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Keywords:

  • Bacteroides fragilis;
  • DNA damage;
  • transcriptional regulator;
  • metronidazole;
  • mitomycin C

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

A putative transcriptional regulator of the AraC/XylS family was identified in a genomic genebank of Bacteroides fragilis Bf-1, which partially relieved the sensitivity of Escherichia coli DNA repair mutants to the DNA-damaging agents, metronidazole and mitomycin C. A homologue of this gene with the same phenotype was identified as BF638R3281 in B. fragilis 638R. Transcription of BF638R3281 was constitutive with respect to exposure to sublethal doses of metronidazole. BF638R3281 was interrupted by single cross-over gene-specific insertion mutation, and the gene disruption was confirmed by PCR and DNA-sequencing analysis. The mutant grew more slowly than the wild type, and the mutation rendered B. fragilis more sensitive to metronidazole and mitomycin C. This indicates that the BF638R3281 gene product plays a role in the survival of B. fragilis following DNA damage by these agents.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bacteroides species are Gram-negative obligate anaerobes residing in the gastrointestinal tract of mammals, and accounting for approximately one-third of the colonic microbiota (Salyers, 1984). Bacteroides fragilis is a medically important opportunistic pathogen, causing more than 80% of anaerobic infections including abscess formation and septicaemia. One of the drugs of choice in treating B. fragilis infections is metronidazole, and the level of resistance is currently <5% worldwide (Falagas & Siakavellas, 2000). However, there are indications that this is increasing, and a small number of clinical isolates are resistant to metronidazole and other therapeutic agents simultaneously (Turner et al., 1995).

Metronidazole enters cells as an inactive prodrug and is activated intracellularly via anaerobic reduction to form the reactive intermediate, which is a DNA-damaging agent (Edwards, 1977). The mechanisms of resistance to metronidazole described thus far in Bacteroides include active drug efflux (Pumbwe et al., 2006), the presence of nim genes that code for drug-inactivating nitroreductases (Löfmark et al., 2005), as well as alteration of electron flux by modulation of pyruvate breakdown (Narikawa et al., 1991; Diniz et al., 2004; Gal & Brazier, 2004).

Studies have shown that impairment of DNA repair mechanisms caused increased sensitivity of bacteria to metronidazole, whereas overproduction of the recA gene from a plasmid caused increased resistance to the drug (Chang et al., 1997). Escherichia coli recA and nucleotide excision repair mutants are more sensitive to metronidazole than the wild-type isogenic strain (Jackson et al., 1984; Yeung et al., 1984), as are recA mutants of Bacteroides thetaiotaomicron, Helicobacter pylori and Mycobacterium bovis (Thompson & Blaser, 1995; Cooper et al., 1997; Sander et al., 2001). The elucidation of the DNA repair response of Bacteroides to metronidazole exposure could provide valuable information regarding the potential alternative mechanisms of resistance or susceptibility of the bacteria to metronidazole.

In the present study, a twofold approach was used to identify candidate genes involved in the cellular response of B. fragilis to metronidazole and mitomycin C (MTC) damage. A genomic B. fragilis genebank was screened in a suitable drug-sensitive DNA repair mutant E. coli host for a candidate gene conferring resistance. The gene of interest was then specifically mutated in B. fragilis, by insertional inactivation, to determine its functional role in the cell response to DNA damage. Here, the isolation and characterization of a putative transcriptional regulator that increased the resistance of E. coli DNA repair mutants to metronidazole and MTC is reported, and it is shown that this regulator was also required for wild-type cell responses to these DNA-damaging agents in B. fragilis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bacterial strains and plasmids

