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Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630erm) and demonstration that the conjugative transposon Tn916E enters the genome of this strain at multiple sites

Division of Microbial Diseases, Eastman Dental Institute for Oral Health Care Sciences, University College London, 256 Gray's Inn Road, London WC1X 8LD, UK

Correspondence Peter Mullany pmullany{at}eastman.ucl.ac.uk

Received June 24, 2004
Accepted August 11, 2004

Erythromycin resistance in Clostridium difficile strain 630 is conferred by a genetic element termed Tn5398 which contains two erm(B) genes: erm1(B) and erm2(B). An erythromycin-sensitive derivative of strain 630 (designated 630erm) was generated by spontaneous mutation after continuous subculture for 30 days. This strain had lost the erm2(B) gene from within Tn5398 but retained erm1(B). However, the strain could revert to erythromycin resistance at a frequency of 2.79 x 10^–8, although it still contained the deletion of erm2(B). The availability of C. difficile 630erm allowed the behaviour of Tn916E to be investigated in this strain. This element entered the genome at multiple sites indicating that it could be useful as an insertional mutagen.

Abbreviation: CDAD, Clostridium difficile-associated disease.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is an important nosocomial enteric pathogen that causes a range of diseases from the life-threatening pseudomembranous colitis to mild antibiotic-associated diarrhoea. C. difficile accounts for up to 15 % of diarrhoeal disease associated with antibiotic treatment (Spencer, 1998). The main virulence factors in C. difficile-associated disease (CDAD) are thought to be two high-molecular-mass toxins, A and B (Poxton et al., 2001). Once released onto the gut mucosa, they act in concert to produce the characteristic pathology and symptoms of CDAD.

The pathogenesis of C. difficile infection is, however, poorly understood and improvement of the genetic tools of C. difficile pathogenic strains is a priority in C. difficile research, especially as the genome sequence of strain 630 has recently been completed at the Sanger Institute UK (http://www.sanger.ac.uk/Projects/C_difficile/). Towards this end we have investigated the behaviour of the tetracycline-resistance-encoding conjugative transposon Tn916 and its erythromycin-resistant derivative Tn916E (Rubens & Heggen, 1988; Mullany et al., 1991) and the endogenous tetracycline-resistance-encoding conjugative transposon Tn5397 in C. difficile (for a recent review on the properties of these elements see Mullany, 2002, and accompanying articles). All these elements enter the genome of C. difficile CD37 at highly preferred sites (Mullany et al., 1990; Mullany et al., 1991; Wang et al., 2000a). Tn5398 is a transferable genetic element (Mullany et al., 1995); however, it does not contain the genes required for self-conjugation, therefore it is likely to be a mobilizable element (Farrow et al., 2001). In C. difficile CD37, Tn5398 appears to enter the genome at a preferred site (Farrow et al., 2001). This site-specificity limits the use of these elements as insertional-mutagenesis tools in strain CD37, although Tn916 has been used for gene cloning in this host (Mullany et al., 1994; Roberts et al., 2003). In order to investigate the behaviour of these elements in the genome strain and to make this strain more generally amenable to genetic manipulation, the aim of the current work was to isolate spontaneous mutants sensitive to either erythromycin or tetracycline.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Strains, plasmids and culture conditions.

All the bacterial strains and plasmids used in this study are shown in Table 1. Escherichia coli TOP10 was used as the host for transformation. E. coli strains carrying plasmids were grown in Luria–Bertani (LB) medium at 37 °C. Bacillus subtilis was grown on or in brain heart infusion (BHI) agar or broth (Oxoid) in an aerobic environment. Transformation of B. subtilis was carried out according to Hardy (1985). C. difficile strains were grown in BHI agar or broth supplemented with 5 % defibrinated horse blood (E and O Laboratories) and incubated in an anaerobic atmosphere (80 % nitrogen, 10 % hydrogen and 10 % carbon dioxide).

