The development of Clostridium difficile genetic systems

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Anaerobe 10 (2004) 75–84

The development of Clostridium difficile genetic systems Nigel Mintona,*, Glen Cartera, Mike Herbertb, Triona O’Keeffeb, Des Purdyb, Mike Elmoreb, Anna Ostrowskib, Oliver Penningtona, Ian Davisa a

Institute of Infection, Immunity and Inflammation, University of Nottingham, Floor C, West Block, Queens Medical Centre, Nottingham NG7 2UH, UK b Health Protection Agency, Porton Down, Salisbury, Wiltshire SP4 0JG, UK Received 28 July 2003; received in revised form 4 November 2003; accepted 6 November 2003

Abstract Clostridum difficile is a major cause of healthcare-associated disease in the western world, and is particularly prominent in the elderly. Its incidence is rising concomitant with increasing longevity. More effective countermeasures are required. However, the pathogenesis of C. difficile infection is poorly understood. The lack of effective genetic tools is a principal reason for this ignorance. For many years, the only tools available for the transfer of genes into C. difficile have been conjugative transposons, such as Tn916, delivered via filter mating from Bacillus subtilis donors. They insert into a preferred site within the genome. Therefore, they may not be employed for classical mutagenesis studies, but can be employed to modulate gene function through the delivery of antisense RNA. Attempts to develop transformation procedures have so far met with little success. However, in recent years the situation has been dramatically improved through the demonstration of efficient conjugative transfer of both replication-proficient and replication-deficient plasmids from Escherichia coli donors. This efficient transfer can only be achieved in certain strains through negation of the indigenous restriction barrier, and is generally most effective when the plasmid employed is based on the replicon of the C. difficile plasmid, pCD6. r 2003 Elsevier Ltd. All rights reserved. Keywords: Clostridium difficile; Conjugation; Replicon; Restriction modification

1. Introduction Clostridium difficile is the most common cause of nosocomial diarrhoea in the UK and elsewhere [1]. C. difficile-associated disease (CDAD) accounts for up to 15% of diarrhoeal disease associated with antibiotic treatment [2]. Antibiotics disrupt the normal intestinal microflora so that C. difficile is able to rapidly colonise the gut and elaborate toxins. Broad-spectrum cephalosporins and clindamycin are among the antibiotics more commonly associated with CDAD, but most antibiotics have been implicated. The clinical severity of CDAD ranges from a self-limiting ‘antibiotic-associated diarrhoea’ (AAD), through acute and severe AAD, to lifethreatening clinical manifestations such as toxic dilatation and pseudomembranous colitis. Patients over 65 years of age are particularly at risk of CDAD, as are the immuno-suppressed. Elderly people *Corresponding author. Tel.: +44-115-84-67458; fax: +44-115-84-66296. E-mail address: [email protected] (N. Minton). 1075-9964/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2003.11.003

in hospitals, nursing homes, and other chronic-care facilities, are at high risk of cross-infection and can develop CDAD within 5–6 days of contact with an index case [3]. Indeed, C. difficile has become by far the most common enteric pathogen identified in the elderly. The impact of CDAD in hospitals and nursing homes is considerable; patients require isolation (preferably to a separate ward), revised supportive therapy (particularly to antibiotic use) for underlying disease and for CDAD, specific therapy to eliminate C. difficile, scrupulous hygiene in nursing, environmental decontamination, and (in outbreaks) ward closure. With an increasingly ageing population, the incidence of CDAD in the western world is set to continue to rise in the coming years. More effective countermeasures are clearly required. The rational development of therapeutic strategies to counter the threat of a particular bacterial pathogen

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requires a detailed knowledge of the molecular basis of virulence. The pathogenesis of C. difficile infection is, however, poorly understood [4,5]. The availability of genome sequence(s) should provide new insights. However, the genetic tools needed to exploit this information were until recently practically non-existent. For many years, the only tools available for the transfer of genes into C. difficile were conjugative transposons, such as Tn916, delivered via filter mating from Bacillus subtilis donors. These may be employed to deliver cloned DNA into non-toxinogenic strains, but the required procedures are inefficient and cumbersome. Attempts to develop transformation procedures have so far met with little success. However, in recent years the situation has been dramatically improved through the demonstration of efficient conjugative transfer of both replicationproficient and replication-deficient plasmids from Escherichia coli donors.

