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Keywords:

  • Bacteroides fragilis;
  • RecQ;
  • metronidazole

Abstract

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

The inactivation of Bacteroides fragilis genes encoding putative RecQ helicases recQ1, recQ2 and recQ3 (ORFs BF638R_3282, BF638R_3781, BF638R_3932) was used to determine whether these proteins are involved in cell survival following metronidazole exposure. The effects of the mutations on growth, cellular morphology and DNA integrity were also evaluated. Mutations in the RecQ DNA helicases caused increased sensitivity to metronidazole, with recQ1, recQ2 and recQ3 mutants being 1.32-fold, 41.88-fold and 23.18-fold more sensitive than the wild type, respectively. There was no difference in cell growth between the recQ1 and recQ3 mutants and the wild type. However, the recQ2 mutant exhibited reduced cell growth, aberrant cell division and increased pleiomorphism, with an increase in filamentous forms and chains of cells being observed using light, fluorescence and electron microscopy. There was no spontaneous accumulation of DNA single- or double-strand breaks in the recQ mutants, as compared with the wild type, during normal cell growth in the absence of metronidazole. Bacteroides fragilis RecQ DNA helicases, therefore, enhance cell survival following metronidazole damage. The abnormal cellular phenotype and growth characteristics of recQ2 mutant cells suggest that this gene, or the downstream gene of the operon in which it occurs, may be involved in cell division.


Introduction

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

Members of the Bacteroides group, particularly Bacteroides fragilis, form the majority of bacterial isolates from anaerobic extraintestinal infections at a number of body sites, and metronidazole is used extensively to treat these. Metronidazole is activated intracellularly under anaerobic growth conditions, with the formation of toxic nitro radicals that damage DNA (Citron et al., 2007). Although Sigeti et al. (1983) originally reported no chromosomal fragmentation in certain B. fragilis strains in response to metronidazole treatment, it is now generally accepted that both single- and double-strand breaks are generated (Dachs et al., 1995) as well as DNA transversions (GC to CG) (Trinh & Reysset, 1998). Metronidazole, therefore, causes DNA fragmentation and enhances the risk of bacterial genetic mutations. Studies in Escherichia coli with impaired DNA repair systems found increased sensitivity to metronidazole (Jackson et al., 1984), suggesting that DNA repair mechanisms are important for the repair of metronidazole-induced DNA damage.

The DNA repair response of B. fragilis to metronidazole exposure is not well characterized, although putative repair pathways for double-strand break repair and single-strand break repair are evident in the genome annotation. These include a gene coding for recombinase A (recA). Mutation of recA rendered B. fragilis more sensitive to metronidazole, UV light and hydrogen peroxide (Steffens et al., 2010). The annotated genome also revealed the presence of 24 putative DNA helicases, three of which encode putative RecQ orthologues (Cerdeño-Tárraga et al., 2005). RecQ helicases unwind DNA with 5′–3′ polarity during recombinational repair. They are ATP dependent and are found in most prokaryotic and eukaryotic organisms. All RecQ helicases are defined by the presence of three conserved domains namely the helicase domain, the helicase superfamily C-terminal domain (RecQ-ct) and one or more and RNase D C-terminal helicase (HRDC) domains (Bennett & Keck, 2004). Mutation of the recQ gene in E. coli, as well as higher organisms, causes an increase in genetic instability as characterized by increased illegitimate recombination (Hanada et al., 1997; Bachrati & Hickson, 2003). Mutation of the HRDC domain of RecQ in Neisseria gonorrhoeae caused increased sensitivity to hydrogen peroxide (Stohl & Seifert, 2006).

Some eukaryotes encode several RecQ helicases, whereas the prokaryotic bacteria studied thus far have only a single orthologue (Bennett & Keck, 2004; Hartung & Puchta, 2006). The presence of three putative RecQ homologues in the B. fragilis genome is, therefore, novel and of interest. This study aims to investigate the involvement of RecQ proteins in the survival of B. fragilis in response to metronidazole-induced DNA damage, as well as to assess whether there are changes in cellular morphology or DNA integrity in B. fragilis RecQ mutants in the absence of an exogenously added metronidazole.

