide inducible phage reactivation in Bacteroidesfragilis. FEMSMicrobiol. Lett. 20,401-405. SLADE,H. J. K., SCHUMANN,J. P., PARKER,J. R., JONES, D. T., AND WOODS,D. R. (1983b). El& ofoxygen on host cell reactivation in Bacteroidesfragiiis. J. Bacteriol. 153, 1545-1547. TAEIOR,S., AND RICHARDSON,C. C. (1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Nat/. Acad. Sci. USA 82, 1074-1078. TABOR, S., AND RICHARDSON,C. C. (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767-477 I. WEHNERT,G. U., ABRATT, V. R., GOODMAN,H. J. K., AND WOODS,D. R. ( 1990).Cloning of Bacteroidesfiagilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli. Plasmid 23, 155-158. ZABEAU,M., AND STANLEY,K. K. (1982). Enhanced expression of cro-&galactosidase fusion proteins under the control of the PR promoter of bacteriophage X. EMBO J. l(lO), 1217-1224. Communicated by Stuart B. Levy
21,242-245 (1992)
Molecular Analysis of a Gene from Bacteroides fragilis Involved in Metronidazole Resistance in Escherichia co/i GABI U. WEHNERT, VALERIE R. ABRATT, AND DAVID R. WOODS Department of Microbiology, University of Cape Town, P B Rondebosch 7700, South Africa Received June 11, 199I ; revised September 20, 1991 The region of Bacteroidesfragilis DNA on the recombinant plasmid pMT 100 responsible for conferring metronidazole resistancein Escherichia coli strains was characterized. An open reading frame (ORFl) of 195bp encoded a protein of 64 amino acids with a predicted M, of 7.3 kDa. Deletion analysis indicated that ORFl conferred the metronidazole resistance phenotype and encoded a protein with an apparent h4, of approximately 8-10 kDa. o 1992 Academic PWSS, IX.
Investigations have been carried out on DNA damage and repair in the obligate anaerobic bacterium Bacteroidesfragilis (Jones et al., 1980; Jones and Woods, 198 1; Slade et al., 1983a,b, Abratt et al., 1985, 1986; Goodman and Woods, 1990), and a B. fragilis gene affecting metronidazole resistance and ultraviolet survival in Escherichia coli has been cloned (Wehnert et al., 1990). The recombinant plasmid pMT 100 cloned in pEcoR25 1 (Zabeau and Stanley, 1982) conferred increased resistance to metronidazole under aerobic conditions in E. coli AB1157 and E. coli AB 1886 (uvrA). pMT 100 also conferred increased sensitivity to far-ultraviolet irradiation under aerobic conditions in these E. coli strains. The loci affecting metronidazole resistance and uv sensitivity were located on a 5kb DNA fragment which originated from the small cryptic plasmid pBFC 1 from B. fragilis Bf-2. The region of pMT100 encoding increased resistance to metronidazole in E. coli was located on a MluI-Hind111 restriction endonuclease fragment (Wehnert et al., 1990). The role of pMT 100 in B. fragilis BF2 is not known since it has not been possible to cure the strain of pBFC 1. The MIC of B. fragilis BF2 and the metronidazole-sensitive B. fragilis 638 strain (Breuil et al., 1989) to metronidazole has been determined and under test conditions the B. fragilis BF2 strain was resistant to approximately twice the concentra0147-619X/92 $5.00 Copyright 0 1992 by Academx Press, Inc. All rights of reproduction in any form reserved
tion of metronidazole as the B. fragilis 638 strain. To determine whether expression of the gene(s) encoding increased metronidazole resistance was controlled by the X rightward promoter of the cloning vector pEcoR25 1, the metronidazole resistance of E. coli AB1157 (pMT 100) in the presence and absence of the plasmid ~~1857 (Remaut et al., 1983), which carries the gene encoding the X repressor, was investigated. The presence of ~~1857 repressed the increased metronidazole resistance phenotype in E. coli AB 1157 (pMT100). It was therefore concluded that the gene(s) on pMT 100 was expressed from the X promoter of pEcoR251 in E. coli ABI 157 (pMT100) cells. To facilitate nucleotide sequencing the Ml&Hind111 fragment of pMT 100 was subcloned into the EcoRV-Hind111 sites of Bluescript plasmid SK (Stratagene, San Diego, CA). The subclone, pMT 115, was shortened from both the 5’ and the 3’ ends of the insert using exonuclease III (Henikoff, 1984) and a range of nested deletions spanning the entire insert was chosen (Fig. 1). The nucleotide sequence of both strands of the pMT 115 insert DNA was determined using the dideoxynucleotide triphosphate chain termination method of Sanger et al. (1977), according to the protocol of Tabor and Richardson (1987) using the Sequenase DNA Sequenc-
242
243
SHORT COMMUNICATIONS
.
. .
.
7
pMTll6
.,.
.
1822
0 ~ 182
pMTll~3 pMTll@
532
.._..._._._._,_.
