FEMS Microbiology Letters 229 (2003) 103^110

www.fems-microbiology.org

Gene transfer into Clostridium dicile CD630 and characterisation of its methylase genes
Michael Herbert a , Triona A. OKeee a , Des Purdy a , Michael Elmore a , Nigel P. Minton a;b;
b a Health Protection Agency, Porton Down, Salisbury, Wiltshire SP4 0JG, UK Institute of Infection, Immunity and Inammation, University of Nottingham, Floor C, West Block, Queens Medical Centre, Nottingham NG7 2UH, UK

Received 15 September 2003; received in revised form 8 October 2003 ; accepted 16 October 2003 First published online 7 November 2003

Abstract
Ignorance of pathogenesis in Clostridium difficile may be attributable to a lack of effective genetic tools. We have now shown that oriTbased shuttle vectors may be conjugated from Escherichia coli donors to the C. difficile strain CD630, at frequencies of around 1036 transconjugants per donor cell. Transfer is unaffected by either sequences present on the vector or its methylation status. Whilst the genome of this strain carries five methylase genes, there is no in silico or experimental evidence for cognate restriction enzymes. It would seem that the identified methylases do not participate in restriction-modification, and must, therefore, fulfil another role. A similar situation most likely applies to other clostridia. 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Gene transfer; Restriction; Methylase ; Conjugation; Clostridium dicile

1. Introduction The spore-forming anaerobe Clostridium dicile is a major cause of healthcare associated disease [1,2]. Despite its importance, the organism remains poorly characterised. This may be largely attributable to a lack of eective genetic tools and transfer systems. The development of gene transfer procedures in C. dicile has, however, proven problematic. Until recently, the only means of introducing DNA was based on the transfer of conjugative transposons, such as Tn916, from Bacillus subtilis donors using lter mating [3]. Transfer frequencies are, however, relatively low (1038 per donor) and the transposon has limited utility as it always inserts into a single site within the genome [4]. In recent years, attention has focussed on using oriT-mediated plasmid mobilisation from Escherichia coli donors. In the initial study, the vectors employed were replication minus and were deliberately designed to integrate into the genome of the non-toxinogenic strain CD37

by single crossover [5]. Subsequently, replication procient plasmids have also been transferred to this strain [6]. In the case of two toxinogenic strains of C. dicile, CD3 and CD6, successful high frequency transfer (1.0U1036 and 5.7U1035 transconjugants per donor) of oriT-based, replication procient vectors from E. coli was dependent on the circumvention of host restriction/modication (RM) systems [7]. Such measures were not necessary in the case of strain CD37 due to the absence of eective RM systems [7]. In the present study we have turned our attention to the toxinogenic C. dicile strain CD630. The genome sequence of this strain has recently been completed at the Sanger Institute UK (http:// www.sanger.ac.uk/Projects/C_dicile/). The availability of gene transfer systems for this particular strain is therefore a priority.

2. Materials and methods 2.1. Bacterial strains and plasmids

* Corresponding author. Tel. : +44 (115) 84 67458 ; Fax : +44 (115) 84 66296. E-mail address : nigel.minton@nottingham.ac.uk (N.P. Minton).

Plasmids and strains used in this study are summarised in Table 1.

0378-1097 / 03 / $22.00 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00795-X

104 Table 1 Bacterial strains and plasmids Organism/plasmid Strains C. dicile CD630 C. dicile CD6 (R8575) C. dicile CD3 (R7201) C. dicile CD37 C. dicile VPI 10463 E. coli Top10 E. coli GM2163

M. Herbert et al. / FEMS Microbiology Letters 229 (2003) 103^110

Feature genome strain PCR type 12 PCR type 2 PCR type 14

Source/reference P. Mullany, UCL J. Brazier, PHL, Cardi J. Brazier, PHL, Cardi P. Mullany, UCL J. Brazier, PHL, Cardi Invitrogen Novagen

E. coli HB101 Plasmids R702 pCR2.1-TOPO-KmR , ApR pJIR418-EmR , CmR pACYC177-ApR , KmR pACYC184-CmR , TetR pCR2.1:M.Cdi25-ApR pACYC177 :M.Cdi1226:M.Cdi25-ApR pACYC177 :M.Cdi25-ApR pMTL20 pMTL20:M.Cdi1226 pACYC184 : :Sau5-CmR pCD35EC-EmR , ApR , CmR pCD35ECoriT-EmR , ApR , CmR pMTL930-EmR pMTL9301-EmR pMTL93011-EmR , CmR pMTL93012-EmR , CmR pMTL93013-EmR , CmR

