Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Penglais SY23 3DD, UK1
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France2
Université de Provence, Marseille, France3
Author for correspondence: Michael Young. Tel: +44 1970 622348. Fax: +44 1970 622307. e-mail: miy{at}aber.ac.uk
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ABSTRACT |
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Keywords: conjugation, electro-transformation, methylation, cellulosome
a Present address: Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes, UK.
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INTRODUCTION |
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Gene transfer to many species of Clostridium is now possible using a variety of methods, including protoplast transformation, electro-transformation and conjugation (reviewed by Young & Cole, 1993 ; Young et al., 1999
). Moreover, allelic replacement, which is essential for the analysis of gene function in vivo, has been achieved in three species, C. beijerinckii (Wilkinson & Young, 1994
), C. acetobutylicum (Green et al., 1996
) and C. perfringens (Awad et al., 1995
).
An ever-growing range of clostridia has been electro-transformed (Young et al., 1999 and references therein). The activity of host restriction enzymes remains a significant barrier to electro-transformation with double-stranded DNA. If the nature of the restriction system is known, transforming DNA may be protected by methylation in vivo or in vitro (Mermelstein & Papoutsakis, 1993
; Davis et al., 2000
). As an alternative to electro-transformation, the extremely broad-host-range conjugation machinery of IncP plasmids may be employed to mobilize plasmids from Escherichia coli to a wide range of Gram-positive bacteria (Trieu-Cuot et al., 1987
). Vectors containing the cis-acting oriT site (origin of transfer) of the IncP plasmid RK2 are mobilized if all other conjugation functions are provided in trans on an IncP plasmid or as part of the donor chromosome. This method was initially optimized for C. beijerinckii (Williams et al., 1990a
, b
) and it has since been employed for transferring DNA to other clostridia (Young et al., 1999
and references therein).
Conjugative transposons are useful tools for insertional mutagenesis and mutational cloning (Clewell & Flannagan, 1993 ; Young, 1993a
, b
; Scott & Churchward, 1995
). The most widely used representatives are the broad host-range streptococcal transposons, Tn916 (Franke & Clewell, 1981
) and Tn1545 (Courvalin & Carlier, 1987
). One or other of these elements has been transferred to a wide range of clostridia (Young et al., 1999
and references therein).
Although the number of clostridia that are amenable to genetic analysis is constantly increasing, there have been no reports so far of gene transfer to cellulolytic species. [Note, however, that Tn1545 has been transferred to Eubacterium cellulosolvens (Anderson et al., 1998 ), which is a very close relative of the clostridia.] We describe herein the development of conjugation and electro-transformation procedures for Clostridium cellulolyticum ATCC 35319, which we have employed to establish a range of plasmids as well as a conjugative transposon in this organism. They represent a first step towards functional analysis of the cellulosome in clostridio.
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METHODS |
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Filter mating procedure.
The optimized procedure was adapted from that of Williams et al. (1990a ) as follows. Donor strains of E. coli and Enterococcus faecalis were grown overnight, at 37 °C in aerobic conditions, to stationary phase in brain heart infusion broth (BHIB, Oxoid) supplemented with 50 µg ampicillin ml-1 and 10 µg erythromycin ml-1, selective for their mobilizable plasmid or conjugative transposon, respectively. The following morning, donor strains were diluted back ten-fold and regrown to OD600 >1·0 (about 5x108 c.f.u. ml-1). Alternatively, they were diluted with an equal volume of pre-warmed BHIB. Donor cells were washed twice in pre-warmed BHIB just before mating, to remove traces of the selective antibiotic. All manipulations involving the recipient, C. cellulolyticum ATCC 35319, were carried out at 34 °C in an anaerobic workstation (Don Whitley Scientific). The recipient was grown for 1724 h in GS medium to late exponential phase at OD600 0·5-1·0 (about 5x106 c.f.u. ml-1). The low number of c.f.u. recovered is due, in part, to a tendency to grow in filaments, but this organism also shows a low plating efficiency. Donor and recipient cells were mixed at a 10:1 ratio by pipetting 0·2 ml donor culture into 2 ml recipient culture. The bacteria were harvested by filtration through a nitrocellulose filter (Whatman WCN, pore size 0·45 µm, 25 mm diameter). Filters were incubated overnight at 34 °C, bacteria uppermost, on plates spread with 2 mg catalase (Sigma). For matings with E. coli donors, GS medium was used; for matings with En. faecalis donors, VM medium was used. Bacteria were harvested from the filters the following morning by vortex mixing in 0·5 ml holding buffer (1 mM MgSO4, 25 mM potassium phosphate, pH 7·0) and serial dilutions were prepared in this buffer. Viable counts of donor and recipient bacteria were obtained by plating on BHIB and either GS (for E. coli matings) or VM (for En. faecalis matings), respectively. Recipient bacteria were counter-selected by aerobic incubation whereas donor bacteria were counter-selected by incorporation of 10 µg trimethoprim ml-1 (E. coli) or the absence of a fermentable carbon source (En. faecalis). Transconjugants were selected anaerobically on the above media supplemented with 10 µg erythromycin ml-1. Transconjugants appeared 45 d after plating. Transfer frequencies were expressed as the number of transconjugants per recipient colony formed after the mating period.