Materials and methods

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

Bacterial strains, plasmids and growth conditions

The bacterial strains and plasmids used in this study are shown in Table 1. Bacteroides fragilis 638R and derived strains were cultured in a supplemented brain–heart infusion broth (BHISB) or agar (BHISA) under anaerobic conditions (Casanueva et al., 2008). Escherichia coli strains were cultured aerobically on Luria agar or in Luria broth (Sambrook et al., 1989). Media were supplemented with antibiotics where necessary: gentamicin (200 μg mL−1), erythromycin (10 μg mL−1) and ampicillin (100 μg mL−1).

Table 1.   Bacterial strains and plasmids used in this study
 GenotypeReferences
  1. Gent, gentamicin; Amp, ampicillin, Ery, erythromycin; Rif, rifampicin; R, resistant.

Bacterium
 B. fragilis 638RGentR, RifRPrivitera et al. (1979)
 B. fragilis RecQ1B. fragilis 638R derivative, with BF638R_3282 mutationThis study
 B. fragilis RecQ2B. fragilis 638R derivative, with BF638R_3781 mutationThis study
 B. fragilis RecQ3B. fragilis 638R derivative, with BF638R_3933 mutationThis study
 E. coli S17-1recA Strr Tp, RP4 integrated into genomeSimon et al. (1983)
 E. coli JM109endA1, recA1, gyrA96, thi, hsdR17 (rk, mk+), relA1, supE44, Δ(lac-proAB), [F´traD36, proAB, laqIqZΔM15]Yanisch-Perron et al. (1985)
Plasmid
 pGERMSuicide vector, AmpR, EryRSalyers et al. (2000)
 pGQ1pGERM containing the internal fragment BF638R_3282This study
 pGQ2pGERM containing the internal fragment of BF638R_3781This study
 pGQ3pGERM containing the internal fragment of BF638R_3933This study

Bioinformatic analysis

RecQ sequences from the B. fragilis strains 638R (GenBank accession number CBW23724.1) and NCTC 9343 (GenBank accession number NC_003228) were used to search for the presence of putative recQ homologues in the genomes of other members of the Bacteroides group (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). Sequences were analysed using blast (Altschul et al., 1997), clustalx (Thompson et al., 1997) and mega version 4 (Tamura et al., 2007). RNA was explored for secondary structure using mfold (Zuker, 2003), while the presence of potential known riboswitches was investigated using RibEX (Abreu-Goodger & Merino, 2005) and RFAM (http://www.sanger.ac.uk/Software/Rfam/search.shtml).

DNA and RNA extraction and reverse transcriptase (RT)-PCR

Extraction of genomic DNA was performed as described by Casanueva et al. (2008). RNA extraction and RT-PCR analysis of the recJ, recQ and tpr transcripts were performed as described by Patel et al. (2008) using the primer sets indicated (Supporting Information, Table S1).

Construction of recQ mutants

Primers specific for the B. fragilis ORFs BF638R_3282, BF638R_3781 and BF638R_3932 were used to PCR amplify internal fragments of the recQ genes Q1, Q2 and Q3, respectively (Table S1). The PCR products were blunt-end ligated into the SmaI site of pGERM (Table 1) and the DNA transformed into E. coli JM109 (Salyers et al., 2000). Recombinant plasmids pGQ1, pGQ2 and pGQ3 (Table 1) were sequenced to verify the identity of all inserts. Plasmids were transformed into E. coli S17-1, before mating with B. fragilis 638R (Hooper et al., 1999). Bacteroides fragilis transconjugants were selected on BHISA containing gentamicin (200 μg mL−1) and erythromycin (10 μg mL−1). Interruptions of the target genes were confirmed by PCR using primers external to each gene (Table S1) in combination with M13 primers that recognize the pGERM vector (Casanueva et al., 2008), followed by nucleotide sequencing of PCR products.

