Conditional vectors and uses thereof

Abstract

The present invention now provides a conditional vector comprising DNA encoding for: (i) an inducible expression cassette comprising an inducible promoter operably linked to a plasmid replication region; and (ii) a selectable marker.

Claims

1. A conditional vector comprising DNA encoding for: (i) a genetically engineered inducible expression cassette comprising an inducible promoter operably linked to a plasmid replication region, wherein the inducible promoter and expression cassette are heterologous, and wherein the plasmid replication region consists of a sequence of SEQ ID NO: 7; and (ii) a selectable marker.

2. The conditional vector of claim 1, further comprising a transposable element.

3. The conditional vector of claim 2, wherein the transposable element is the mini-transposon, Himar1C9.

4. The conditional vector of claim 1, further comprising a transposase operably linked to a promoter.

5. The conditional vector of claim 1, wherein the inducible promoter is selected from a P.sub.fac, P.sub.fet or P.sub.xylA promoter.

6. The conditional vector of claim 1, wherein the selectable marker encodes resistance to erythromycin, tetracycline, spectinomycin or thiamphenicol.

7. The conditional vector of claim 1, further comprising a group 5 RNA polymerase sigma factor.

8. The conditional vector of claim 7, further comprising a promoter recognised by the group 5 RNA polymerase sigma factor.

9. The conditional vector of claim 7, wherein the group 5 RNA polymerase sigma factor is TcdR.

10. The conditional vector of claim 8, wherein the promoter recognised by the group 5 RNA polymerase sigma factor is tcdA or tcdB.

11. A method of delivering a transposon into a bacterial host genome comprising introducing the conditional vector of claim 1 into a bacterial cell.

12. A method of delivering a transposon into Clostridia comprising contacting the conditional vector of claim 1 to Clostridia.

13. A bacterial cell comprising a conditional vector of claim 1.

14. The bacterial cell of claim 13, wherein the bacterial cell is selected from C. acetobutylicum, C. difficile, C. beijerinckii, C. ljungdahlii, C. kluyveri, C. botulinum, C. autoethanogenum, C. saccharobutylicum, C. carboxidovorans, C. sporogenes, C. phytofermentans, C. ragsdalei, C. tyrobutyricum, C. perfringens, C. butyricum, C. cellulolyticum, C. formicaceticum, C. novyi, C. scatologenes, C. septicum, C. sordellii, C. sticklandii, C. tetani, C. thermocellum, C. thermosaccharolyticum, C. paprosolvens, C. scindens, C. pasteuranium or C. bifermentans.

15. The bacterial cell of 14, wherein the bacterial cell is C. acetobutylicum.

Description

(1) Preferred embodiments of the present invention will now be described, merely by way of example, with reference to the following drawings and examples.

(2) FIG. 1—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 16), of the IPTG inducible expression vector pMTL-YZ006.

(3) Key: ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; repH, replication region of the Clostridium butyricum plasmid pCB102; catP, encoding chloramphenicol acetyltransferase, isolated from plasmid pC194; Pfac, the promoter of the Clostridium pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; traJ, transfer function of the RP4 oriT region; Pptb, the promoter of the Clostridium beijerinckii gene encoding phosphotransbutyrylase; LacI, the E. coli gene encoding LacI repressor; ColE1, the replication origin of plasmid ColE1, and; bla, the pBR322 gene encoding beta-lactamase

(4) FIG. 2—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 19), of the IPTG inducible expression vector pMTL-YZ007.

(5) Key: T2, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene; LacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the Clostridium beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the Clostridium pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; catP, encoding chloramphenicol acetyltransferase, isolated from plasmid pC194; T1, transcriptional terminator of the ferredoxin gene of Clostridium pasteurianum; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region.

(6) FIG. 3—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 20), of the lac-based, IPTG inducible expression cassette.

(7) Key: LacI is the LacI repressor protein gene. LacI binds to the indicated lacO region, blocking transcription from the P.sub.fac promoter. The −35 and −10 regions of the P.sub.fac promoter are underlined in the illustrated sequence. The P.sub.ptb promoter (derived from the Clostridium beijerinckii gene encoding phosphotransbutyrylase) directs the transcription of the LacI gene.