F3 mcrA v(mrr-hsdRMS-mcrBC) P80lacZvM15vlacX74 recA1 deoR araD139 v(ara-leu)7697 galU galK rpsL (strR ) endA1 nupG F3 ara-14 leuB6 thi-1 fhuA31 lacY tsx-78 galK2 galT22supE44 hisG4 rpsL136 (StrR ) xyl-5 mtl-1 dam13: :Tn9 (CamR ) dcm-6 mcrB1 hsdR2 (rK 3mK +) mcrA F3 hsdS20 (r3B m3B ) supE44 recA13 ara-14 proA2 lacY1 galK2 rpsL20 (strR ) xyl-5 mtl-1 Tra Mob IncP KmR TcR SmR SuR HgR PCR cloning vector, ColE1, KmR E. coli/Clostridium shuttle (pIP404 replicon) E. coli cloning vector E. coli cloning vector pCR2.1 TOPO vector carrying M.Cdi25 gene pACYC177+M.Cdi1226 gene and M.Cdi25 gene pACYC177 carrying M.Cdi25 gene E. coli cloning vector pMTL20 carrying M.Cdi1226 gene pACYC184+M.Sau961 gene pCD35E+catP (from pJIR418) pCD35EC+oriT (from pMTL30) pMTL28+pCD6 replicon (from pCD35EC) pMTL930+oriT (from pMTL30) pMTL9301+CatP at StuI site pMTL93011 minus SmaI site pMTL93012+BalI site

M. Young, UCW, Aberystwyth

M. Young, UCW, Aberystwyth Invitrogen [8] [21] [22] This study This study This study [23] This study [7] [7] [7] [7] [7] This study This study This study

2.2. Culture media and growth conditions E. coli strains were cultured at 37C in 2UYT medium [7]. C. dicile was grown in supplemented BHI medium (Oxoid brain heart infusion, 37 g l31 ; yeast extract, 5 g l31 , cysteine-HCl, 0.5 g l31 , haemin, 5 mg l31 , vitamin K1, 1 mg l31 , resazurin, 1 mg l31 ) and was cultivated at 37C in an atmosphere of 87% N2 , 9% CO2 and 4% H2 using a Don Whitley Mk III anaerobic cabinet. Media were

placed in the anaerobic environment for 24^36 h to become free of oxygen. Antibiotics used for E. coli were erythromycin (400 Wg ml31 ), ampicillin (50 Wg ml31 ), kanamycin (50 Wg ml31 ) and chloramphenicol (10 Wg ml31 ), and for C. dicile thiamphenicol (15 Wg ml31 ). 2.3. Cloning and analysis of methylase genes DNA cloning and manipulation was carried out as de-

Table 2 Primer sequences used to amplify putative methylase genes from CD630 Methylase gene M.Cdi633 M.Cdi587 M.Cdi1226 M.Cdi25 M.Cdi824 Primers P#633MF: 5P-GATAGTTACTTGTCCCCATTGTG-3P P#633MR: 5P-GTAAAATTAACTCTCATTACTCTCTCC-3P P#587MF: 5P-TTATCTAAATATGAAATGGATATGGA-3P P#587MR: 5P-CTTAAAGCTTCTTTACTTCCATATG-3P P#1226MF : 5P-TATTTGAATAGATTATCCAGAAGAC-3P P#1226MR: 5P-GACCGTTTTCAAGCTCAAATTTTGCAT-3P P#25MF: 5P-GAAGATGGCAAGACATACATG-3P P#25MR: 5P-TCCTTTCATATTATCACCTACTATATC-3P P#824MF: 5P-TACGCAGAACGTCAAAG-3P P#824MR: 5P-TTCGTTTACCTCGATAAG-3P PCR fragment size (bp) 1053 1373 1962 2238 1298 ORF size (bp) 806 1022 1625 1733 959 Predicted protein size (aa) 269 358 547 578 320