2. Conjugative transposons In the years that immediately followed the realisation that C. difficile was the principal aetiological agent of antibiotic-associated diarrhoea, a number of studies set out to investigate the genetic basis of antibiotic resistance. In an initial analysis [6] it was established that resistance to tetracycline (Tc) was readily transferable between strains of C. difficile by conjugation, but not clindamycin (Clin) and erythromycin (Em). It soon became clear that a plasmid was not involved in the transfer of Tc, and that the determinant responsible was chromosomally located [7]. The frequencies of Tc transfer were relatively low, residing within the range of 10
6–10
7 transconjugants per donor cell. Similar frequencies of transfer were subsequently obtained by Wust . and Hardegger [8]. However, these workers were additionally able to show that Clin and Em could also be transferred, albeit at a lower frequency (10
8 transconjugants per donor cell). They again concluded that transfer did not correlate with plasmid DNA. One of the two donor strains utilised in the conjugation studies of Wust . and Hardegger [8] was shown to possess two plasmid species of 5 and 22 MDa, respectively. This isolate is in fact the strain, CD630, whose genome has just been completed at the Sanger Institute in the UK. This analysis (http://www.sanger.ac.uk/ Projects/C difficile/) indicates the strain harbours just one circular element in addition to the genome. This element is 7881 bp in size, and therefore equates to the 5 MDa plasmid identified by Wust . and Hardegger [8]. Intriguingly, none of the ORFs carried by this element encode products that share homology with known proteins traditionally associated with plasmids, i.e., replication proteins. The nature of the second circular molecule noted by these workers in cell lysates remains

to be determined, but could represent a transient circular form of one of the mobile elements or prophage that are present in this strain. Its presence in lysates was not reported in a later study by H.achler et al. [9] who noted the presence of a single plasmid, estimated to have a molecular weight of 8 kb. Having established the likely presence of conjugative transposons in C. difficile [9], H.achler and co-workers were able to demonstrate that the chromosomal DNA of C. difficile strains carrying the TcR element, including strain CD630, possessed demonstrable DNA homology with the TcR gene of the Enterococcus faecalis transposon, Tn916. This finding led Mullany et al. [10] to clone the TcR from strain CD630 and used it in dot-blot hybridisation studies, demonstrating that the gene belonged to hybridisation class M. 2.1. Nucleotide sequence analysis Eventually designated Tn5397, nucleotide sequence analysis has now been employed to define the exact relationship between this transposon and Tn916. Preliminary sequencing [11] established that the element contained a group II intron that interrupted a gene in Tn5397 that was almost identical to orf14 from Tn916. This was the first group II intron to be found in Grampositive bacteria and was shown, by DNA hybridisation, to be present in related elements carried by five other C. difficile strains from different geographical locations. The Tn5397 group II intron was shown to be functional, being spliced in both C. difficile and B. subtilis and through the demonstration that non-spliced RNA is also present [12]. However, splicing is not required for the conjugative transfer of Tn5397 between bacteria. Thus, mutational disruption of the open reading frame within the intron prevented splicing but did not prevent the formation of the circular form of the conjugative transposon (the likely transposition intermediate) or decrease the frequency of intergeneric transfer of Tn5397 [12]. The completion of the entire sequence of Tn5397 [13] has now enabled a more precise comparison to be made to Tn916 and to a partial sequence of a C. perfringens conjugative transposon, CW459tet(M). This analysis has indicated that these elements have a modular structure, comprising: (i) Tn916-like conjugation region; (ii) resistance gene, tet(M), (iii) regulatory region; and (iv) the region responsible for insertion/excision of the element. All three elements contain the tet(M) resistance gene and have sequence similarity throughout their central region. However, they have very different integration/excision modules. Tn5397 has a large resolvase gene, tndX, which is essential for excision and transposition of the element [14]. In contrast, Tn916 carries the smaller int and xis genes. CW459tet(M) encodes a putative integrase, Int459, that demonstrates

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only distant homology to Int from Tn916. On the basis of their comparative analysis, these authors [13] concluded that the clostridial elements were derived independently from distinct mobile genetic elements.