E. coli donor strains were constructed by introducing the plasmids to be mobilized into a strain of HB101 in which the IncPß plasmid, R702 (Thomas & Smith, 1987 ), was already present.
Characterization of the restriction system.
Crude extracts of C. cellulolyticum were prepared from a 20 ml late-exponential-phase culture, using the method described by Azeddoug & Reysset (1991) . The soluble cellular extract was adjusted to 50% (v/v) glycerol and stored at -20 °C. For restriction assays, 350400 ng DNA substrate was mixed with 2·5 µl of crude extract (approx. 10 µg protein) and incubated for 2 h at 37 °C in 25 µl of 10 mM Tris/HCl, pH 7·5, buffer containing 50 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol, 0·01% BSA. The products were analysed by electrophoresis through agarose (1·2%, w/v). For methylation protection assays, HpaII methylase (M.HpaII) and MspI methylase (M.MspI) (New England Biolabs) were used according to the suppliers instructions. Methylated DNA was purified using QIAEX II (QIAGEN) before incubation with restriction endonucleases.
Electro-transformation procedures.
C. cellulolyticum was grown for 1724 h in 50 ml cultures in GS medium to late exponential phase, i.e. OD600 0·51·0 (about 5x106 c.f.u. ml-1). Bacteria were harvested by centrifugation in sealed tubes for 10 min at 6000 g and 4 °C in a Hettich EBA 12 R centrifuge. Cells were washed twice with 10 ml ice-cold electroporation buffer (270 mM sucrose; 1 mM MgCl2; 5 mM sodium phosphate buffer, pH 7·4) in the anaerobic chamber and resuspended in a final volume of 1 ml of this buffer. Plasmid DNA (12 µg) was added to pre-chilled electroporation cuvettes (Equibio, 0·2-cm inter-electrode distance) followed by 200 µl cell suspension, and the cuvettes were incubated on ice for 10 min.
In some experiments, bacteria were pulsed once at 1·5 kV, 25 µF and 100 using a Bio-Rad Gene Pulser electroporation apparatus. The resulting pulse duration was 1·92·0 ms. Fresh GS medium (1 ml) was added to the cuvette immediately after electroporation and the cell suspension was transferred to a further 1 ml GS for overnight incubation. The following morning, bacteria were harvested by centrifugation, resuspended in 200 µl GS and spread on two GS agar plates supplemented with 10 µg erythromycin ml-1. Transformation efficiencies were expressed as the number of transformants per µg DNA. Since an 18 h period was allowed for phenotypic expression, during which two to three bacterial generations may have occurred, the transformation frequencies are probably overestimated by between four- and eightfold.