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scribed by Sambrook et al. [9]. All DNA modifying enzymes were obtained from New England Biosciences. CD630 chromosome DNA was prepared as in Homan and Winston [10]. DNA fragments encompassing the putative methylase genes were amplied from genomic DNA by polymerase chain reaction (PCR) (see Table 2 for primers), using Expand High Fidelity polymerase (Roche). In all ve cases, to maximise the likelihood of subsequent gene expression, the region amplied covered both the structural gene and the 5P non-coding region extending up to the translation stop of the adjacent gene, i.e. the most likely location of promoter elements. The extents of these non-coding regions were 264 bp (M.Cdi25), 131 bp (M.Cdi587), 215 bp (M.Cdi633), 279 bp (M.Cdi824) and 159 bp (M.Cdi1226). The DNA fragments obtained were cloned into pCR2.1 TOPO (Invitrogen) and sequence veried prior to further use. Sodium bisulte treatment was carried out as described in Davis et al. [11]. Mutated DNA was amplied by PCR for sequencing purposes using primers P#BIS-1 (5P-GGTAGAGGAAGTGTATTAGTTTT-3P) and P#BIS-2 (5P-ATACCTACTACTAAATAACCTACA-3P). Wild-type DNA was detected using primers P#BISWT-1(5P-GTACCTGCTGCTGGATAACCTGC-3P) and P#BISWT-2 (5-P-GGCAGGAGGAAGTGCATTAGCCC-3P).

2.4. Vector construction Plasmid pMTL93011 was obtained by ligating a 1.159kb SnaBI-DpnI fragment carrying the pJIR418 catP gene into the StuI site of pMTL9301 (Fig. 1). Plasmid pMTL3012 was generated by blunting the XmaI site of pMTL93011 with Klenow DNA polymerase. Primers BalFw (5P-AGTTAACTTCAGGTTTGTCTGATTACGC-3P) and BalRv (5P-GATCGGTGCTGGCCACTTCGCTATTACGC-3P) were used to amplify a 433-bp region, anked by HpaI and PvuI, between the CmR and EmR genes of pMTL93011 changing a Sau96I site into a BalI site. The PCR product was ligated into pMTL93011 via a HpaI and PvuI digest to form pMTL93013. pACYC177:M.Cdi25 was obtained by isolating the 2.1kb EcoRV and XbaI fragment carrying M.Cdi25 from pCR2.1:M.Cdi25, and inserting it between the SmaI and NheI sites of pACYC177. To construct a plasmid carrying both M.Cdi25 and M.Cdi1226, the two genes were rst sequentially cloned into pACYC184. The M.Cdi25 gene was isolated from pCR2.1:M.Cdi25 as a 2.1-kb RsaI/ XhoI fragment and inserted between the EcoRV and SalI sites of pACYC184, whereas a 2.4-kb PvuII fragment encompassing the M.Cdi1226 gene (derived from pMTL20:M.Cdi1226, which included the promoter/opera-

Fig. 1. Schematic diagram of constructed plasmids. pMTL930 was generated by cloning the replication region of C. dicile plasmid CD6 into pMTL28, a derived vector of pMTL23E having no CdiI sites, and lacking bla [7]. The SmaI site of pMTL93011 was removed by cutting with XmaI and Klenow lled to generate pMTL93012, plasmid not shown. pMTL93013 was then generated from pMTL93012 by the PCR mutagenesis of a Sau96I site to a BalI site.

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tor region of the E. coli lac operon) was inserted at the unique NruI site. The now adjacent two genes were then excised as a 4.5-kb XbaI fragment and inserted into the NheI site of pACYC177 to give pACYC177 :M.Cdi25: M.Cdi1226. In the nal vector, the M.Cdi25 gene resided immediately downstream of the tet promoter (from pACYC184), whereas the M.Cdi1226 gene was preceded by the lac promoter (from pMTL20). 2.5. Conjugal transfer from E. coli All manipulations of clostridia were undertaken anaerobically in a Don Whitley Mk III anaerobic workstation and at an incubation temperature of 37C. E. coli were transformed by standard procedure [9]. Conjugations were carried out as previously described [7], except that harvested cells of the E. coli donor were resuspended in a total volume of 100^200 Wl of an overnight culture of C. dicile grown in BHI broth. In addition, growth resulting from overnight incubation of the mating mixture on BHI agar was subsequently resuspended in 500 Wl, prior to plating onto selective agar. E. coli donors were counterselected for by the addition of D-cycloserine (250 Wg ml31 ), cefoxitin (8 Wg ml31 ) and trimethoprim (10 Wg ml31 ), and
Table 3 Methyltransferase genes of C. dicile CD630 and their identied homologues Name M.Cdi633 M.BalI* M.Cdi587 Methyltransferase* M.DsaV M.EaeI M.ScrFI M.SsoII M.SenPI M.Cdi1226 Methyltransferase* M.DdeI M.HphI M.Bpu10I M.XorII M.Cdi25 M.BstVI* Methyltransferase Methyltransferase M.BstLVI M.BseCI M.BssSI M.BanIII M.Cdi824 M.Cfr9I* M.Pac25I M.XmaI M.XcyI M.SmaI Target site TGGCCA TGGCCA CCNGG CCNGG YGGCCR CCNGG CCNGG CCNGG GCWGC CTNAG GGTGA CCTNAGC CGATCG Unknown CTCGAG Methylation Cytosine Cytosine Cytosine Cytosine Cytosine Cytosine Cytosine Cytosine Cytosine Cytosine specic specic specic specic specic specic specic specic specic specic Host (strain)