Growth curves

Bacteroides fragilis strains 638R and recQ mutant strains RecQ1, RecQ2 and RecQ3 were grown for 16 h in 10 mL BHISB. The cultures were subinoculated into fresh BHISB at a starting OD600 nm of 0.1 and incubated anaerobically. Growth was measured as the increase in OD600 nm over an 8-h period using a Beckman DU530 spectrophotometer. Three independent experiments were performed for each strain.

Metronidazole sensitivity

Metronidazole (final concentration 6 μg mL−1) was added to mid-log-phase BHISB cultures (OD600 nm 0.4–0.5) of B. fragilis 638R and recQ mutants RecQ1, RecQ2 and RecQ3. Aliquots were removed from the cultures at 0, 30 and 60 min after the addition of metronidazole, dilutions were made and the cells were plated on BHISA. Colonies were counted after 2 days of anaerobic growth at 37 °C. The mean cell survival was calculated from three independent experiments. Sensitivity to metronidazole was also evaluated using E-test strips on BHISA according to the manufacturer's instructions (AB Biodisk, Solna, Sweden).

Visualization of the cell phenotypes and DNA strand breaks in the recQ mutants in the absence of metronidazole

Bacteroides fragilis 638R and recQ mutant cells were grown on BHISA before colonies were scraped off and resuspended in phosphate-buffered saline (PBS). Aliquots of these suspensions were fixed in glutaraldehyde (2% v/v in PBS) and prepared for transmission electron microscopy (TEM) (Simpson et al., 2006). Ultrathin sections were viewed using a JEOL 1200EX II TEM. Further cell samples were either Gram stained or the DNA and membranes were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg mL−1) and FM4-64 (1 mM), respectively. Fluorescence microscopy was performed at × 1000 magnification using a Zeiss Axiovert 200 microscope and photographed using a Zeiss Axiocam. Pictures were analysed using axiovision 4.6. To visualize DNA strand breaks, genomic DNA was extracted and the presence of double- and single-strand breaks was analysed by neutral and alkaline agarose gel electrophoresis, respectively (Abratt et al., 1990; Dachs et al., 1995).

Results and discussion

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

Bioinformatic analysis

Analysis of the B. fragilis 638R genome sequence revealed the presence of three putative RecQ genes, identified by ORF numbers BF638R_3282 (Q1), BF638R_3781 (Q2) and BF638R_3932 (Q3). These ORFs encoded deduced proteins of 607 amino acids (aa), 634 aa and 726 aa in length, respectively. These B. fragilis 638R loci corresponded to BF3249, BF3706 and BF3892, respectively, from strain NCTC 9343. Q1 was most similar to E. coli RecQ (43.7% aa identity), while Q2 and Q3 showed 39.6% and 38.9% aa identity to it, respectively.

The presence of multiple RecQ homologues in prokaryotes had not been described before this study on B. fragilis, although it is well documented in higher eukaryotes. For example, the human genome encodes five RecQ proteins (BLM, WRN, RecQL1, RecQL4, RecQL5), while Arabidopsis thaliana encodes seven RecQ homologues (Hartung & Puchta, 2006). Our analysis revealed that the annotated genomes from 14 members of the genus Bacteroides encoded multiple putative RecQ homologues (Fig. 1a; Table S2). The significance of multiple RecQ homologues in Bacteroides is unclear. All the B. fragilis 638R RecQ homologues contained two of the signature amino acid domains representative of all known RecQ helicases, namely a helicase domain (with the essential DEXX motif [X representing alanine (A), histidine (H), serine (S) or aspartate (D) residues]) as well as a helicase C-terminal domain (Fig. 1b). The helicase domain from all three homologues further contained the conserved regions 0 to VI (Bennett & Keck, 2004). The BF638R_3932 (Q3) homologue contained an incomplete HRDC domain, whereas the RecQ coded by BF638R_3781 (Q2) was unusual as it completely lacked an HRDC domain. The effect of the absence of this domain is not known, but mutation of any of the three HRDC domains of the RecQ from Deinococcus radiodurans affected substrate binding and specificity (Killoran & Keck, 2008), the ATPase activity of the protein and the ability to resist DNA damage (Hua et al., 2008).