In some experiments, a JOUAN PS15 electropulsator (JOUAN, France) was used. This apparatus delivers square wave pulses, which provide a constant electric field of pre-programmed duration and voltage. The mixture containing 109 cells and 1010 plasmid molecules (e.g. 80 ng pCTC1) was subjected to a 5-ms pulse at 6·5, 7, or 7·5 kV cm-1. The electroporated cells were incubated overnight in 10 ml fresh GS medium before plating on solid medium and transformation efficiencies were evaluated as described above. Alternatively, after an overnight phenotypic expression period without antibiotic, the electroporated cells were subcultured in 100 ml GS liquid medium supplemented with 10 µg erythromycin ml-1. Growth of the transformed bacteria was monitored by following the OD600. Apparent transformation efficiencies were derived by extrapolating the growth curves to zero time (assuming exponential growth throughout). This gave a theoretical initial OD600 for the transformed fraction of the bacterial population. This value was converted into the theoretical number of transformants at t0 (1 OD600 unit corresponds to a total count of 2·3x108 cells ml-1 culture), from which the apparent number of transformants per µg DNA was derived. (Note that apparent transformation efficiencies will be underestimated if there is a lag before growth commences.)
Before electroporation, plasmid DNA was purified from E. coli DH5 and then subjected to methylation in vitro using MspI methylase. In some experiments DNA was methylated in vivo using the Bacillus subtilis site-specific BsuFI methylase (Walter et al., 1990
; Davis et al., 2000
). Both of these enzymes methylate the sequence CCGG on the external cytosine. The gene encoding this enzyme was cloned in plasmid pACYC184 by T. Davis (CAMR, Porton Down), who kindly provided the resulting plasmid, pM.BsuFI, in the TOP10 strain of E. coli. This strain was transformed with the target plasmids. Total plasmid DNA was then extracted and used directly for electroporation experiments.
Molecular methods.
The methods employed for DNA extraction and manipulation were those described by Sambrook et al. (1989) . Large-scale plasmid extraction from E. coli was achieved using the QIAGEN Midi Prep Kit according to the manufacturers instructions. For small-scale plasmid extraction from C. cellulolyticum, 6 ml of an overnight culture was subjected to the extraction procedure described previously by Williams et al. (1990a
) for Clostridium beijerinckii NCIMB 8052. Total DNA was extracted using a method adapted from that of Noirot et al. (1987)
.
Methylases (New England Biolabs), restriction endonucleases (Promega, New England Biolabs) and T4 DNA ligase (Promega) were used according to the manufacturers instructions. DNA fragments were purified using the QIAEX agarose extraction kit (QIAGEN). Transformation of E. coli strains DH5, HB101 and TOP10 was carried out as described by Hanahan (1985)
.
Standard procedures were employed for Southern hybridizations (Southern, 1975 ). DNA fragments were transferred to a positively charged nylon membrane (Boehringer) and hybridized with digoxigenin-labelled probes (Boehringer). Plasmid pAT187 (Trieu-Cuot et al., 1987
), which contains an aphA-3 gene very similar to that of Tn1545 (Caillaud et al., 1987
), was employed to detect Tn1545.
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RESULTS |
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Transfer of Tn1545 to C. cellulolyticum by conjugation
In mating experiments with the En. faecalis BM4110 donor, which contains multiple copies of Tn1545 (Woolley et al., 1989 ), EmR transconjugants were obtained at frequencies of between 1·7x10-6 and 3·8x10-8 per recipient. DNA was extracted from two independent transconjugants, digested with HindIII, EcoRI and PstI and the presence of Tn1545 was verified by Southern hybridization. A Tn1545-specific probe hybridized with multiple HindIII fragments in DNA isolated from both transconjugants, whereas no signal was detected with wild-type DNA (Fig. 1
). Since HindIII does not cleave Tn1545 between the aphA-3 gene detected by hybridization and the left end of the element (Caillaud et al., 1987
), hybridizing bands should correspond to junction fragments containing the transposon end and adjacent clostridial DNA. The presence of multiple bands normally reflects multiple sites of transposon insertion into the bacterial chromosome. However, the various fragments in Fig. 1
are not all of equivalent intensity, suggesting that some of them may be partial digestion products. The digestions with EcoRI and PstI alone are not informative in this regard, but the samples digested with PstI plus HindIII show simplified hybridization patterns, indicating that Tn1545 has probably inserted into at least three different sites in the two strains (strain 2 probably harbours two copies of Tn1545).