thiamphenicol (15 Wg ml31 ) to select for the transferred plasmid. Plates were incubated at 37C for 24^72 h.

3. Results 3.1. Conjugative transfer to CD630 Vector pMTL9301 was shown to transfer to the toxinogenic C. dicile strain CD3 [7] after extensive modication to remove the sequence motif 5P-CATCG-3P recognised by the identied restriction enzyme of this strain, CdiI. It was, therefore, of interest to see whether pMTL9301 could be conjugated into strain CD630 from the E. coli donor CA434 (HB101 carrying R702). As strain CD630 is resistant to erythromycin, pMTL93011 (pMTL9301+catP) was employed in these conjugation experiments. Thiamphenicol resistant transconjugants were found to reproducibly arise at frequencies of between 1036 and 1037 transconjugants per donor cell. The colonies were shown to be true transconjugants by transforming E. coli cells with C. dicile lysates, and authenticating the inherited plasmids by appropriate restriction digests. In general, the transfer frequencies observed were approximately one order of mag-

Identity to E value CD630 gene (%) 100 41 100 40 29 42 29 30 30 100 33 34 33 28 29 100 37 37 24 24 22 23 100 48 47 46 46 47 3e-53 4e-51 1e-27 1e-27 2e-27 2e-26 2e-26 3e-31 7e-27 4e-24 3e-20 2e-19

Accession number

Cytosine specic Cytosine specic Cytosine specic Cytosine specic Unknown Adenine specic

ATCGAT ATCGAT CACGAG ATCGAT CCCGGG CCCGGG CCCGGG CCCGGG CCCGGG CCCGGG

Adenine specic Adenine specic Adenine specic Adenine specic Cytosine specic Cytosine specic Cytosine specic Cytosine specic Cytosine specic Cytosine specic

Clostridium dicile CD630 Curtobacterium albidum Clostridium dicile CD630 Streptococcus agalactiae 2603V/R Dactylococcopsis salina Enterobacter aerogenes Lactococcus lactis subsp. cremoris Shigella sonnei Salmonella enteritidis Clostridium dicile CD630 Helicobacter pylori J99 Desulfovibrio vulgaris Haemophilus parahaemolyticus Bacillus pumilus Xanthomonas oryzae pv. oryzae Clostridium dicile CD630 Bacillus stearothermophilus V Clostridium tetani E88 Clostridium acetobutylicum Bacillus stearothermophilus LV Geobacillus stearothermophilus Geobacillus stearothermophilus Aneurinibacillus aneurinilyticus Clostridium dicile CD630 Citrobacter freundii Pseudomonas alcaligenes Xanthomonas campestris Xanthomonas campestris pv. cyanopsidis Serratia marcescens

BAA11514.1 NP_688295.1 P50185 AAB95336.1 A48966 P34879 AAC45971.1 AAD06007.1 CAA68505.1 P50192 CAA74996.1 S46293

2e-77 8e-73 2e-18 3e-18 3e-14 5e-14 1e-73 9e-69 3e-68 1e-67 4e-65

AAO36732.1 AAK80265.1 AAF04626.1 CAA56041.1 AAM21167.1 P22772 S07540 AAD40332.1 AAC08981.1 P30774 P14230

*Indicates the top BLASTP hit. The target recognition sequence, methylation type and name of bacterial host are presented. The identities and E values refer to the values generated by the BLASTP search.

