You will be shown a paragraph from a biomedical research paper, followed by a question about the paper. Please extract text from the paragraph to answer the question. Do not include any text in your repsonse other than the answer.

Paragraph: Its main virulence factors are the major clostridial toxins A and B  [29, 30] . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin [31, 32] . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow [24, 25] . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores [33] and the gene is required for persistence and transmission in mice [34] . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A [2] . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A [35] . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum [36] . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter [37] . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes. Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta(DE3) pLysS (Novagen). C. difficile strains were grown in a glucose-free trypton-yeast based medium (TTY; 3% w/v bactotrypton (BD), 2% yeast extract (Fluka), 0.1% w/v thioglycollate (Sigma) pH 7.4), supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates (Biomerieux). All plasmids are listed in Table 1 . Primers (obtained from Sigma Aldrich) are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase (Fermentas) according to the instructions of the manufacturer. Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO (Invitrogen), yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE (20 mM Tris Acetate, 0.5 mM EDTA) gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation (using a GeneJET Gel Extraction kit, Fermentas) and cloned into similarly digested pMF14 [10] that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 (see below). Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 (see below). Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit (Macherey Nagel) according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry (Invitrogen) on an ABI3130 sequencer (Perkin Elmer), according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix (Invitrogen) in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid (Invitrogen) at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 (SciEd) and Geneious version 5.6.2 (Biomatters Ltd). Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta(DE3) pLysS (Novagen). Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described [10] . In short, cells were disrupted in 4 mL lysis buffer (2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry (Clontech) in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column (BioRad) and washed with 2 mL wash buffer (20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9). The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column (identical to wash buffer but with 50, 100, 250 or 500 mM imidazole). The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT) using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa (Pierce). Proteins were stored at 280uC in storage buffer (identical to dialysis buffer but containing 20% glycerol). Protein concentrations were determined using Bradford reagent (BioRad), according to the manufacturer's instructions. DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase (Promega) and chromosomal DNA from B. subtilis JH642 (Bacillus Genetic Stock Center 1A96; http://www.bgsc.org), plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm [38] as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS) and a QIAExII kit (Qiagen), according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase (Invitrogen) according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter (Beckman). EMSA conditions were based on previous studies [10] . In short, binding reactions were carried out in binding buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol) in the presence of 200 mg/mL bovine serum albumin (NEB) and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer. Electrophoresis was carried out for 120 min at 85 V. After vacuum drying the gels onto filter paper, they were imaged after overnight exposure on Phosphorimager screens on a Typhoon instrument (GE Healthcare). The toxic effects of C. difficile culture supernatants on Vero cells (a kind gift of Eric Snijder [39] ) were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium (Dulbecco modified Eagle medium (Lonza) supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum), before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin (Techlab) was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB (Techlab) was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects (cell rounding) were observed. Statistical significance was evaluated with an independent sample t-test. Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute (Hinxton, UK). Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer (10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC (Roche)). After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer (0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB) was added, and samples were heated to 96uC for 5 mins. Total cell lysates (amounts corrected for OD 600 ) were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in PBST buffer (phosphate buffered saline with 0.1% v/v Tween-20) containing 5% membrane blocking reagent (Amersham Biosciences). To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection (Amersham Bioscience), or a goatanti-mouse-biotin-conjugated secondary antibody (Dako) followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody (Jackson). Detection was done using on a Typhoon instrument (GE Healthcare). Background corrected peak volumes were quantified using ImageQuant TL (Amersham Biosciences). Alignments of B. subtilis and C. difficile spo0A were made using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) on the basis of the published genome sequences, Genbank accession numbers AL009126 and AM180355, respectively, and the 630Derm spo0A sequence as determined in this study. The sequence for spo0A of C. difficile strain 630Derm was deposited in Genbank (accession no JX050222). Consensus Spo0A boxes were identified using a Single string Search command in Genome2D [40] , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel. The results were manually inspected for those boxes within 500 bp upstream of a gene on the same strand. Figures for publication were prepared using ImageQuant TL (Amersham Biosciences), Adobe Photoshop CS3 (Adobe Systems Inc) and Corel Graphics Suite X5 (Corel Corporation). In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain (DBD) were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli (Fig. 1A) and purified to near homogeneity using metal affinity chromatography ( Fig. 1A ; lanes P).

Question: What are the main virulence factors in C. difficle?
toxins A and B