1271
pMT121
1528
B ECORI “l”dlll sty, pMT100
sty,
PatI , SlYl
Sfml
M’“’
’ met resistant
7 pMT106 pMTlO7 pMT108
+
pMT100 pMT110 pMT111
+
I-
1 440 ORFl
FIG. 1. (A) Plasmid pMTl15 contained the 1.622-kb metronidazole resistance-encoding B. fiugih fragment from pMTlO0 subcloned into Bluescript SK. The exonuclease IIIderived shortenings are shown as arrows above the restriction map of pMTll5. The arrows indicate the direction and extent of sequence derived from the different nested deletions. Plasmids pMTll6, pMT 118, pMT I 19, and pMT 121 contain exonuclease III-shortened DNA fragments of pMT 115. The lengths of the B. frugilis inserts in nuc&otide basesare indicated. The location and direction of transcription of the T7 promoter are indicated (T7 ). (B) Plasmids pMT106 to pMTll1 were derived by subcloning Bluescript SK exonuclease III-shortsed insert DNA Fragments(pMTll6 to pMT12 1) into pEcoR25 1 adjacent to the X rightward promoter (P ). At the top the figure the &&I-~/u1 DNA fragment of pMTlO0 is shown for comparison. The metronidazole resistance phenotypes conferred in E. coli AB 1I57 by pMT106 to pMTll1 are indicated: +, metronidazole resistance; -, parental level of metronidazole sensitivity. The 440-bp region containing ORFl (0) is shown. Bluescript SK DNA (. . .); pEcoR25 1 DNA (-); B. frudis DNA (I).
ing kit (US Biochemical Corp., Cleveland, OH) (Fig. 2). The 1.6-kb nucleotide sequencecontained several small open reading frames (ORF) and therefore a smaller DNA region encoding metronidazole resistance had to be defined. The exonuclease III-shortened insert DNA fragments (pMTl16 to pMT121) were subcloned back into pEcoR251 to make use of the X promoter for expression studies (Fig. 1). The resulting plasmids (pMT106 to
pMTll1) were transformed into E. co/i ABl157 and the transformants were plated onto different concentrations of metronidazole to determine their minimum inhibitory concentration under aerobic conditions. As controls E. coli ABl157 transformed with pMT 100 and the deletion plasmid pMT 104, where the entire insert was deleted (Wehnert et al., 1990), were used. Only E. coli AB 1157 (pMT108) and ABl157 (pMT111) showed increased resistance to metronidazole similar
244
SHORT COMMUNICATIONS
fiG. 2. Nucleotide sequence of the 440-bp DNA region of pMT 115 containing ORFl _The deduced amino acid sequence for ORFI is given in single-letter code below the nucleotide sequence.
to that of AB 1157(pMT 100), whereasE. coli AB1157 transformed with the other subclones showed parental levels of metronidazole resistance(Fig. 1). This indicated that metronidazole resistance was encoded by gene(s)contained within a 440-bp DNA region. This region contained only one complete open reading frame (ORFI) of 195 bp which encoded a protein of 64 amino acids (Fig. 2). ORFl was initiated by an ATG start codon and terminated by a TAG stop codon. A consensusribosome binding site (Shine and Dalgamo, 1976)upstreamof the start codon was not detected, but an ATGA sequence was located 10 bp upstream of the ATG start. The nucleotide sequenceof the pMTl15 insert DNA was analyzed on a VAX 6000330 computer using the University of Wisconsin GeneticsComputer Group suite of sequence analysis programs (Devereux et al., 1984).The programsTESTCODE, which detects possible protein coding sequencesby plotting a measureof the nonrandomnessof the composition at everythird base,and CODONFREQUENCY, which recognizespossible protein coding sequencesby comparing the codon preferenceof the query sequence to a codon frequency table, were used to ob-
tain further evidence for the presence of a functional ORFl . Both programspredicted a coding region in the area of ORFl (results not shown). The deduced amino acid sequence(64 aa) of ORFl was used to search the GenBank, EMBL, SWISS-Protein, and NBRF-Protein databasesfor related amino acid sequences using the FASTA and TFASTA computer programs. Although limited similarity was demonstratedto a number of unrelated gene products no specific regions of homology or similarity were apparent and the similar amino acids were distributed throughout the proteins. The DNA and amino acid sequenceshavebeendepositedin the GenBank data bank (AccessionNo. M7655 1). The synthesisof proteins from the insert in pMT 115 was investigatedusing the T7 promoter-directed expression system of Tabor and Richardson (1985), which is an in vivo system designed to express only plasmidborne genes.The following constructs were transformed into E. coli AB1157 and used for the T7 promoter-directed expressionsystem: Bluescript SK, pmTl16 and pmT 119, both deleted for ORFl ; and pMT 118 and pMT121, both containing ORFl (Fig. 1). Under aerobic conditions E. coli AB1157 (pMT118) and E. coli AB1157 (pMT121) produced a pronounced protein band with an apparent h& of approximately 8 kDa and a lessintenseprotein band with an apparent M, of approximateIy 10 kDa (Fig. 3). Ahhough the insert DNA of pMT 118 has the coding capacity only for ORF 1,it produced two proteins with apparent M, of 8 and 10 kDa. It is suggestedthat in E. coli AB1157 the 8-kDa protein is a processedor degradation product of the lo-kDa protein. The predicted M, of the protein produced by ORFl is 7.3 kDa. Although this is lessthan the 10-kDa protein observedon the SDS-PAGE gels,it is within the range of accuracy of the gel techniques. These proteins were not produced by E. coli AB1157 (pMT116) or E. coli AB1157 (pMTl19). These results indicated that ORF 1 encodeda protein in E. coli cells.This protein with an apparent A& of approxi-
SHORT COMMUNICATIONS
FIG. 3. T7 promoter-dire&d expression of the ORFI protein. Lanes A to Don the radiograph of the polyacrylamide gel contained cell extracts of the following: (A) E. coli ABI 157 (pMTl16), (B) E. coli AB1157 (pMTl18), (C) E. coli ABI 157 (pMTIl9), and (D) E. coli ABl157 (pMTl2 1). The positions of the proteins encoded by ORFI and ORF2 are indicated by arrows. Approximate M, values are indicated in kilodaltons.