image

Figure 1.  Analysis of RecQ homologues from Bacteroides fragilis 638R. (a) Phylogenetic relationship of RecQ helicases from the Bacteroides group. B.ste, B. stercoris ATCC 43183; B.egg, B. eggerthii DSM 20697; B.uni, B. uniformis ATCC8492; B.cel, B. cellulosilyticus DSM 14838; B.int, B. intestinalis DSM 17393; B.theta, B. thetaiotaomicron VPI-5482; B.fin, B. finegoldii DSM 17565; B.cacc, B. caccae ATCC 43185; B.ova, B. ovatus ATCC 8483; B.dor, B. dorei DSM 17855; B.ple, B. plebeius DSM 17135; B.cop, B. coprocola DSM 17136; B.cop1, B. coprophilus DSM 18228B. (b) Structure of B. fragilis 638R RecQ proteins: helicase domain, black rectangle; RecQ-Ct, oval; HRDC domain, rounded rectangle; incomplete HRDC domain, rectangle with dented sides. Q1, BF638R_3282; Q2, BF638R_3781; Q3, BF638R_3932. The arrows indicate where the protein sequence was interrupted in the amino acid (AA) sequence as a result of the insertional inactivation.

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Bioinformatic analysis of the gene context suggested that the BF638R_3781 (Q2) gene was part of an operon with the upstream BF638R_3780 gene (encoding a putative RecJ exonuclease) and the downstream BF638R_3782 [encoding a hypothetical protein containing three tetratricopeptide repeat regions (TPR)]. The putative recJ and recQ2 genes overlapped, with the last four nucleotides of the first gene, recJ, constituting the first four bases of recQ2, and 67 bp separated recQ2 from BF638R_3782 (Fig. 2b). Further bioinformatic analysis assigned a GTG as the putative start codon for the recQ2 gene. The gene arrangement was confirmed by RT-PCR with ORFs BF638R_3780, BF638R_3781 and BF638R_3782 being cotranscribed (Fig. 2a). Amplification of the intergenic regions yielded less PCR product than from the coding regions (Fig. 2), and this could be due to inhibition of the RT-PCR reaction due to the presence of an mRNA secondary structure as analysed using the mfold software. The proximity of the genes might be important, as it is known that RecQ and RecJ collaborate in the E. coli RecFOR pathway, assisting with the repair of stalled replication forks (Courcelle & Hanawalt, 1999). The third gene of the operon, BF638R_3782, encodes a hypothetical protein containing three TPR. TPR proteins are found in prokaryotes and eukaryotes, and function as effectors of protein–protein interactions. A typical TPR motif consists of a degenerate set of approximately 34 aa containing the core sequence -W-LG-Y-A-F-A-P- within the motif (Das et al., 1998; Blatch & Lässle, 1999). These proteins play a role in cell division (Sikorski et al., 1993; Das et al., 1998; Mesak et al., 2004). Human TPR proteins interact with recombination repair proteins such as the tumour suppressor protein BRCA2, an important protein involved in the repair of double-strand breaks (Wilson et al., 2010). Bacterial TPR proteins are involved in pilin formation (Rodriguez-Soto & Kaiser, 1997; Kim et al., 2006), fruiting body and spore development (Nariya & Inouye, 2005), photosystems I complex formation (Wilde et al., 2001) and the delivery of proteases into hosts (Sun et al., 2008). The role of the BF638R_3782 putative TPR protein in B. fragilis is not yet known.