The plasmids and strains used in this study are described in Table 1. Genbank analysis and primer extension experiments were performed with DNA and RNA derived from B. fragilis Bf-1 (Mossie et al., 1979). All subsequent experiments were performed with B. fragilis 638R (Privitera et al., 1979), because B. fragilis Bf-1 cannot be manipulated genetically. Bacteroides fragilis strains were grown at 37 °C in supplemented brain heart infusion (BHIS) medium (Holdeman & Moore, 1972) in an anaerobic chamber (Model 1024, Forma Scientific Inc., Marietta, OH) containing an atmosphere of oxygen-free N2, CO2 and H2 (85 : 10 : 5 by volume). Escherichia coli strains were grown aerobically at 37 °C on Luria–Bertani (LB) broth or agar (1.5% w/v) medium (Sambrook et al., 1989) with the addition of ampicillin (100 μg mL−1) where applicable.

Table 1.   Bacterial strains and plasmids
StrainsRelevant characteristicsSources or references
B. fragilis Bf-1Metronidazole and MTC susceptibleMossie et al. (1979)
B. fragilis 638RMetronidazole and MTC susceptiblePrivitera et al. (1979)
B. fragilis 638R regMutant created by insertional inactivation of BF638R3281.This study
E. coli JM109recA1, Δ(lac-proAB)[F′,traD36,proAB,lacIZΔM15]Setlow et al. (1963)
E. coli AB1157uvr+ ATCC29055Bachmann (1987)
E. coli AB1886uvrA6, derived from AB1157Howard-Flanders et al. (1966)
E. coli AB1885uvrB5, derived from AB1157Howard-Flanders et al. (1966)
E. coli AB1884uvrC34, derived from AB1157Howard-Flanders et al. (1966)
E. coli S17-1recA derivative of E. coli 294 (Fthi pro hsdR) carrying a modified derivative of IncPα plasmid pRP4 (Aps Tcs Kms) integrated in the chromosome, TprSimon et al. (1983)
Plasmids
 pEcoR251E. coli suicide vector,Zabeau & Stanley (1982)
 pMT104pEcoR251 with a noncoding insertWehnert et al. (1990)
 pAN2pEcoR251 with 4.5-kb B. fragilis genomic fragmentThis work
 pGERMBacteroides insertion vector containing ermG and oriTRK2 that is mobilized by RP4 transfer functions in E. coli S17-1 chromosome; (Emr Rep) AprCheng et al. (2000)
 pGARpGERM-derivative with BF638R3281 internal fragmentThis work

Isolation of a plasmid conferring resistance to DNA-damaging agents

Plasmid pAN2 harbouring 4.5 kb of B. fragilis Bf-1 chromosomal DNA was isolated from a gene bank (Southern et al., 1986) by screening on Luria agar, containing ampicillin and MTC (range 0.6–1 μg mL−1). This plasmid, or control plasmid pMT104, was transformed into the relevant E. coli DNA repair mutant strains and analysed for increased resistance to DNA-damaging agents.

Plasmid constructions

Localization of the functional region of pAN2 was determined by subcloning and deletion analysis. Deletion plasmids pANBB, pANC and pANH (Fig. 1) were constructed by subcloning the 2.5-kb HindIII–BglII fragment, the 1.4-kb HindIII–PvuII fragment and the 1.9-kb HindIII–BamHI fragment from pAN2, respectively, into pEcoR251. Plasmids pRecQ and pE2Reg were constructed by cloning the amplified 2300 and 900-bp fragments from pAN2 into pEcoR251, using primer pairs 1 and 2, respectively (Table 2).

image

Figure 1.  Genomic organization of pAN2 and deletion plasmids. MTC and metronidazole (MTZ) resistance phenotypes in both Escherichia coli AB1886 (uvrA) and AB1885 (uvrB) are indicated: +, increased resistance as compared with E. coli transformed with control plasmid pMT104; −, same level of sensitivity as with pMT104.