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Transfer of plasmids to C. cellulolyticum by electro-transformation
The ability to protect DNA from C. cellulolyticum endonuclease activity by methylation opened up the possibility of using electro-transformation as a method for transferring DNA to C. cellulolyticum. Electro-transformation of appropriately modified DNA (see Methods) was employed to introduce plasmids from several different Gram-positive bacteria into C. cellulolyticum (Table 3). As expected from the conjugation experiments (see above), pCTC1 containing the pAMß1 replicon was successfully transferred. In addition, several other plasmids containing the replicons of the Bacillus subtilis plasmid, pIM13 (Monod et al., 1986
; Azeddoug et al., 1992
), the C. butyricum plasmid, pCB102 (Minton & Morris, 1981
; Collins et al., 1985
), the C. perfringens plasmid, pIP404 (Garnier & Cole, 1988
) and the Lactococcus lactis subsp. cremoris plasmid, pWV01 (Leenhouts et al., 1991
) also generated transformants. No transformants were obtained with plasmid pCTC511 containing the replicon of the C. butyricum plasmid, pCB101 (Collins et al., 1985
; Brehm et al., 1992
), nor were there any antibiotic-resistant colonies in controls lacking plasmid DNA.
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Transformants obtained using pIM13- and pAMß1-based plasmids were verified by extracting plasmid DNA from selected transformants and digesting it with diagnostic restriction endonucleases (Fig. 3). These data showed unequivocally that the pIM13 and pAMß1 replicons function in C. cellulolyticum. Extraction yields of pJIR418 (pIP404-based plasmid) and pGK12 (pWV01-based plasmid) from C. cellulolyticum were much lower than those of pIM13- and pAMß1-based plasmids, suggesting that these are maintained at lower copy numbers. This was not surprising, especially in the case of pGK12 which was maintained in Lactococcus lactis (formerly Streptococcus lactis) and Bacillus subtilis at three and five copies per cell, respectively (Kok et al., 1984
).
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DISCUSSION |
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Although the frequencies of conjugative plasmid transfer to C. cellulolyticum reported here were sometimes as high as 10-5 per recipient, reproducibility was poor. Previous experience (Williams et al., 1990a ) indicated that high-efficiency, reproducible conjugation only occurs under conditions permitting good growth of both donor and recipient as they are co-cultured during the mating period. Since E. coli is a copiotroph and C. cellulolyticum is essentially an oligotroph, co-culture was always likely to prove problematic. Moreover, the low plating efficiency of C. cellulolyticum effectively meant that many potential transconjugants were lost in each experiment (although this is unlikely to affect the conjugation frequency). Similar problems pertained to the conjugative transfer of Tn1545 from En. faecalis to C. cellulolyticum, where even fewer transconjugants were often recovered. They may also account for the low frequencies of transfer of Tn1545 to Eu. cellulosolvens that were reported recently (Anderson et al., 1998
). A single strand of DNA appears to be transferred during intergeneric conjugation (Young et al., 1993
). Therefore, DNA that is transferred by conjugation does not normally have to run the gauntlet of the restriction endonucleases that may be present in the recipient (see below). If an alternative oligotrophic donor in which IncP plasmids can replicate is identified in the future, higher numbers of recombinants and elevated transfer frequencies may be obtained. This may ultimately permit the use of conjugative transfer of non-replicative plasmids to effect allelic exchange.
Insertional mutagenesis using Tn1545 is similarly limited by low transfer frequencies from the copiotrophic En. faecalis donor. The element apparently inserted into three different sites in the C. cellulolyticum chromosome (Fig. 1). The possibility that there is a preferred site for insertion in this organism (cf. the behaviour of the related element, Tn916, in C. difficile: Mullany et al., 1991
) cannot be excluded until larger numbers of transconjugants have been analysed.