3. Transfer of replication deficient plasmids from B: subtilis donors During the analysis of the transfer of Tn5397 into C. difficile from either C. difficile or B. subtilis donors, it was noted that all 20 transconjugants analysed exhibited the same DNA/DNA hybridisation patterns. One explanation for this observation was that the transposon has a preferred point of insertion in the C. difficile genome. Subsequently it was shown that Tn916 (and a derivative carrying an erm gene in place of tet(M), Tn916DE) also inserted into a ‘hotspot’ within the chromosome of CD37 when transferred from a B. subtilis donor [15], at least in the eight transconjugants analysed. This preferred site of insertion was later characterised at the nucleotide sequence level and designated att916 [16]. Interestingly, during the course of this study, an environmental isolate of C. difficile was found which contained an element, Tn916CD, which was indistinguishable from Tn916. This element was also inserted at att916. 3.1. Use in gene cloning The non-random insertion of elements such as Tn5397 and Tn916 into the genome of C. difficile means that they may not be used for mutant generation. However, it was suggested by Mullany et al. [15] that it may be possible to use them to introduce cloned DNA into this Clostridium. Thus, Casey et al. [17] had previously used a pBR322-based plasmid (pCI195) containing a region of homology to the conjugative transposon Tn919 to clone genes into Tn919 via cointegration and thence introduce them into the chromosome of Lactococcus lactis. Mullany et al. [18] followed a similar strategy with C. difficile CD37. The region of Tn919 carried by pCI195 is highly homologous to Tn916. They were therefore able to clone a region of the C. difficile tcdB gene into plasmid pCI195, and thence introduced it into a B. subtilis donor strain carrying Tn916DE. As pCI195 is not able to replicate in B. subtilis, chloramphenicol-resistant (CmR) colonies that arose represented cells in which the plasmid had inserted into the chromosomally located transposon by homologous recombination. They then went on to show that the co-integrate transposon could be subsequently transferred from the B. subtilis donor to C. difficile CD37 by filter mating at frequencies of 10
8 per donor. Whilst somewhat cumbersome and inefficient in nature,

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these experiments represented the first example of gene cloning in this organism. 3.2. Use in antisense RNA delivery This strategy for introducing DNA into C. difficile may theoretically be employed in the inactivation of gene function through the delivery of antisense RNA. To test this feasibility, we constructed a novel cointegrative expression vehicle, pMTL900. The initial plasmid constructed was pMTL4::tet(M), in which a region of the Tn916 tet(M) gene was inserted into pMTL4 (composed of the Gram-negative ColE1 replicon and bla gene [19]). A DNA fragment encompassing the expression cassette of pMTL540FT (viz., the pMTL20 lacZ0 gene and multiple cloning sites flanked by the C. pasteurianum ferredoxin (Fd) gene promoter and transcriptional terminator [20]) and the catP gene of the C. perfringens plasmid pJIR418 was then centrally inserted into the tetM gene of pMTL4::tetM. Plasmid pMTL900 is, therefore, only able to replicate in E. coli and not a Gram-positive host. The presence of the tetM region will, however, allow it to establish in B. subtilis in the presence of chloramphenicol, through its recombination with the chromosomally located Tn916. To date two genes have been investigated using this system. In one experiment, a 350 bp fragment encompassing the 50 -end of the C. difficile virR gene (a) homologue of the C. perfringens virR virulence transcriptional factor [21]), including its ribosome binding site (see Fig. 4), was PCR amplified and cloned adjacent to the Fd promoter such that its transcription would result in an antisense molecule. The final plasmid (pMTL900antivirR) was integrated into Tn916 in a B. subtilis donor, and the cointegrate obtained transferred into C. difficile strain 630 by filter mating. Whilst only a single putative CmR transconjugants was obtained, extensive analysis (including direct sequencing of PCR products) demonstrated that all of the expected components had integrated into the genome. Moreover, upon initial isolation, RT-PCR experiments demonstrated that antivirR mRNA was being produced by this transconjugant (Fig. 1). It was further noted that the strain exhibited impaired growth characteristics, in terms of an extended lag phase, reduced growth rate, and a reduced final cell density. However, upon subsequent subculture and/or storage of the strain in Robinson Cooked meat, this impairment in growth rate was no longer apparent. Furthermore, antisense RNA could no longer be detected. It was concluded that the transconjugant was unstable. The apparent reduction in growth rate brought about by this production was intriguing. Activation of the VirR regulator of C. perfringens is mediated by the VirS sensor, following its interaction with an exogenous autoinducer (AI). This AI is believed to be a peptide

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78 RNA

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To date the experiments with antisense RNA delivery have proven disappointing. However, the discovery that we can now delivery autonomously replicating vectors (see below) now provides a more effective route to more fully explore such a strategy.

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Fig. 1. Demonstration of ‘antivirR’ antisense RNA production in C. difficile. Total RNA was prepared from strain CD630 early (A) and late (C) growth phase and early (B) and late (D) growth phase of FM18A. s-virR=‘sense’ virR RT-PCR (250 bp), as-virR=‘antisense’ virR RT-PCR (350 bp). No RT=control PCR on RNA in the absence of reverse transcriptase. ToxA and VirR=PCR on chromosomal DNA to demonstrate presence of (a) Toxin A gene and (b) ‘sense’ virR gene [21].