Media were supplemented when required with antibiotics at the following concentrations: 100 µg ampicillin ml^–1, 40 µg chloramphenicol ml^–1, 50 µg kanamycin ml^–1, 10 µg thiamphenicol ml^–1 and 30 µg clindamycin ml^–1 (Sigma). Plasmid DNA was isolated from bacteria with Qiagen Mini Prep Kits. DNA fragments for subcloning were isolated from agarose gels with QIAEXII gel extraction kit (Qiagen).

Generation of strain 630 mutants.

A single colony of C. difficile 630 was picked from an agar plate containing tetracycline and erythromycin and incubated in 10 ml of BHI broth without antibiotic selection. The bacterial cultures were grown overnight anaerobically at 37 °C. Ten millilitres of BHI broth was inoculated with 100 µl of the overnight culture and incubated anaerobically at 37 °C. This step was repeated 30 times. After the thirtieth subculture the culture was diluted and plated on antibiotic-free BHI agar and incubated anaerobically at 37 °C. Single colonies were replica-plated on BHI agar containing either erythromycin or tetracycline.

Filter-mating.

Filter-mating experiments were performed as described previously (Roberts et al., 2001b) with the following modifications. Both B. subtilis donor and C. difficile recipient strains were grown on BHI agar plates for 18 h. The C. difficile cells were scraped off the plates and resuspended in 20 ml of BHI broth and B. subtilis were subcultured in 100 ml BHI broth. The cultures were grown at 37 °C until mid-exponential phase (optical density at 600 nm of 0.45). B. subtilis cells were centrifuged and washed twice with BHI broth. Cultures of donor and recipient were mixed and centrifuged in an anaerobic environment to form a cell pellet. The pellet was resuspended in 1 ml of BHI broth, and 100 µl was spread on nitrocellulose 0.45-mm-pore-size filters on BHI agar plates, which were incubated for 18 h at 37 °C in an anaerobic environment. The filters were removed from the agar plates and placed in 20 ml bottles and vigorously washed with 1 ml BHI broth. Aliquots (100 µl) were spread on BHI agar supplemented with the appropriate antibiotics and C. difficile-selective supplement (Oxoid) and incubated anaerobically for 48 h. Putative transconjugants were subcultured on fresh selective plates and incubated for a further 48 h.

Nucleic acid techniques.

Standard recombinant techniques used throughout this work are as described by Sambrook et al. (1989). Genomic DNA extractions for C. difficile and B. subtilis were performed using the Gram-positive and Yeast DNA Isolation kit (Gentra Systems, supplied by Flowgen). PCR was carried out using Taq DNA polymerase in the buffer supplied by the manufacturer. All primers used are shown in Table 2. The typical PCR program is as follows: 94 °C for 4 min followed by 25–30 cycles of 94 °C for 30 sec, 50–60 °C for 1 min 30 s and 72 °C for 1–3 min, followed by a final incubation at 72 °C for 10 min and a rapid thermal ramp and hold at 4 °C until analysis. PCR products were cloned into the pCRII-TOPO vector by using the TOPO TA cloning kit (Invitrogen). The DNA sequence of the PCR-amplified fragment was determined at this stage.

Southern blotting, hybridization and detection were carried out using the ECL kit (Amersham) according to the manufacturer's instructions. The probe containing a fragment of the xis and int genes from Tn916 in pAM120 was produced using the primers xis/int1 and xis/int2 as described previously (Roberts et al., 2001a).

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Isolation of antibiotic-sensitive mutants

After 30 days of continuous subculture 500 well-isolated colonies growing on antibiotic-free medium were replica-plated onto either tetracycline- or erythromycin-containing agar. Forty colonies were erythromycin sensitive (8 % of the population) and all of the 500 remained tetracycline resistant. No erythromycin-sensitive mutants were observed when cells were grown for 10–14 days.