Electroporation proved to be a more reliable method for obtaining gene transfer, but only once the incoming DNA was protected from restriction. C. cellulolyticum ATCC 35319 protects its own DNA from restriction by methylation, and donor DNA that had been modified using an appropriate DNA methylase (M.BsuF1 or M.MspI [5'-m5CCGG-3']) gave reproducible electro-transformation. Vectors containing the replication functions of five different plasmids, originating from five different organisms, were able to function in C. cellulolyticum. Plasmids containing the replication regions of the B. subtilis ssDNA plasmid pIM13, the L. lactis ssDNA plasmid pWV01, the streptococcal theta-replicating plasmid pAMß1 (Bruand et al., 1993 ), the C. perfringens theta-replicating plasmid pIP404 (Garnier & Cole, 1988
) and pCB102 from C. butyricum, with its unknown mode of replication (Minton et al., 1993
), were established with approximately equal efficiency. Although pCB101 is a ssDNA plasmid like pIM13 and, like pCB102, it originates from C. butyricum, we were unable to establish vectors (pCTC501 and pCTC511) containing this replicon in C. cellulolyticum, using either conjugation or electro-transformation.
The apparent transformation efficiencies for the various plasmids were rather low (approx. 102 transformants per µg DNA, see Table 3) when transformants were selected on solid medium. The reason for this seems to be the low plating efficiency of this strain. Substantially higher apparent efficiencies were obtained when a non-conventional method was employed to derive the number of transformants originally present. Essentially, the growth kinetics of transformed bacteria in liquid medium were measured and used to extrapolate the number of transformants initially present. These results open up the possibility of effecting allelic replacement and insertional inactivation of genes in C. cellulolyticum, using suicide plasmids. Stepwise gene replacement using replicative, but segregationally unstable vectors (Maguin et al., 1992
; Biswas et al., 1993
) could also provide a useful alternative. We therefore explored the segregational stability of three representative plasmids in C. cellulolyticum.
Plasmids that have been transferred to an organism in which they do not normally reside are frequently unstable (Gruss & Ehrlich, 1988 ; Ehrlich et al., 1991
; Minton et al., 1993
). This instability may result from structural changes or from segregation of plasmid-less cells owing to inefficient plasmid replication and/or partitioning at cell division (Gruss & Ehrlich, 1989
). Instability of plasmids containing two of the replicons employed here has previously been reported in other clostridia (Oultram et al., 1988
; Azeddoug et al., 1992
). Frequencies of segregation of plasmid-less cells were 4·1x10-2 per generation for a pAMß1-based plasmid in C. beijerinckii (Oultram et al., 1988
) and 2·3x10-2 per generation for a pIM13-based plasmid in Clostridium saccharoperbutylacetonicum NI-4081 (Azeddoug et al., 1992
). In C. cellulolyticum however, both the pAMß1 (theta-replication) and the pIM13 (rolling circle, ssDNA plasmid) replicons were moderately stable in the absence of selection and segregated plasmid-less cells at frequencies of about 5x10-3 per generation. In view of the behaviour of pIM13-based plasmids in other organisms (Azeddoug et al., 1992
), the comparative stability of plasmids containing this replicon was unexpected. To explore the phenotypic consequences of disruption of cipC, which encodes the scaffoldin component of the C. cellulolyticum cellulosome, several derivatives of the pIM13-based plasmid, pECII, (e.g. pECN2) were constructed with the intention of obtaining cipC disruption mutants of C. cellulolyticum. The comparative stability of these plasmids in this organism prevented us from pursuing this line of enquiry. In view of their reduced stability, it might be provident to employ pCB102-based plasmids for this purpose in the future.
The results we have obtained demonstrate that C. cellulolyticum ATCC 35319 is amenable to genetic analysis. Significant difficulties still to be circumvented are the low frequency of conjugative gene transfer, which is probably related to the oligotrophic nature of this organism, and the low plating efficiency. This makes it difficult to recover cells stressed by conjugative transfer, electroporation, or possibly even the presence of a plasmid per se and we strongly suspect that most potential transformants/transconjugants are lost when bacteria are plated on agar-solidified medium. Reliable electro-transformation, in combination with a segregationally unstable replicon, such as that of pCB102, may yet provide a way of obtaining detectable homologous recombination and thus facilitate functional analysis of the bacterial cellulosome in vivo. It has recently been shown that UV irradiation of plasmid DNA enhances homologous recombination with the Mycobacterium tuberculosis chromosome (Hinds et al., 1999 ). It will be of interest to determine whether a similar effect is observed with C. cellulolyticum.
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ACKNOWLEDGEMENTS |
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Received 22 June 2000;
revised 3 August 2000;
accepted 30 August 2000.