pheromone termed substance ‘A’ [22]. However, recent analysis has shown [23] that the VirR/S system is additionally involved in the negative regulation of luxS (=ygaG). This gene encodes a second autoinducer, AI2, which is produced by both Gram-negative and Grampositive bacteria [24]. Thus, the C. difficile virR gene may play a role in the production of AI-2. This observation is consistent with attenuation of luxS function as effects on growth rate have recently been described in a luxS mutant of S. pyogenes, [25] and is consistent with the fact that AI-2 is known to be involved in the regulation of many different genes, i.e., 10% of the genes in E. coli O157:H7, [26]. However, this phenotypic trait proved unstable due to loss of the ability of the transconjugant to produce antisense RNA. The reason for this loss was unclear. The use of a Tn916specific probe indicated that the strain contained two or more copies of the gene. Strain CD630 is also known to contain a closely related transposon, Tn5397 [14]. Moreover, analysis of the genome sequence has now shown that there are three other distinct putative transposons related to Tn5397. The presence of these other elements could have contributed to the observed instability. The second gene to be analysed has been an adhesin gene cwp66 of the toxinogenic strain C. difficile 79-685 [27]. In contrast to the situation with strain CD630, the transfer of the co-integrate constructed from B. subtilis proved more effective with several transconjugants being obtained per experiment. In common with the virR experiments, production of antisense RNA was clearly evident. However, no reduction in the levels of Cwp66 were noted, and no effect on the adhesive properties of the transconjugants compared to wild type were noted.

All of the early work with C. difficile involved the use of B. subtilis donors for the conjugative transfer of DNA. In other clostridial species, the conjugative mobilisation of oriT-based vectors from E. coli donors had proven remarkably effective [28,29]. Thus, a number of shuttle vectors have been made by cloning clostridial replicons into the ColE1-based vector pMTL31, and the plasmids obtained shown to transfer to clostridial donors at appreciably frequencies. Moreover, plasmid pMTL31 has also been shown to represent an effective suicide vector for bringing about the integration of the plasmid into the clostridial genome by single crossover. Thus, by cloning a fragment from the recipient’s chromosome into the polylinker of this plasmid, it has proven possible to obtain a number of different gene knock-outs in C. beijerinckii, including that of gutD and spoOA [30,31]. This strategy was first applied to C. difficile by Liyanage et al. [32], as part of an investigation concerned with methylglyoxal detoxification. In these experiments, a central portion of the gldA gene (encoding glycerol dehydrogenase) was inserted into pMTL31, and the resultant plasmid conjugated from the conjugation proficient E. coli donor CA434 (HB101 carrying R702) to the non-toxinogenic strain C. difficile CD37. However, disruption of gldA apparently proved lethal, and the transconjugants obtained developed no farther than pinpoint colonies. Nevertheless, convincing evidence was obtained that the plasmids had indeed integrated, through the generation of DNA fragments by PCR of a size consistent with integration. Moreover, integrants were also generated in which inactivation of gldA was deliberately not attained. DNA fragments consistent with integration of the plasmid by single crossover were also obtained using appropriate primers in a PCR assay and chromosomal DNA from these clones. However, in neither case was the frequency of transfer quantified.

5. Conjugative transfer of replication-proficient plasmids from E: coli donors In a parallel study, we also investigated the potential of mobilising oriT-based vectors from E. coli donors to C. difficile [33]. In this case, however, we sought to

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introduce shuttle vectors capable of autonomous replication. 5.1. A novel C. difficile plasmid replicon To guarantee that the plasmid utilised would be able to replicate, we elected to base our vectors on a replicon derived from an indigenous C. difficile plasmid. Accordingly, we screened a total of 30 strains of C. difficile for the presence of plasmids and identified a plasmid of some 7 kb in size in the lysates of strain CD6 which we designated pCD6. The cloning and analysis of both restriction fragments and PCR products derived from this plasmid enabled the identification of the likely replication region of this plasmid, as being confined to a 3994 bp fragment. This putative assignment was then confirmed through the demonstration that the insertion of this fragment into the replicon cloning vector pMTL20E [34] resulted in a plasmid (pCD35E) that was able to transform C. beijerinckii NCIMB 8052. The isolated pCD6 replicon is the first such element to be described from C. difficile. Its closest relative appears to be that of the C. perfringens plasmid pIP404, as evidenced by the presence of a gene predicted to encode a large polypeptide (RepA) essential for replication which exhibits a distant similarity to the pIP404 RepA protein, and the presence of an extensive ‘downstream’ repeat region [35]. Although nothing is known of the mechanism by which either pIP404 or pCD6 replicate, it seems likely that the repeat region represents an origin of replication. Repeat elements (iterons) are commonly found within plasmid replicons and are particularly evident in plasmids that replicate by a theta mechanism, but may also be found in plasmids that replicate via strand displacement or rolling circle [36]. A second gene (ORF B) found 50 to RepA exhibits no similarity to any known protein, and does not appear to be required for replication. Thus, the introduction of a frameshift after codon position 95 (total codons 215) in pCD35E produced a plasmid that was still able to transform C. beijerinckii. However, when ORF B was entirely deleted, EmR colonies of C. beijerinckii cells transformed with the resultant plasmid took 48 h to attain a size similar to that seen in 24 h with cells carrying pCD35EC [33]. The region encompassing ORF B does, therefore, contribute to an unknown aspect of effective plasmid maintenance. 5.2. Restriction-modification systems Our initial attempts to introduce plasmids by either electroporation (using the electroporation protocol employed to transform C. beijerinckii [37]), or by conjugation using a number of different toxinogenic strains as recipients, were unsuccessful. In other clostridia, the presence of restriction activities have been found to prevent the introduction of plasmid