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Table 2.   Oligonucleotide primers
PairNameSequence (5′–3′)PurposeOrigin
 PERCY5GTC GTA CAA TTC ATC TGCPrimer extensionThis study
1Re1CCT TAG CTG AAT AGT CCGPCR of full length ORF1This study
Re2CCT ATT GCA ATT GGC AGC  
2Re3GGT TGT GGA AGA TCT CTT CCPCR of full length ORF2This study
Re4CAA GAT TAC GGT TGC AGC This study
2AI1ACA AAA CCC AGG AAG AAA CTC CTo obtain internal fragment from 638RBF3281This study
AI2GCA GCG TAA AAA GAC TGA CGG This study
3Re3As aboveTo confirm insertional mutation of 638RBF3281This study
M13RGTT TTC CCA GTC ACG AC Yanisch-Perron et al. (1985)
4Re4As aboveTo confirm insertional mutation of 638RBF3281This study
M13FCGC CAG GGT TTT CCC AGT CAC GAC Yanisch-Perron et al. (1985)

Metronidazole and MTC resistance assays in E. coli repair mutants

The agar dilution method was used for drug resistance assays. For aerobic growth, stationary-phase cultures of E. coli strains AB1157, AB1886, AB1885 and AB1884, containing pAN2 or pMT104, were diluted, and 103 cells were plated onto LB–ampicillin agar plates containing either MTC (range 0–2.4 μg mL−1) or metronidazole (range 0–1000 μg mL−1). For anaerobic growth, the same E. coli cultures were plated on prereduced yeast–tryptone (YT) agar, containing the antibiotics, and also supplemented with 0.5% glucose and 0.2% sodium nitrate. The minimum inhibitory concentration (MIC) was determined after a 24-h incubation under aerobic or anaerobic conditions.

DNA sequencing and computer analysis

Sequencing reactions were performed using the DYEnamic ET Dye terminator Cycle sequencing Kit for MegaBACE (Molecular Dynamics), based on dideoxynucleotide chain termination chemistry (Sanger et al., 1977). The nucleotide sequences were analysed using the megabace 500 sequence analyser v2.4 software and the software program dnaman, version 4.13 (Lynnon BioSoft). Sequence similarity searches were performed using the NCBI blast program (Altschul et al., 1997).

RNA extraction and analysis

Total RNA was extracted as described previously (Aiba et al., 1981). Primer extension analysis was performed to identify the transcriptional start of ORF2. RNA (70 μg) was annealed with Cy5-labelled primer PER (Table 1) at 42 °C overnight. The annealed mixture was subjected to primer extension with 20 U of AMV Reverse Transcriptase (Promega) at 42 °C for 2 h. The primer extension products were sequenced using the ALFexpress Automated DNA Sequencer (Pharmacia Biotech) and analysed together with the sequence products using the same primer PER. For transcriptional analysis of BF638R3281, total RNA was extracted from mid-logarithmic phase cultures of B. fragilis 638R exposed to a sublethal dose (1 μg mL−1) of metronidazole. Equal amounts of RNA (4 μL each, containing 10 μg of RNA per spot) were spotted onto nylon membranes and UV cross-linked (Hoefer UVC Cross-linker, Amersham Biosciences). The DNA probe used was an internal fragment of BF638R3281 obtained by PCR using primer pair 2 (Table 2) and digoxigenin-labelled using Digoxigenin (Roche Diagnostics). Hybridization was performed overnight at 50 °C using EasyHyb hybridization buffer and chemiluminescence detected using CSPD (Roche Diagnostics).

Construction and characterization of a B. fragilis BF638R3281 mutant

An internal fragment of BF638R3281 was amplified by PCR from B. fragilis 638R, using primer pair 2 (Table 2). The fragment was cloned into suicide vector pGERM (Cheng et al., 2000) to create plasmid pGAR, which was transformed into E. coli S17-1 and transferred via aerobic mating into B. fragilis 638R as described previously (Hooper et al., 1999). Putative mutants were selected anaerobically on BHIS agar containing gentamycin 200 μg mL−1) and erythromycin (10 μg mL−1). The mutation locus was confirmed by PCR using primer pairs 3 and 4 (Table 2) to obtain fragments of DNA flanking the plasmid insertion in BF638R3281. The identity of the PCR fragments obtained was verified by sequencing. The mutant was named B. fragilis 638R reg.