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nitude lower than than seen when strain CD3 was used as the recipient [7]. The studies with strain CD3 had established that the presence of a single recognition site on the plasmid being transferred for the restriction endonuclease present in this strain (CdiI) reduced transfer frequencies by approximately one order of magnitude [7]. Thus, one possible explanation for the relatively lower frequency of plasmid transfer observed in strain CD630 compared to CD3 was the presence of a restriction barrier. 3.2. Identication and characterisation of methylase genes in CD630 Whilst restriction enzymes do not possess generalised homology, methylase enzymes share conserved primary amino acid sequences. A BLASTP search of the translated DNA of all six reading frames of the CD630 genome sequence (incomplete at the time) was therefore undertaken for homology to known methylation enzymes. This analysis indicated that ve genes were present encoding putative methylases (Table 3). Accordingly, a DNA fragment encoding each gene was amplied from the CD630 chromosome using PCR and cloned into pCR2.1-TOPO. DNA extracts (plasmid and chromosome) from a sequence veried clone of each gene were then tested to see if they had become resistant to cleavage with any particular commercially available endonuclease. Plasmid DNA carrying the M587 gene was no longer cleaved by the enzyme Sau96I, indicating that restriction sites in the plasmid corresponding to 5P-GGNCC-3P had been methylated at the rst C position. The enzyme was therefore designated M.Cdi587. Its activity corresponds to that of M.CdiII found in strain CD6 [7]. Chromosomal DNA prepared from CD630 was, however, found to be only partially resistant to digestion with Sau96I, suggesting that not all of the target sites were methylated. This was conrmed by sequencing a PCR amplied region of the chromosome following treatment with sodium bisulte, as previously described for strain CD6 [7]. Only one of the two Sau96I sites present in the amplied fragment was found to be methylated. A high degree of similarity (46^49%) was evident between M824 and a group of enzymes which all recognise and methylate the sequence 5P-CCM CGGG-3P, including M.SmaI. In keeping with this observation, plasmid DNA carrying the gene could no longer be cleaved by SmaI. Additionally, some partial protection was evident against SacII. Chromosomal DNA of CD630 was shown to be wholly resistant to cleavage with SmaI, partially digested with SacII and cleaved by StyI. When the M824 gene was inactivated through the introduction of a frameshift in the coding sequence, the DNA of the plasmid carrying the mutant gene became fully sensitive to SmaI and SacII. The specicity of the identied enzyme (designated M.Cdi824) was therefore deemed to be 5P-CCM SSGG-3P. The DNA of plasmids carrying the M1226 gene was

found to be resistant to digestion with TseI, and was designated M.Cdi1226. The specicity of this enzyme therefore equates to 5P-GM CWGC-3P. Consistent with this conclusion was the observation that chromosomal DNA of strain CD630 was not cleaved by TseI. Furthermore, plasmid DNA became sensitive to digestion with this enzyme following the introduction of a frameshift into the M.Cdi1226 gene. M633 exhibits a high degree of similarity (42%) to M.BalI. The plasmid carrying this gene possessed two BalI sites and was observed to undergo partial digestion with BalI, with the majority of the DNA being present in a mixture of uncut and linearised, with only a small portion of the DNA present as the predicted two linear fragments. Following the introduction of a frameshift into the M633 structural gene, however, the plasmid became more sensitive to digestion with complete elimination of the uncut form. However, complete digestion still did not occur, with the products being evenly split between the linearised partial (cut once) and the predicted two fragments (cut twice). Further screening of plasmid DNA carrying the functional gene was undertaken with the following enzymes: MscI, ScrFI, BstNI, Cac8I, ClaI, HaeIII and BssKI. No evidence for methylation protection was, however, obtained. The enzyme was designated M.Cdi633, and most likely has the same specicity as M.BalI. Whilst the M25 predicted polypeptide showed the highest degree of similarity to a number of adenosine methylase enzymes (e.g. M.BstLVI, M.BseCI, M.BanIII), the protein also shared similarity with a number of restriction enzymes, such as Eco57I [12]. It therefore appears to belong to a class of enzymes that possess both activities. However, screening of DNA of the plasmid carrying the functional gene failed to identify a restriction enzyme which was no longer able to cut. The enzymes screened were: AatII, AccI, AgeI, AlwNI, ApaLI, AvaII, BclI, BglI, BsgI, BsrBI, BstBI, BstNI, BstUI, Cac8I, ClaI, Csp45I, MboI, DpnI, DraI, Eco47III, Fnu4HI, HaeII, HhaI, HincII, MseI, MspI, NheI, HpaI, NaeI, NruI, NsiI, PstI, PvuI, RsaI, SacI, SacII, SalI, Sau3AI, Sau96I, ScrFI, SgfI, SmaI, SmlI, TaqI, TseI and XhoI. This included the use of MboI. The identied methylase does not, therefore, appear to correspond to the M.CdiIII activity identied in strain CD6, equivalent to the E. coli Dam system. 3.3. Distribution of methylase genes in other C. dicile strains Having characterised the methylase genes of strain CD630, it was of interest to determine the distribution of the genes in other strains of C. dicile, and in particular strains CD3 and CD6. Accordingly, Southern blots were performed using chromosomal DNA prepared from the C. dicile strains CD630, CD3, CD6, VPI 10463 (the type strain) and CD37 (a non-toxinogenic strain), and probes based on the ve CD630 methylase genes encoding