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vectors [29,38,39]. Successful transfer has only proven possible through circumvention of this restriction barrier following appropriate methylation of the vector DNA to be introduced. We therefore sought to characterise the restriction activities of two potential C. difficile recipients. In the case of the one strain, CD3, our analysis of restriction activities indicated the presence of a single restriction endonuclease, designated CdiI, which cleaved marker plasmid DNA at the non-palindromic sequence 50 -CATCG-30 [33]. Further analysis showed that cleavage by CdiI produces a blunt-ended double-stranded (ds) DNA fragment, by cutting between the 4th and 5th nucleotide of the sequence 50 -CATCG-30 , and between the first and second nucleotides of the complimentary sequence, 50 -CGATG-30 . The enzyme therefore belongs to the Type IIs family, and at the time of discovery was the first example of an enzyme exhibiting this particular sequence specificity. Whilst type IIs enzymes were formerly defined as enzymes which cut at some distance to the recognition sequence [40], there are now an increasing number of examples of enzymes which like CdiI cleave within the DNA motif recognised, e.g., BsiI, SimI and BtrI. Analysis of a second strain, CD6, demonstrated that this strain contained one restriction enzyme which cleaved the recognition sequence 50 -GGNCC-30 and a second enzyme that cleaved at the sequence 50 -GATC-30 [33]. These two enzyme activities were designated CdiII and CdiIII, respectively. Moreover, the strain also possesses corresponding methylase activities, designated M.CdiII and M.CdiIII, with a methylation specificity of 50 -GGNCMC-30 and 50 -GAMTC-30 , respectively. From a practical perspective, the presence of CdiIII does not affect gene transfer from E. coli, as the resident dam system will appropriately modify vector DNA. 5.3. Conjugative transfer of autonomous plasmids to C. difficile strains As no methylase is currently known that is capable of protecting the recognition sequence of CdiI, to circumvent this barrier we constructed a clostridial vector lacking its recognition sequence. This was achieved by derivatising pMTL23E, which carries four CdiI sites, through three sequential deletions to give plasmid pMTL28, and then inserting a number of fragments carrying different clostridial replication regions [33]. These replicons were derived from pCD6 (C. difficile) the C. butyricum plasmid pCB102 [41], and the C. perfringens plasmid pIP404 [34]. Each of the three plasmids obtained were then further modified by insertion of the oriT region of plasmid RK2. The final plasmids obtained, and the replicons they carried, were pMTL9301 (pCD6), pMTL9401 (pCB102) and pMTL9611 (pIP404). All of the inserted fragments