Analysis of growth of B. fragilis strains 638R and B. fragilis 638R reg mutant

The growth of the wild-type and mutant strains in BHIS broth was monitored. The OD600 nm of the cultures was measured every 2 h for a period of 24 h (Spectrawave S1000, Biochrom). Four independent experiments were performed for each strain.

Sensitivity assays with DNA-damaging agents

Metronidazole (5 μg mL−1) was added to mid-logarithmic cultures of the B. fragilis 638R and B. fragilis 638R reg mutant. Aliquots were removed from cultures at times from 0 to 40 min. Viable cell numbers were determined under anaerobic conditions by diluting aliquots in anaerobic water, plating on BHIS agar and enumeration of colonies after two days of growth at 37 °C. Survival after 40 min of exposure was expressed relative to survival at 0 min of exposure.

For MTC assays, overnight cultures were centrifuged, and cells were resuspended in prereduced Ringers solution to obtain an OD600 nm of 0.8. MTC was added to a final concentration of 1 μg mL−1. Aliquots were removed from cultures at times from 0 to 30 min. Dilutions of aliquots were made and plated on BHIS agar to obtain viable cell numbers. For both damaging agents, three independent experiments were performed and the mean percentage survival was calculated (SE in parentheses).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Identification of a gene involved in metronidazole and MTC resistance

A B. fragilis Bf-1 gene library was screened for genes conferring increased metronidazole and MTC resistance in E. coli uvr repair mutants. Escherichia coli AB1886 uvrA transformants with increased MTC resistance relative to the parent strain were isolated on plates containing 0.6 μg mL−1 MTC. These were all found to harbour the same plasmid, carrying a 4.5-kb DNA fragment from the B. fragilis chromosome. The recombinant plasmid was designated pAN2 (Fig. 1).

The presence of pAN2 also increased the survival of the E. coli uvrA mutant following metronidazole treatment. Similarly, the uvrB mutant containing pAN2 survived better than the control after metronidazole and MTC exposure. pAN2 did not affect the E. coli uvrC mutant survival following either treatment (Table 3). The improved survival conferred by pAN2 on E. coli uvrA and uvrB mutants after metronidazole exposure was also present under anaerobic conditions. The fact that the increased survival of metronidazole-treated cells was seen under both aerobic and anaerobic conditions suggested that the cloned gene product might be linked to cell survival following DNA damage caused by the activated metronidazole nitro radicals formed under anaerobic conditions, as well as the reactive oxygen species (ROS) formed during oxidation of metronidazole in the presence of O2 (Edwards, 1977). This might also indicate that the B. fragilis ORF2 is involved in a general stress tolerance mechanism in the heterologous E. coli.

Table 3.   Susceptibility of Escherichia coli strains, transformed with pAN2, to metronidazole and MTC
E. coli strainRelevant repair characteristicsMitomycin C MIC* (μg mL−1)Metronidazole MIC* (μg mL−1)
O2AnO2
  • A representative set of data is shown from 10 experiments.

  • *

    The MIC was determined on LB agar plates under aerobic conditions.

  • The MICs were determined under aerobic conditions (O2) or anaerobic conditions in prereduced media (AnO2).

  • ND, not determined.