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Fig. 2. Southern hybridisation of the strain CD630 methylase genes against ve dierent strains of C. dicile. HindIII digested chromosomal DNA (2 Wg) was electrophoresed on a 1% agarose gel and transferred to Hybond H nylon membrane by an alkali blot. The membrane was probed using labelled EcoRI fragments of each methylase gene. Hybridised DNA was detected using the AlkPhos Direct Labelling and Detection system (Amersham Biosciences). Blots were developed with Kodak developing reagents. Lanes are: 1 and 9, 1-kb DNA marker, 2 and 8, genomic DNA marker (Fermentas); 3, CD630 ; 4, CD3; 5, CD6; 6, CD37; 7, VPI 104633.

M.Cdi25, M.Cdi1226, M.Cdi587, M.Cdi633 and M.Cdi824. The results (Fig. 2) demonstrated that M.Cdi25 and M.Cdi1226 are conserved in all ve strains, M.Cdi587 is present in three strains (CD630, CD3 and CD37), M.Cdi633 is carried by two strains (CD630 and VPI 10463), and M.Cdi824 is carried by strain CD630 alone. Intriguingly, although M.Cdi587 was shown in this study to possess equivalent activity to that previously experimentally shown to be present in CD6 [7], its DNA probe did not hybridise to the CD6 chromosome. The M.CdiII of CD6 must therefore be entirely distinct from M.Cdi587. 3.4. Conjugative transfer into strain CD630 Having derived data on the specicity of a number of the methylase enzymes present in strain CD630, we investigated the possibility that the presence of sequence motifs that are the substrates of these methylases may be aecting transfer frequency. Two types of experiment were undertaken. In the one case, restriction sites corresponding to the target site of the identied methylase were introduced into the vector employed in conjugation experiments. Thus, plasmid pMTL93011 was modied such that either a SmaI was removed (pMTL93012) or a BalI site was added (pMTL93013). In both cases, there was no signicant dierence in the frequency of transfer to C. dicile CD630 compared to pMTL93011. In the second series of experiments methylase genes were cloned into plasmid pACYC177, and then the plasmid obtained introduced into the CA434 donor strain along with pMTL93011. In the one instance, the compatible pACYC177 plasmid carried both the gene encoding M.Cdi1226 (positioned 5P to a copy of the E. coli lac promoter) and the M.Cdi25 (positioned 5P to the promoter of the pACYC184 tet gene). In the other instance, the

plasmid employed was the previously constructed vector pACYC184: :Sau5-CmR [7], which carries the M.Sau96I gene. In no instance did the presence of a methylase gene in the donor cell signicantly increase the transfer frequency of pMTL93011.

4. Discussion Our analysis of strain CD630 has indicated that the organism possesses at least four genes encoding cytosine methylases. The enzyme M.Cdi587 appears functionally equivalent to M.Sau96I and CdiII. However, M.Cdi587 is not the same enzyme as M.CdiII [7], as a DNA probe against the latter does not hybridise against the chromosome of CD6. Within strain CD630, M.Cdi587 does not appear to eectively methylate all of its target sites. This may indicate that the enzyme is relatively ineective, either intrinsically or perhaps as a consequence of poor expression of the gene. The enzymes M.Cdi824 and M.Cdi1226, or at least the recombinant enzymes when produced in E. coli, appear to eciently methylate 5P-CCM SSGG-3P and 5P-GM CWGC-3P, respectively. The situation with the other two enzymes is less clear. The evidence obtained in this study suggests that M.Cdi633 has a similar specicity to M.BalI. In the case of M.Cdi25, no conclusion may be drawn as regards its activity save that it is likely, based on homology to other such proteins, to possess both restriction endonuclease and methylase activities. The results with conjugation experiments suggest that there is no barrier to DNA transfer in strain CD630. Genes encoding cognate restriction and methylase activities of a bacterial RM system either are located immediately adjacent to one another, or in rare cases are separated by one or two intervening genes (Rich Roberts, personal communication). Now that the genome sequence