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lacked CdiI recognition sites. Prior to their use in experiments involving C. difficile, each vector was shown to be replication proficient through their successful transformation into C. beijerinckii by electroporation. The three constructed plasmids, together with pCD35ECoriT, were introduced into the E. coli donor strain CA434 and conjugations undertaken using a plate mating procedure [33]. Plasmid pMTL9301 (pCD6 replicon) was found to transfer at the highest frequency (5.7  10
5 per donor cell) with the transfer of pMTL9401 (pCB102) with an approximate ten-fold less efficiently. Moreover, colonies that arose in the case of pMTL9401 took 48 h to achieve an equivalent size to that observed after 24 h with pMTL9301. A similar reduced colony size was also evident with pMTL9611, but in this case colonies, albeit of a small size, were only evident after at least 96 h of incubation. Moreover, the developed colonies could not be further subcultured, suggesting that this particular plasmid functioned extremely inefficiently in these strains of C. difficile. Not unexpectedly, plasmid pCD35EcoriT, which carries eight CdiI sites, was not transferred, underlining the importance of restriction as a barrier to gene transfer. Indeed, the sequential addition of CdiI sites to pMTL9301 caused a cumulative reduction in transfer frequencies, such that by the time four sites were added transfer of plasmid to CD3 could not be detected. Although transfer of plasmid to strain CD3 could be obtained, through the prior removal of CdiI sites, it would be more desirable to utilise a C. difficile strain where any endogenous restriction activity could be negated in the E. coli donor through the provision of an appropriate methylase gene. Such an approach is possible with strain CD6, as methylase enzymes are readily available capable of protecting DNA from its two restriction enzymes CdiII and CdiIII, namely the product of the E. coli dam gene and M.Sau96I. Thus, an E. coli donor (CA434) was chosen which was dam+ and which carried plasmid pACYC184::Sau5 (encoding the M.Sau96I methylase gene), and used as a donor to introduce plasmids pMTL9301, 9401 and 9611 into C. difficile CD6 by conjugation [33]. All three plasmids were found to transfer at low frequencies. Whilst the frequency observed with pMTL9401 and pMTL9611 was broadly similar to that seen with strain CD3, the transfer frequency of pMTL9301 was some two orders of magnitude lower. This was subsequently shown through curing experiments to be largely due to the presence of plasmid pCD6, which is incompatible with pMTL9301 as they share the same replicon. Transfer to the cured strain occurred at frequencies of between 2  10
7 and 1.0  10
6 per donor. In both strains, plasmid pMTL9301 was found to be reasonably segrationally stable, with only 8% of the colonies tested being found to have lost the plasmid

after 32 generations in the absence of antibiotic selection. This contrasted with pMTL9401, which was lost from 96% of the cells after growth under the same conditions [33]. 5.4. The restriction barrier and its presence in other strains As plasmid DNA is transferred during conjugation in the single-stranded (ss) form, there is an argument that the process should be little affected by RM systems, which require ds DNA as a substrate. Such an explanation is invoked to explain, for instance, why plasmids conjugated from E. coli substantially overcome the low frequencies obtained in Neisseria transformation [42]. However, numerous studies contradict this observation. Thus, the presence of unmodified restriction sites in a plasmid caused a 3000-fold reduction in transfer frequency to B. subtilis [43] and in Gramnegative bacteria [44]. Elhai et al. [45] noted an almost 100-fold reduction in the frequency of transfer per restriction site of a plasmid from E. coli to Anabaena, while Butler and Gotschlich [46] showed that the appropriate methylation of a plasmid increased the frequency of conjugal transfer to Neisseria gonorrhoeae by four orders of magnitude. Clearly, as recently transferred ss DNA is converted to ds, a competition must ensue between cleavage by endogenous restriction enzymes, and protection by the cognate methylase. It would appear that the latter process must, at least in the documented examples, operate less efficiently than restriction. Thus, comparatively speaking, methylase enzymes exhibit poorer kinetics for their substrate than restriction enzymes. Given the importance of restriction in the two strains CD3 and CD6, what of the situation in other strains? During the course of this study, and given the results of Liyanage et al. [32], we took the opportunity of examining the non-toxinogenic strain CD37. Transfer of plasmid pMTL9301 was found to occur from E. coli donors at high frequencies, equivalent to those observed with strain CD3. Moreover, no restriction activity could be detected in lysates of this strain. This may indicate that this strain possesses no restriction barrier to gene transfer. Indeed, subsequent to our study, the highfrequency transfer of a shuttle vector based on the replicon of pIP404 to this strain has been reported [47]. In the case of the genome strain CD630, our recent work has shown that it too lacks a restriction barrier, and DNA may be readily introduced by conjugation from E. coli donors [48]. 5.5. Complete nucleotide sequence of pCD6 Subsequent to our characterisation of the pCD6 replication region [33], the nucleotide sequence of the

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penicillin/methicillin binding proteins (PenJ, BlaR, MecR) from Staphylococcus aureus and Bacillus licheniformis [52,53]. Moreover, ORF D is preceded by a small gene (ORF C) encoding a small protein, which shows distant similarity to the MecI protein (Fig. 4) that acts as a repressor of the mec operon of S. aureus [52]. Given the similarity of these gene products to proteins known to be involved in antibiotic resistance we undertook a preliminary screen of the comparative sensitivity of C. difficile CD3 compared to the variant strain, CD6c, from which the plasmid pCD6 has been cured [33]. However, no difference in sensitivity to the four antibiotics ampicillin, penicillin G, oxycillin or amoxicillin could be detected. A more detailed analysis involving the screening of a wider variety of antibiotics is clearly required.