image

Figure 2.  Genomic context of Bacteroides fragilis BF638R_3780, BF638R_3781 and BF638R_3782. (a) Transcriptional analysis of the recJ-recQ2-tpr operon. Lane 1, 100 bp O'gene ruler; lanes 2–6, cDNA used as a template for PCR; lane 7, RNA used as a template for PCR (to check for DNA contamination); lanes 8–12, genomic DNA used as a PCR template; lane 2, recJ; lane 3, fragment spanning recJ-recQ2; lane 4, recQ; lane 5, fragment spanning recQ and tpr; lane 6, tpr; lane 8, recJ; lane 9, fragment spanning recJ-recQ2; lane 10, recQ2; lane 11, fragment spanning recQ2 and tpr; lane 12, tpr reverse transcription was performed using 1.5 μg of RNA. Equal amounts of the cDNA (2 μL) were used for the PCR reactions. Each well contained 10 μL of the PCR reaction, except lanes 3, 5, where 20 μL was loaded. (b) Genomic context of recJ-recQ-tpr genes: the black box indicates the region that overlaps in recJ and recQ: (1) the bases indicated in capital letters (in the text box below) represent the four base pairs that overlap in recJ and recQ; (2) the TGA stop codon of recJ is in underlined; (3) the GTG start codon of recQ is in italics. Primer pair RTJF and RTJR was used to amplify the overlapping region of recJ and recQ. Primer pair RTqF and RTtprR was used to amplify the intergenic region of recQ and tpr.

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Analysis of the mRNA for known riboswitch elements, using RibEX (Abreu-Goodger & Merino, 2005) and RFAM (http://www.sanger.ac.uk/Software/Rfam/search.shtml), yielded no positive result and further studies are necessary to determine whether or not there may be a riboswitch mechanism in this operon. Analysis of the genomic contexts of BF638R_3282 (Q1) and BF638R_3932 (Q3) genes showed that they were transcribed independently and possessed a B. fragilis promoter-like sequence (Bayley et al., 2000).

Confirmation of mutations

Mutations were generated in the helicase domain of each of the three recQ genes (Fig. 1b). Because of the highly conserved nature of the nucleotide sequence of the helicase domain, specific primers were designed to unique regions of the helicase domains of the three genes to ensure amplification of the correct gene target. This strategy resulted in the interruption of the helicase domain as well as its separation from the RecQ C-terminal and HRDC domains. Mutations were confirmed by PCR and sequencing of the products generated by the mutants (Fig. S1).

Assessment of growth and cell morphology of mutants

Growth comparison of B. fragilis 638R wild type and the three mutants showed that mutant RecQ2 exhibited reduced growth after 8 h (OD600 nm=0.5) as compared with the other strains (OD600 nm=0.8). Gram staining (Fig. 3a) and TEM of ultrathin sections (Fig. 3b) revealed that strain RecQ2 was considerably more pleiomorphic than the wild type, displaying extensive elongation (10–20 μm) (Fig. 3b, iv) as compared with the wild type (1–5 μm) (Fig. 3b, i). Chains of short cells were also seen in RecQ2 (Fig. 3b, v), suggesting that the cells did not separate to completion possibly due to a defect in cell division. It is well known that wild-type B. fragilis is intrinsically pleiomorphic and that elongated cells or filaments of attached cells are occasionally seen even in wild-type cultures (Jousimies-Somer et al., 2002). The genetic and biological reasons for this are not known. Cells with mutated recQ2 show an increase in the frequency of this phenomenon and point to an involvement of this RecQ protein in the cell-division process. The phenomenon of elongation, abnormal growth and defective septa formation was reported previously in B. fragilis cells grown in the presence of low doses of clindamycin and cephalosporins (Fang et al., 2002; Silvestro et al., 2006). It is important to note here that the interruption of recQ2 could affect the transcription of tpr, the third gene in the recJ-recQ2-tpr operon, and hence influence the phenotype.

image

Figure 3.  Cell morphology visualized by microscopy. (a) Light micrograph of Gram-stained cells viewed at × 1000 magnification; (i) 638R; (ii) mutant RecQ1; (iii) mutant RecQ3; (iv) mutant RecQ2. (b) Transmission electron micrograph (viewed at × 10 000 magnification): (i) 638R; (ii) mutant RecQ1; (iii) mutant RecQ3; (iv) and (v) mutant RecQ2.