M. Herbert et al. / FEMS Microbiology Letters 229 (2003) 103^110 Table 4 Clostridial RM systems Strain Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium Clostridium dicile CD6 dicile CD3 botulinum ATCC 25765 cellulolyticum ATCC 35319 acetobutylicum ATCC 824 perfringens strain 13 botulinum ATCC 3502 dicile CD630 thermocellum ATCC 27405 tetani E88 Genome sequenced No No No No Yes Yes Yes Yes Yes Yes Methylase 2 2 1 1 6 1 3 5 7 3 (+) (+) (+) (+) Restriction enzyme 2 1 1 1 2 0 0 0 3 1 DNA transfer demonstrated No No No No No Yes Yes Yes No No

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(+) The numbers of methylases given are those activities experimentally shown to be present, and it is assumed (given the data that have emerged from genome sequencing) likely that further enzymes will be present.

of CD630 is complete, inspection of the genes immediately adjacent, or close, to the ve identied methylase genes indicates that there are no candidate genes that could encode a restriction endonuclease. On the basis of this observation, and the experimental data obtained here, one may conclude that the identied CD630 methylase genes do not participate in restriction-modication, and must therefore full another function, most likely in gene regulation or DNA mismatch repair [13]. The lower frequency of plasmid transfer from E. coli donors to CD630, in comparison to CD3 and CD37, must therefore be a consequence of other factors involved in the conjugation process, and may reect the fact that strain CD630 is less able to form the necessary cell^cell contacts with the E. coli donor. The conservation of the M.Cdi1226 and M.Cdi25 gene amongst all ve C. dicile strains suggests that whatever role these enzymes play, they may be universally important in this organism, and in the case of the latter enzyme, clostridia generally. Thus, the closest matches to M.Cdi25 in current database are the putative methylases CAC2309 of C. acetobutylicum and BstVI of C. tetani, which both share 38% identity. The M.Cdi824 gene is interesting for a number of reasons. Although present in three of the ve strains examined, it is clearly of a non-clostridial origin, as its G+C content exceeds 50%, compared to the average 28% of the C. dicile genome. Indeed, analysis of the region of the genome where it is located shows that there is a contiguous region of some 30 kb of DNA which demonstrates an atypical G+C content and encodes genes typical of genetic elements such as Tn1549. It would therefore appear that the M.Cdi824 gene is carried by a conjugative transposon. A DNA cytosine methylase gene has previously been reported on the Bacteroides conjugative transposon Tn5252 [14], where, given that DNA methylation of Tn10 and IS10 is known to play a role in transposition [15,16], it is assumed to have a role in conjugative transfer. It seems likely that the majority of methylase genes in other clostridia also play no role in restriction-modication (Table 4). Thus, the recently completed genomes of

C. botulinum ATCC 3502 and C. perfringens contain orphan copies of methylase genes (three and one, respectively), and are both readily transformable in the absence of any measures to circumvent restriction barriers [17,18]. In the case of C. acetobutylicum, a total of six methylase genes are present, but only two have an adjacent restriction enzyme gene; C. thermocellum has seven methylase genes, but only three restriction enzyme genes; while C. tetani has three methylase genes and a single restriction enzyme gene. The latter two strains have yet to be transformed. C. acetobutylicum, on the other hand, requires that the activity of just the one restriction endonuclease needs to be countered (Cac824I), for successful DNA transfer [19]. The circumvention of just a single restriction activity is similarly required for transformation in C. botulinum ATCC 25765 [11], C. cellulolyticum [20] and C. dicile CD3 [7].

5. Conclusion In summary, we have obtained evidence that the methylase genes of the toxinogenic C. dicile strain CD630 play no role in restriction-modication. This may represent a general feature of clostridial genomes. As a consequence of the absence of restriction activity, oriT-based plasmid DNA vectors may be readily mobilised into strain CD630 from E. coli donors at appreciable frequencies by a lter mating procedure. This gene transfer system should facilitate the exploitation of the data now available from the recently completed genome sequence of this strain (http://www.sanger.ac.uk/Projects/C_dicile/).

Acknowledgements We wish to thank Nicola Minion for typing the manuscript, Jon Brazier, Mike Young and Peter Mullany for supplying bacterial strains, and the nancial support of the UK Department of Health and the BBSRC (Grant 346/E13746).