remainder of the plasmid has now been determined. Plasmid pCD6 proved to be 6830 bp in length and to have a G+C content of 24.5%. Translation of the sequence indicated that five major ORFs were present (Fig. 2), with two additional ORFs (ORF C and ORF D) to those present on the previously sequenced replicon fragment [32]. A fifth gene (ORF E) was also identified, the 30 end of which resided on the replicon fragment and was previously designated ORF X [33]. The polypeptide encoded by ORF E was found to be 187 aa in length and to share similarity with the muramoyl-pentapeptide carboxypeptidase (Fig. 3) of Streptomyces albus [49]. This enzyme catalyses the carboxypeptidation and transpeptidation reaction involved in bacterial cell-wall biosynthesis, and in addition has weak beat-lactamase activity, hydrolysing penicillin into penicilloate. It also shares similarity to the penicillin DD-carboxypeptidases of Nostoc [50] and Myxococcus xanthus [51]. ORF E is also preceded by a gene (ORF D) encoding a protein, which shares homology (Fig. 4) to

6. Conclusions For many years, the inability to efficiently transfer DNA into C. difficile has imposed a major impediment to the molecular analysis of virulence in this organism. That progress that had been made was confined to the non-toxinogenic strain CD37. We have now developed procedures whereby autonomous vectors may be introduced into a variety of strains by conjugative transfer from E. coli donors. In the case of strains CD3 and CD6, our experiments have emphasised the need to counter the restriction barrier if effective transfer of plasmid vectors is to be obtained. This does not appear to be the case with the non-toxinogenic strain CD37, or indeed the genome strain CD630, which apparently possess no restriction barrier to DNA transfer.

ScaI 715

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L Q Q E E Q K

S Q A I T S A

K A A A A A A

V V V V V V V

K M I K K Q Q

S N K A A A R

F F F F F F F

Q Q Q Q Q Q Q

G R R E I R S

S T A Q Q A A

Y N N Q R R Y

G G G N N G G

L L L L L L L

T S T T S Q A

V V A V V V A

D D D D D D D

G G G G G G G

I I I I V I I

V V V V V V A

G G G G G G G

S R S V F P P

A L K Q N K A

ORF E B. halo Nostoc 1 Nostoc 2 Nostoc 3 M. xanth S. albus

176 304 75 220 197 80 111

T T T T T T T

W W W W W W F

K D V Y Y S N

K V A C S A K

F L L L L L I

A F R N Y

S R K S Q

A N V T T A L

T N P T G Q

N T T H G D

L T P S A D

D T T K R G D

-

187 313 84 228 203 91 122

ORF E B. halo Nostoc 1 Nostoc 2 Nostoc 3 M. Xanth S. Albus

(187 (881 (128 (228 (203 (302 (255

aa) aa) aa) aa) aa) aa) aa)

Fig. 3. Alignment of pCD6 ORF E with closest homologues. Proteins, and GenBank Accession numbers ( ) are: B. halo, B. halodurans unknown protein (NP 242161); Nostoc 1, Nostoc sp. unknown (NP 489024); Nostoc 2, Nostoc sp. similar to penicillin-resistant DD-carboxypeptidase (NP 485580); Nostoc 3, Nostoc sp. unknown (NP 487970); M. xanth, Myxococcus xanthus penicillin-resistant DD-carboxypeptidase (BAA83081); and S. albus, Streptomyces albus muramoyl-pentapeptide carboxypeptidase precursor (P00733).

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82

180v 190v 200v 210v 220v 230v 240v 250v 260v INTPSFFGLFDNYILLPPH--NYNLNELHWILKHELIHYKSKDLYIRYLVLFLKCVYFFNPFIYLLDRIIYHSCELHCDEVVLKGCSLENIKSYANTIL I::P FG:: YI:LP : :: :E:. :L HEL H.K.KD: I.Y:: :LK VY.FNP::. L.: . E: CD .VLK. . . .Y::.IL BLPenJ IKSPITFGVIRPYIILPKDISMFSADEMKCVLLHELYHCKRKDMLINYFLCLLKIVYWFNPLVWYLSKEAKTEMEISCDFAVLKTLDKKLHLKYGEEIL 180^ 190^ 200^ 210^ 220^ 230^ 240^ 250^ 260^ 270^ ORFD

180v 190v 200v 210v 220v 230v 240v 250v 260v INTPSFFGLFDNYILLPPH--NYNLNELHWILKHELIHYKSKDLYIRYLVLFLKCVYFFNPFIYLLDRIIYHSCELHCDEVVLKGCSLENIKSYANTIL I::P FG:: YI:LP : :: :E:. :L HEL H.K.KD: I.Y:: :LK VY.FNP::. L.: . E: CD .VLK. . . .Y::.IL BLBlaR IKSPITFGVIRPYIILPKDISMFSADEMKCVLLHELYHCKRKDMLINYFLCLLKIVYWFNPLVWYLSKEAKTEMEISCDFAVLKTLDKKLHLKYGEVIL 180^ 190^ 200^ 210^ 220^ 230^ 240^ 250^ 260^ 270^ ORFD