(8) FIG. 4—shows IPTG induction of CAT production in cells harbouring pMTL-YZ007. Triangles equate to cells which received no IPTG, squares represents samples from cells that were induced with IPTG. Activity is expressed as units of CAT activity per mg or soluble protein.

(9) FIG. 5—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 21), of the Non-Conditional vector pMTL-YZ008.

(10) Key: LacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the Clostridium beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the Clostridium pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; T1, transcriptional terminator of the ferredoxin gene of Clostridium pasteurianum; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region.

(11) FIG. 6—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 22), of the IPTG-induced Conditional vector pMTL-YZ009.

(12) Key: LacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the Clostridium beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the Clostridium pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region.

(13) FIG. 7—shows the stability of pMTL-YZ008 and pMTL-YZ009 in the presence of IPTG. Transformed cells of Clostridium acetobutylicum ATCC 824 containing either pMTL-YZ008 or pMTL-YZ009 were plated at serial dilutions onto CGM agar with and without IPTG (1 mM) and the Colony Forming Units (CFU) estimated.

(14) FIG. 8—shows the effect of IPTG on growth rate of cells harbouring pMTL-YZ008 and pMTL-YZ009. Transformed cells of Clostridium acetobutylicum ATCC 824 containing either pMTL-YZ008 or pMTL-YZ009 were cultured from 5% overnight inoculum in CGM broth with and without IPTG (1 mM). The culture's OD600 were measured at time points of 0, 2 h, 4 h, 6 h, 8 h, 10 h and 24 h.

(15) FIG. 9—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 23), of the tet-based, aTc inducible expression cassette.

(16) Key: TetR is the TetR repressor protein gene. TetR binds to the indicated tetO region, blocking transcription from the P.sub.fet promoter. The −35 and −10 regions of the P.sub.fet promoter are underlined in the illustrated sequence. The P.sub.thl promoter (derived from the Clostridium acetobutylicum gene encoding thiolase) directs the transcription of the TetR gene.

(17) FIG. 10—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 24), of the Vector pMTL-tet3nO carrying the aTc-inducible expression cassette.

(18) Key: T2, transcriptional terminator descented downstream of the Clostridium difficile strain 630 CD0164 gene; TetR, synthetic repressor gene encoding the repressor protein TetR of plasmid R100; P.sub.thl, promoter of the Clostridium acetobutylicum thiolase gene (thl), P.sub.fet, a derivatised P.sub.fdx promoter of the Clostridium sporogenes ferredoxin gene containing a tetO operator sequence; catP, a Clostridium perfringens-derived gene encoding chloramphenicol acetyltransferase; T1, transcriptional terminator of the Clostridium pasteurianum ferredoxin gene; repA and orf2, replication region of the Clostridium botulinum plasmid pBP1; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1, and; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region.

(19) FIG. 11—shows aTc induction of CAT production in cells harbouring pMTL-tet3nO. Circles equate to cells which received no aTc, squares represents samples from cells that were induced with aTc. Activity is expressed as units of CAT activity per mg or soluble protein.

(20) FIG. 12—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 25), of the Non-Conditional vector pMTL-YZ010.

(21) Key: T2, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene; TetR, synthetic repressor gene encoding the repressor protein TetR of plasmid R100; P.sub.thl, promoter of the Clostridium acetobutylicum thiolase gene (thl), P.sub.fet, a derivatised P.sub.fdx promoter of the Clostridium sporogenes ferredoxin gene containing a tetO operator sequence; T1, transcriptional terminator of the ferredoxin gene of Clostridium pasteurianum; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region.

(22) FIG. 13—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 26), of the aTc-induced Conditional vector pMTL-YZ011.