110

M. Herbert et al. / FEMS Microbiology Letters 229 (2003) 103^110 lytic Clostridium botulinum type B strains. J. Mol. Microbiol. Biotechnol. 2, 59^69. Janulaitis, A., Petrusyte, M., Maneliene, Z., Klimsasuskas, S. and Butkus, V. (1992) Purication and properties of the Eco57I restriction endonuclease and methylase ^ prototypes of a new class (type IV). Nucleic Acids Res. 20, 6043^6049. Marinus, M.G. (1987) DNA methylation in Escherichia coli. Annu. Rev. Genet. 21, 113^131. Sampath, J. and Vijayakumar, M.N. (1998) Identication of a DNA cytosine methyltransferase gene in conjugative transposon Tn5252. Plasmid 39, 63^76. Roberts, D., Hoopes, B.C., McClure, W.R. and Leckner, N. (1985) IS10 transposition is regulated by adenosine methylation. Cell 43, 117^130. Yin, J.C.P., Krebs, M.P. and Rezniko, W.S. (1988) Eect of dam methylation on Tn5 transposition. J. Mol. Biol. 199, 35^45. Scott, P.T. and Rood, J.I. (1989) Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene 82, 327^ 333. Mauchline, M.L., Davis, T.O. and Minton, N.P. (1999) Clostridia. In: Manual of Industrial Microbiology and Biotechnology (Demain, A.L. and Davies, J.E., Eds.), pp. 475^492. ASM Press, Washington, DC. Mermelstein, L.D. and Papoutsakis, E.T. (1993) In vivo methylation in Escherichia coli by the Bacillus subtilis phage P3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum. Appl. Environ. Microbiol. 59, 1077^ 1081. Jennert, K.C., Tardif, C., Young, D.I. and Young, M. (2000) Gene transfer to Clostridium cellulolyticum ATCC 35319. Microbiology 146, 3071^3080. Rose, R.E. (1988) The nucleotide sequence of pACYC177. Nucleic Acids Res. 16, 356. Rose, R.E. (1988) The nucleotide sequence of pACYC184. Nucleic Acids Res. 16, 355. Chambers, S.P., Prior, S.E., Barstow, D.A. and Minton, N.P. (1988) The pMTL nic-cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68, 139^149.

References
[1] Wilcox, M.H. and Smyth, E.T.M. (1998) Incidence and impact of Clostridium dicile infection in the UK, 1993^1996. J. Hosp. Infect. 39, 181^187. [2] Cartmill, T.D., Panigrahi, H. and Worsley, M.A. et al. (1994) Management of diarrhoea due to Clostridium dicile. J. Hosp. Infect. 27, 1^15. [3] Mullany, P., Wilks, M. and Puckey, L. et al. (1994) Gene cloning in Clostridium dicile using Tn916 as a shuttle conjugative transposon. Plasmid 31, 320^323. [4] Mullany, P., Wilks, M. and Tabaqchali, S. (1991) Transfer of Tn916 and Tn916vE into Clostridium dicile : demonstration of a hot-spot for these elements in the C. dicile genome. FEMS Microbiol. Lett. 79, 191^194. [5] Liyanage, H., Kashket, S., Young, M. and Kashket, E.R. (2001) Clostridium beijerinckii and Clostridium dicile detoxify methylglyoxal by a novel mechanism involving glycerol dehydrogenase. J. Appl. Environ. Microbiol. 67, 2004^2010. [6] Mani, N., Lyras, D., Barroso, L., Howarth, P., Wilkins, T., Rood, J.I., Sonenshein, A.L. and Dupuy, B. (2002) Environmental rsponse and autoregulation of Clostridium dicile TxeR, a sigma factor for toxin gene expression. J. Bacteriol. 184, 5971^5978. [7] Purdy, D., OKeee, T.A.T., Elmore, M., Herbert, M., LcLeod, A., Bokori-Brown, M., Ostrowski, A. and Minton, N.P. (2002) Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium dicile through circumvention of the restriction barrier. Mol. Microbiol. 46, 439^452. [8] Sloan, J., Warner, T.A., Scott, P.T., Bannam, T.L., Berryman, D.I. and Rood, J.I. (1992) Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid. Plasmid 27, 207^219. [9] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [10] Homan, C.S. (1987) A ten-minute DNA preparation from yeast eciently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267^272. [11] Davis, T.O., Henderson, I. and Minton, N.P. (1997) Development of a transformation and gene reporter system for group II, non-proteo[12]

[13] [14]

[15]

[16] [17]

[18]

[19]

[20]

[21] [22] [23]