AB1886 (pAN2)uvrA0.740050
AB1886 (pMT104)uvrA0.530035
AB1885 (pAN2)uvrB1.1950250
AB1885 (pMT104)uvrB0.7600220
AB1884 (pAN2)uvrC0.7300ND
AB1884 (pMT104)uvrC0.7300ND

Deletion analysis of the 4.5-kb insert showed that only the plasmids pANBB and pE2Reg (Fig. 1) yielded increased resistance to the E. coli uvrA and uvrB mutants. This suggested that the gene involved in the resistance phenotype was found within the common region on these fragments. The DNA insert in plasmid pAN2 that conferred resistance to MTC and metronidazole was fully sequenced in both directions. This region contained an incomplete ORF1, truncated at the C-terminus, and two complete ORFs (ORF2 and ORF3) (Fig. 1). Disruption of ORF2 by deletion analysis caused loss of activity, suggesting the involvement of the ORF2 gene product in resistance. This was confirmed by the fact that the PCR-cloned full-length ORF2 alone caused the improved survival phenotype in E. coli uvrA and uvrB mutants (data not shown). This gene was designated reg.

Sequence analysis and primer extension of reg

The transcription start point of reg was determined using primer extension analysis. A single primer extension product identified the transcriptional start site to be an adenine 59 bp upstream of the putative ATG start codon of reg (Fig. 2). The promoter region revealed the presence of sequences similar to the B. fragilis promoter consensus sequences (Bayley et al., 2000) −7 (TGAAATTTG) and −33 (GTTG). Three sets of inverted repeats were identified in the promoter region. One set was found upstream of the −33 promoter sequence, while the others were located near the −33 and −7 promoter regions, which may involve a regulatory role. The reg gene encoded a putative protein of 154 amino acids with a deduced molecular weight of 18.1 kDa and sequence similarity at the amino acid level to members of the AraC/XylS family of transcriptional regulators. AraC/XylS regulators typically contain a highly conserved region of 100 amino acid residues that constitutes the DNA-binding domain (Gallegos et al., 1997; Rhee et al., 1998), and the B. fragilis Reg contained most of the amino acids of this consensus sequence. The homologues of reg in other B. fragilis strains are BF3248 in B. fragilis NCTC 9343 (Accession number NC_003228) and BF638R3281 in B. fragilis 638R (http://www.sanger.ac.uk), and all three genes are identical in nucleotide sequence and gene arrangement on the chromosome.

image

Figure 2.  Identification of the Bacteroides fragilis reg transcriptional start site by primer extension analysis. Nucleotide sequence of the putative promoter region of reg: the ATG start codon (box) is at position 181, the transcription start site is at (+1) and the putative −7 and −33 motifs are in bold and underlined. The three sets of inverted repeats are shown by matching arrows. The reverse primer used is shown in bold italics.

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The identification of a regulator from this family with potential involvement in DNA repair is significant because a number of these transcriptional regulators have been involved in bacterial virulence (Frota et al., 2004), multidrug resistance systems and in response to alkylating agents (Tanaka et al., 1997). The best-characterized members of this family are the E. coli MarA and Rob proteins, both of which have been implicated in multidrug resistance (Gallegos et al., 1997; Kwon et al., 2000). The DNA-binding regulatory domain of these two proteins contains seven α-helices that have been shown to form two helix–turn–helix (HTH) motifs involved in binding to adjacent grooves of the DNA (Bustos & Schleif, 1993). The deduced amino acid sequence of the B. fragilis Reg putative protein similarly contained eight α-helices: one outside the conserved domain and seven within the DNA-binding domain, with two putative HTH motifs.

Mutation of the BF3248 homologue in B. fragilis 638R

In order to investigate the involvement of the putative regulator in cell survival following DNA damage in B. fragilis, a mutation in BF638R3281 (the BF3248 homologue in strain 638R) was achieved by gene-specific insertional inactivation. The B. fragilis Bf-1 strain could not be used for this purpose because it cannot be genetically manipulated. The site of insertion was confirmed by PCR analysis of the insertion junction using M13 primers (annealing to the inserted plasmid) in combination with B. fragilis-specific primers flanking the mutated gene. The PCR products obtained were sequenced, and this confirmed the insertion of plasmid pGAR into the target gene. Sequence data also revealed that the internal gene fragment, cloned into the suicide vector to facilitate homologous recombination, was now duplicated and flanked the inserted plasmid. The mutant was named B. fragilis 638R reg.