mately 10 kDa appeared to be responsible for increased resistance to metronidazole in E. co/i AB1157. The results from the TESTCODE analysis predicted a second ORF downstream of ORFl (results not shown) which encoded a protein with a predicted &I, of 14.7 kDa. A protein with an approximate && of 16 kDa was produced in E. coli AB 1157 (pMT119) and E. coli AB1157 (pMT121), but not in E. coli AB1157 (pMTl16) or E. coli AB1157 (pMTl18) (Fig. 3). The presence or absence of ORF2 had no affect on the metronidazole phenotype of the cells. E. coli AB 1157 transformed with Bluescript SK did not produce either the 8 to 10 kDa or the 16-kDa proteins. REFERENCES ABRATT, V., JONES,D. T., AND WOODS,D. R. (1985). Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroidesfragilis. J. Gen. Microbial. 131,2479-2483. ABRATT, V. R., LINDSAY, G. L., AND WOODS,D. R. (1986). Pyrimidine dimer excision repair of DNA in Bacteroidesfragils wild-type and Mitomycin C-sensitive mutants. J. Gen. Microbial. 132,2577-258 1.
245
BREIJIL,J., DUBLAN~HET,A., TRUFFAUT,N., AND SEBALD, M. (1989). Transferable 5nitroimidaxole resistance in the Bacteroidesfragilis group. Plasmid 21, 151-154. DEVERBUX,J., HAEBERII, P., AND SMITH~S, 0. (1984). A comprehensive set of sequence analysis programs for the vax. Nucleic Acids Res. 12, 387-395. GOODMAN,H. J. K., AND WOOD$ D. R. (1990). Moleeular analysis of the Bacteroidesfragiiis recA gene. Gene 94,77-82. HENIKOFF,S. (1984). Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28,35 l-359. JONES,D. T., ROBB,F. T., AND WOODS,D. R. (1980). E&et of oxygen on Bacteroides fragilis survival after far-ultraviolet irradiation. J. Bacterial. 144(3), 11791181. JONES,D. T., ANJJWOODS,D. R. ( 1981). Effect of oxygen on liquid holding recovery of Bacteroidesfragilis. J. Bacterial. 145(l), l-7. REMAUT,E., TSAO,H., AND FIERS,W. (1983). Improved plasmid vectors with thermo-inducible expression and temperature regulated runaway replication. Gene 22, 103-I 13. SANGER,F., NICKLEN, S., AND COIJISON,A. R. ( 1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463. SHINE, J., AND DALGARNO,L. (1976). Determinant of cistron specificity in bacterial ribosomes. Nature 254, 34-38. SLADE,H. J. K., S~HIJMANN,J. P., JONES,D. T., AND WOODS,D. R. (1983a). Peroxide inducible phage reactivation in Bacteroidesfragilis. FEMSMicrobiol. Lett. 20,401-405. SLADE,H. J. K., SCHUMANN,J. P., PARKER,J. R., JONES, D. T., AND WOODS,D. R. (1983b). El& ofoxygen on host cell reactivation in Bacteroidesfragiiis. J. Bacteriol. 153, 1545-1547. TAEIOR,S., AND RICHARDSON,C. C. (1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Nat/. Acad. Sci. USA 82, 1074-1078. TABOR, S., AND RICHARDSON,C. C. (1987). DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84, 4767-477 I. WEHNERT,G. U., ABRATT, V. R., GOODMAN,H. J. K., AND WOODS,D. R. ( 1990).Cloning of Bacteroidesfiagilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli. Plasmid 23, 155-158. ZABEAU,M., AND STANLEY,K. K. (1982). Enhanced expression of cro-&galactosidase fusion proteins under the control of the PR promoter of bacteriophage X. EMBO J. l(lO), 1217-1224. Communicated by Stuart B. Levy