180v 190v 200v 210v 220v 230v 240v 250v 260v INTPSFFGLFDNYILLPP---HNYNLNELHWILKHELIHYKSKDLYIRYLVLFLKCVYFFNPFIYLLDRIIYHSCELHCDEVVLKGCSLENIKSYANTIL I:.P FGL .: I:LP. :. N :E::.I: HEL H KS.DL :. L : :K ::.FNP :Y: . :: :.CE CD VLK : :: .Y:::IL SAMecR IDNPMVFGLVKSQIVLPTVVVETMNDKEIEYIILHELSHVKSHDLIFNQLYVVFKMIFWFNPALYISKTMMDNDCEKVCDRNVLKILNRHEHIRYGESIL 170^ 180^ 190^ 200^ 210^ 220^ 230^ 240^ 250^ 260^ ORFD

180v 190v 200v 210v 220v 230v 240v 250v 260v INTPSFFGLFDNYILLPP---HNYNLNELHWILKHELIHYKSKDLYIRYLVLFLKCVYFFNPFIYLLDRIIYHSCELHCDEVVLKGCSLENIKSYANTIL I:.P FGL .: I:LP. :. N :E::.I: HEL H KS.DL :. L : :K ::.FNP :Y: . :: :.CE CD VLK : :: .Y:::IL SAMecR1 IDNPMVFGLVKSQIVLPTVVVETMNDKEIEYIILHELSHVKSHDLIFNQLYVVFKMIFWFNPALYISKTMMDNDCEKVCDRNVLKILNRHEHIRYGESIL 170^ 180^ 190^ 200^ 210^ 220^ 230^ 240^ 250^ 260^ ORFD

ORFC SAMecI

10v 20v 30v 40v 50v 60v 70v 80v MKKKFSSACKNSYSNAYECSIITELVL-KISVRIKLDIYSTYYFLSFFIKALKDANSSFPIFSLLTESTKLTIESLCTFFTKSDVANSK . . . A . :II.E: : K . .: . T : . FI. KD N. F. :SL:.ES. .S : . . : :. MDNKTYEISSAEWEVMNIIWMKKYASANNIIEEIQMQKDWSPKTIRTLITRLYKKGFIDRKKD-NKIFQYYSLVEESDIKYKTSKNFINKVYKGGFNSLVL 10^ 20^ 30^ 40^ 50^ 60^ 70^ 80^ 90^ 100^

Fig. 4. Alignment of ORFD and ORF C with closest homologues. Proteins, and GenBank Accession numbers ( ) are: BLPenJ, Bacillus licheniformis penicillnase antirepressor (I39942); BLBlaR, B. licheniformis penicillin binding protein (P12287); SAMecR, Staphylococcus aureus methicillin resistance protein (T44117); and SAMecR1, S. aureus methicillin resistance protein (AAB03636). pCD6 OrfC alignment with the S. aureus MecI, SAMecI (X63598) repressor protein.

The two immediate challenges to further progress will involve the derivation of transformation procedures, whereby vectors may be directly introduced, and, more importantly, the development of efficient procedures for the directed inactivation of chromosomal genes. The former will require the formulation of an effective electroporation procedure. This will need to overcome the inherent fragility of C. difficile cells to harvesting and resuspension, and the electric pulse itself. One report of the successful electroporation of a nontoxinogenic strain has appeared [54]. However, the data presented are not sufficiently complete to make an objective assessment of the validity of the results obtained. The availability of autonomously replicating vectors, which were unavailable to these authors, should facilitate future progress. The derivation of gene knockout procedures, however, represents the most pressing need, if maximum benefit is to be extracted from the recently completed genome sequence. In this light, it is interesting to note the recent study of Haraldsen and Sonenshein [55], who conjugated a Tn916 derivative carrying a copy of the C. difficile sigK gene from B. subtilis to strain C. difficile CD196. Unexpectedly, the derivatised transposons did not enter the chromosome at the preferred hotspot, rather it integrated through homologous recombination between the two copies of sigK. It will be interesting to see whether this system has general utility for the generation of knock-outs in C. difficile.

Acknowledgements We wish to thank Nicola Minion for typing this manuscript, and the financial support of the UK

Department of Health and the BBSRC (Grant No. 346/E13746). The nucleotide sequence of plasmid pCD6 has been deposited in GenBank under Accession number AY350745.

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