Growth analysis

The growth of B. fragilis 638R and B. fragilis 638R reg in BHIS broth (Fig. 3) showed that the mutant grew more slowly than the parent strain, as measured by OD600 nm. This was most noticeable at the later growth stages, and after 24 h the parent and mutant showed OD600 nm values of 1.048 (±0.031) and 0.863 (±0.014), respectively. This suggested that the Reg protein is required throughout growth, but might become more important as the age of the culture increases. If Reg is a global regulator, a number of essential cell functions could be affected by its absence. However, it is interesting to note that a slow growth phenotype has been described in certain clinically isolated metronidazole-resistant B. fragilis isolates (Gal & Brazier, 2004). It was, therefore, important to determine whether the reg gene was itself regulated at the transcription level by exposure to sublethal concentrations of metronidazole, and whether the B. fragilis 638R reg mutant was more or less sensitive to metronidazole that the parent strain.

image

Figure 3.  Analysis of growth of Bacteroides fragilis 638R and B. fragilis 638R reg. The growth of the wild-type B. fragilis 638R and B. fragilis 638R reg mutant strains in BHIS broth was monitored using the OD600 nm of the cultures. Four independent experiments were performed for each strain. ▪, Bacteroides fragilis 638R; □, B. fragilis 638R reg.

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Regulation of reg transcription by exposure to metronidazole

Transcription of the reg gene in B. fragilis 638R was analysed during normal growth and after exposure to a sublethal dose of metronidazole (1 μg mL−1). There was no change in the level of transcription of this gene upon exposure to metronidazole relative to the untreated control (data not shown). This indicated that the putative regulator was expressed constitutively under both these conditions.

Sensitivity to metronidazole and MTC

The B. fragilis 638R reg mutant was more sensitive than the parent strain to both metronidazole and MTC, suggesting that the gene product is involved in protecting the cells against these DNA-damaging agents. This confirmed the results obtained when the cloned gene was introduced into the E. coli repair mutants. After 40 min of exposure of mid logarithmic phase cultures to metronidazole, 91.38 (±18.5)% of B. fragilis 638R parent cells remained viable, compared with only 8.5 (±4.2)% of the reg mutant strain. A similar pattern was observed on exposure to MTC, where only 0.009 (±0.063)% of the mutant cells survived exposure to MTC, compared with 7.62 (±2.75)% of the B. fragilis 638R parent strain. These results suggest that reg may perhaps be involved in modulating cell responses that repair single-stranded breaks (caused by metronidazole) and cross-linked DNA (caused by MTC).

DNA single- and double-strand breaks are repaired in Gram-negative bacteria via the RecFOR and RecBCD pathways, respectively, through the helicase and exonuclease functions of the proteins involved. Mutations in these pathways cause increased sensitivity of E. coli to DNA-damaging agents such as MTC (Keller et al., 2001). Sensitivity to MTC in Gram-negative bacteria is caused by mutations in recA, defective nucleotide excision repair, as well as defective recBCD and recFOR repair pathways (Keller et al., 2001; Zuniga-Castillo et al., 2004). No information has been published to date regarding the sensitivity of B. fragilis recBCD mutants or recFOR mutants to metronidazole. This report, therefore, describes a novel role for a putative AraC/XylS transcriptional regulator with regard to the damage induced by the DNA-damaging agents, metronidazole and MTC. Further studies will elucidate which genes are regulated by the Reg protein, and whether they are damage specific or linked to general cell stress responses. The work will provide valuable information concerning the ways in which B. fragilis manages the DNA damage caused by metronidazole and MTC exposure.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

This study was supported by a Wellcome Trust Sanger Grant (070375/Z/03/Z). The authors thank Prof. A.A. Salyers and N.B. Shoemaker (Urbana, IL) for providing the pGERM plasmid strain. A.C. also acknowledges financial support from the University of Cape Town.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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