Characterization of the erythromycin-sensitive mutants

Two erythromycin-sensitive mutants were selected for further study. As erythromycin resistance in C. difficile 630 is contained within a genetic element called Tn5398, a series of PCR amplifications with primers binding in this region were conducted on genomic DNA isolated from these strains to determine if the mutation was associated with a loss of all or part of Tn5398. Tn5398 contains two ermB genes separated by two orfs, ORF3 and ORF298 (Fig. 1a). The location of the primers used in these assays is shown in Fig. 1 and Table 2. The results demonstrated that there had been a deletion of the erm2(B) gene and some of the flanking regions. The two clones gave identical results; therefore, one was selected for further study and designated C. difficile 630erm.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Diagram showing the structure of Tn5398 before and after deletion of erm2(B). (a) Structure of Tn5398 as determined by Farrow et al. (2001). The directions of the putative ORFs are shown by arrowed boxes. Regions incorporating a direct repeat sequence, originally identified from C. perfringens (Berryman & Rood, 1995), are shown as black boxes. The binding sites and direction of priming of the oligonucleotides used in PCR analysis are shown by the arrows. The scale in kbp is shown. (b) Structure of Tn5398E. The direct repeats flanking the deletion point are underlined and in bold. The erythromycin-resistance genes are shaded in grey.

A PCR and sequencing strategy was used to define more precisely the deleted region in Tn5398. Amplification of genomic DNA from the wild-type strain 630 and strain 630erm was carried out using oligonucleotides at positions 2820 and 3800 and at positions 10050 and 11340 (Tables 2 and 3, Fig. 1a). The 630erm products all had a deletion of 2.4 kbp compared to the products obtained from strain 630.

View this table: [in this window] [in a new window]   Table 3. PCR analysis of the genetic organization of C. difficile 630 and the erythromycin-sensitive mutant C. difficile 630erm

A 3.8 kbp PCR product amplified from genomic DNA of strain 630erm using oligonucleotides 3800 and 10050 was cloned into the pCRII-TOPO vector and the DNA sequence was determined. The results revealed an internal deletion of 2.4 kbp, removing most of ORF298, and all of erm2(B) and ORF3 (Fig. 1b), but leaving the erm1(B) leader peptide and the erm1(B) gene intact. The left end of the deleted region was shown to be 32 bp upstream of the start codon of ORF298 and delineated by the sequence AAATCACAAGTGATT (with representing the start point of the deletion); the right end of the deletion was shown to be in the intergenic space between ORF3 and ORF13, 648 bp upstream of the ORF13 start codon and delineated by the sequence TGTGATTGTTGATGA (with representing the end point of the deletion) (Fig. 1b). The deletion point is flanked by directly repeated hexanucleotide sequences (underlined). We have previously found 6 bp direct repeats flanking putative deletions of part of the tetracycline-resistance transposon Tn5397 and the non-mobile element CWP459tet(M) from Clostridium perfringens (Roberts et al., 2001a); however, the significance of these repeats remains to be determined.

Interestingly the continued presence of erm1(B) does not confer erythromycin resistance upon C. difficile 630erm. This indicates that erm2(B) is solely responsible for erythromycin resistance. However, the putative promoter upstream of this gene is deleted in strain 630 (Farrow et al., 2000), therefore this gene must be expressed from another upstream promoter.

The erm1(B) gene from the 630erm strain was sequenced in its entirety and found to be identical to erm2(B). However, erythromycin-resistant revertants appeared at a frequency of 2.79x10^–8 revertants per 630erm c.f.u. Again the erm1(B) gene and leader region was sequenced in these strains and no difference could be seen between strain 630erm, the revertant or the wild-type 630. The revertants also still possessed the deletion. No explanation can be offered at this time for the reversion back to erythromycin resistance but it almost certainly involves erm1(B) as the revertants have the MLS (macrolide-lincosamide-streptogramin B resistance) phenotype and there are no other erm(B) homologues present in the genome sequence of the wild-type 630 (www.sanger.ac.uk).