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Cells were stained with DAPI to investigate whether the double-stranded integrity of the genetic material in the RecQ2 mutant was affected, and the cell membranes were further stained with FM4-64 to visualize the individual cell boundaries. Fluorescence microscopy of the stained cells confirmed the Gram stain and TEM results. The cells of the wild type, mutant RecQ1 and mutant RecQ3 had a similar appearance (short individual rods with a compact chromosome), whereas the filaments of mutant RecQ2 consisted of chains of long and short cells that failed to separate into single cells (Fig. S2). All strains showed equivalent fluorescence intensity of the DAPI stain, indicating equivalent amounts of double-stranded DNA. DNA from the strains was further analysed by standard and alkaline gel electrophoresis to detect the presence of single- and double-strand breaks (respectively), but no difference could be observed between the mutants and the wild-type strains (Fig. S3). It may, therefore, be concluded that, in the absence of exogenous DNA damage, the lack of any of the individual RecQ homologues in B. fragilis does not significantly increase the presence of DNA strand breaks. This is in contrast to what was observed in a B. fragilis recA mutant, where the absence of the RecA protein led to an increase in the presence of single- and double-strand breaks in DNA (Steffens et al., 2010).

Response of RecQ mutants to lethal doses of metronidazole

The recQ mutant strains showed varying levels of increased sensitivity to metronidazole (Table 2). At 60 min, the wild type survived 1.32-fold, 41.88-fold and 23.18-fold better than mutants RecQ1, RecQ2 and RecQ3, respectively. These results confirmed that these proteins are needed for cell survival following metronidazole damage in B. fragilis, although their exact roles have not yet been elucidated. The extreme sensitivity of strain RecQ2 to metronidazole highlights the fact that the absence of this particular homologue (and/or the downstream Tpr protein) causes significant stress in the bacterium, as evidenced by elongated cells and defective growth. The E-test results confirmed that the mutants were more sensitive to metronidazole, with B. fragilis minimum inhibitory concentration values of 0.25 μg mL−1 for strain 638R, compared with 0.125 μg mL−1 for strains RecQ1 and RecQ3, and <0.016 μg mL−1 for RecQ2. This suggests that a RecA-positive background supports metronidazole damage repair in the absence of RecQ1 and RecQ3, but is insufficient in the absence of RecQ2 and possibly its downstream gene product.

Table 2.   Survival of Bacteroides fragilis after exposure to metronidazole
B. fragilis strain% of cells survivingFold survival (of the wild type/mutant)
  1. Exponential-phase cells (approximately 108 cells) were exposed to 5 μg mL−1 metronidazole. % Survival=(100 × [CFU mL−1 at tx minutes after treatment/CFU mL−1 at t0]). Fold survival: % of 638R cells surviving divided by % mutant cells surviving. Three independent experiments were performed for each strain. Values in brackets represent the SE of the mean.

638R26.09 (18.30)1
RecQ319.70 (8.19)1.32
RecQ20.62 (0.51)41.88
RecQ11.13 (0.50)23.18

In this study, it has been shown that mutations in the RecQ helicases render B. fragilis more sensitive to metronidazole and that these proteins are, therefore, important for the cellular response to metronidazole-induced cell damage. The most sensitive mutant strain, RecQ2, exhibited severe growth defects, defective cell division and aberrant cell morphology, possibly due to polar effects on ORF638R_3782, which encodes a putative TPR protein and may be implicated in cell division. Further studies are needed to establish the precise function of each RecQ homologue in maintaining B. fragilis viability following metronidazole challenge.

Acknowledgements

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

This study was supported by grants from the Wellcome Trust (070375/Z/03/Z), the South African Medical Research council and a South Africa–Sweden Collaborative Research Grant (through the National Research Foundation). C.E.N. acknowledges a grant from the Swedish Research Council (348-2006-6862). We thank A.A. Salyers and N.B. Shoemaker (Urbana, IL) for providing the pLYL01 and pGERM plasmids, and acknowledge G. Blakely for useful discussions.

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

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

Fig. S1. Confirmation of insertional mutation of recQ genes.

Fig. S2. Visualization of Bacteroides fragilis cells using fluorescence microscopy.

Fig. S3. Visualization of DNA double- and single-strand breaks.

Table S1. Primers used in this study.

Table S2. RecQ homologues from the Bacteroides groupNB.

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