This low level of reversion to erythromycin resistance should not compromise the use of erythromycin-resistance-encoding vectors in C. difficile 630erm as the level of reversion is at least an order of magnitude lower than the transfer frequencies of most vectors.

Behaviour of Tn916E in C. difficile 630erm

The availability of an erythromycin-sensitive derivative of C. difficile 630 enables the behaviour of Tn916E in this strain to be analysed. Ten independently derived transconjugants (all from different filters) from the filter-mating between B. subtilis BS59A (containing Tn916E; Table 1) and 630erm were analysed further. A Southern blot on HincII-digested genomic DNA was probed with a PCR product derived from the integrase gene as described previously (Roberts et al., 2001a). The results in Fig. 2 show that Tn916E inserted into the strain 630erm genome apparently at random, with no two transconjugants sharing a common insertion (a HincII digest will release a genome/transposon junction fragment so insertion at the same place should result in hybridizing bands of the same size). This is completely different from the behaviour in CD37 where Tn916E enters the CD37 genome at the same site in the majority of transconjugants (Mullany et al., 1991; Wang et al., 2000a). This allows the use of Tn916E as an insertional mutagen in the derivative of the genome strain, 630erm. However, care must be exercised as it can be seen from Fig. 2 that four out of ten (40 %) of the transconjugants contain more than one copy of Tn916E (Fig. 2).

View larger version (94K): [in this window] [in a new window]   Fig. 2. Southern blot showing the insertion of Tn916E in C. difficile 630erm. DNA present in lanes as follows: 1–10, independent isolates from different filter matings. DNA was digested with HincII and probed with a PCR-amplified fragment of Tn916 from the xis/int region of the transposon. M, 1 kbp ladder marker; WT, C. difficile 630erm.

Previous studies (Wang et al., 2000b) have shown that if Tn916E is introduced into C. difficile CD37 containing Tn5397 then a percentage of the cells would lose Tn5397, presumably by some trans-acting factors from Tn916E promoting the instability of Tn5397. To see if this was also the case in C. difficile 630erm, Tn916E was introduced into this strain by filter-mating from a B. subtilis donor strain containing Tn916E (strain BS59A).

Erythromycin-resistant strains of 630erm were obtained at a similar frequency reported previously for CD37 (3x10^–7 transconjugants per donor c.f.u.) (Wang et al., 2000b). None of the transconjugants were tetracycline sensitive when replica-plated on BHI agar containing tetracycline. Both Tn916E and Tn5397 were still present (conferring erythromycin and tetracycline -resistance, respectively) in all of the 300 colonies after serial subculture for 20 days on media containing no antibiotics. Therefore, unlike the case in CD37, introduction of Tn916E does not induce the loss of Tn5397. This again illustrates the point that conjugative transposons behave differently in different strains of C. difficile.

This study has resulted in a defined deletion mutant that has lost the erm2(B) gene and an area of flanking DNA including most of ORF298 and all of ORF3. It has also lost the phenotypic resistance to erythromycin and clindamycin. The fact that there is an intact (and identical) copy of erm1(B) still present suggests that this gene is usually silent. Furthermore this work extends our observations that Tn916, in some strains, can enter the C. difficile genome at multiple sites (Roberts et al., 2003). In fact only C. difficile CD37 has an obvious hot spot for the element (Wang et al., 2000a).

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This work was supported by the BBSRC (grant number 364/E13746). We thank Abraham Linc Sonenshein for strain CD196.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 

Mullany, P., Wilks, M. & Tabaqchali, S. (1991). Transfer of Tn916 and Tn916E into Clostridium difficile: demonstration of a hot-spot for these elements in the C.difficile genome. FEMS Microbiol Lett 79, 191–194.[CrossRef]

Rubens, C. E. & Heggen, L. M. (1988). Tn916E: a Tn916 transposon derivative expressing erythromycin resistance. Plasmid 20, 137–142.[CrossRef][Medline]