(23) Key: T2, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene; TetR, synthetic repressor gene encoding the repressor protein TetR of plasmid R100; P.sub.th, promoter of the Clostridium acetobutylicum thiolase gene (thl), P.sub.fet, a derivatised P.sub.fdx promoter of the Clostridium sporogenes ferredoxin gene containing a tetO operator sequence; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region.

(24) FIG. 14—shows the stability of pMTL-YZ010 and pMTL-YZ011 in the presence of aTc. Transformed cells of Clostridium difficile strain 630 containing either pMTL-YZ010 or pMTL-YZ011 were plated at serial dilutions onto BHIS agar with and without aTc (300 ng per ml) and the Colony Forming Units (CFU) estimated.

(25) FIG. 15—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 27), of the modular vector, pMTL-YZ012, carrying the IPTG-based Conditional Plasmid replication region.

(26) Key: T2, transcriptional terminator descented downstream of the Clostridium difficile strain 630 CD0164 gene; LacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the Clostridium beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the Clostridium pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; T1, transcriptional terminator of the ferredoxin gene of Clostridium pasteurianum; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; ColE1, the replication origin of plasmid ColE1, and; traJ, transfer function of the RP4 oriT region.

(27) FIG. 16—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 30), of the Conditional mariner Transposon Vector pMTL-YZ013.

(28) Key: LacI, the E. coli gene encoding LacI repressor; Pptb, the promoter of the Clostridium beijerinckii gene encoding phosphotransbutyrylase; Pfac, the promoter of the Clostridium pasteurianum ferredoxin gene derivatised to include an E. coli lac operator; T1, transcriptional terminator of the ferredoxin gene of Clostridium pasteurianum; repH, replication region of the Clostridium butyricum plasmid pCB102; ermB, the macrolide-lincosamide-streptogramin B antibiotic resistance gene of plasmid pAMβ1; ColE1, the replication origin of plasmid ColE1; traJ, transfer function of the RP4 oriT region; IR1 & IR2, inverted repeat regions flanking the mini-transposon element; T1, transcriptional terminator of the Clostridium pasteurianum ferredoxin gene; catP, a Clostridium perfringens-derived gene encoding chloramphenicol acetyltransferase, Himar1 C9, mariner transposase gene, and; po, the promoter region of the Clostridium difficile tcdB gene.

(29) FIG. 17—shows a schematic view, and the nucleotide sequence (SEQ ID NO: 31), of the plasmid pMTL-ME6c

(30) Key: LHA, left hand homology arm encompassing a foreshortened Clostridium acetobutylicum pyrE gene lacking its first 13 codons; LacZ′, incorporating multiple cloning sites; T1, transcriptional terminator of the Clostridium pasteurianum ferredoxin gene; RHA, right hand homology arm composed of 1200 bp from immediately downstream of the Clostridium acetobutylicum pyrE gene, encompassing the hydA gene, CAC0028; RepL, the replication protein of plasmid pIM13; catP, a Clostridium perfringens-derived gene encoding chloramphenicol acetyltransferase, and; ColE1, the replication origin of plasmid ColE1.

(31) FIG. 18—shows a schematic representation of the genome of Clostridium acetobutylicum strain CRG3011. A promoter-less copy of the tcdR gene (including its ribosome binding site, RBS) of Clostridium difficile strain 630 has been inserted into the genome using ACE technology immediately downstream of the pyrE gene (CAC0027). Illustrated are the surrounding genes, and the position of the promoter responsible for expression of tcdR, and the position of the tcdR RBS. The illustrated terminator is the T1 transcriptional terminator of the Clostridium pasteurianum ferredoxin gene.

(32) FIG. 19—shows agarose gel electrophoresis of the inverse PCR DNA fragments generated from the chromosome of 31 randomly selected putative transposon mutants. Genomic DNA from 31 individual transposon mutants was isolated and digested with HindIII restriction endonuclease and the resultant DNA circularised by subsequent incubation with T4 DNA ligase. Lane M, Molecular Weight marker; lane C, wild-type C. acetobutylicum CRG3011, and; lanes 1 to 31, pMTL-YZ013-derived Thiamphenicol resistant clones 1 to 31.

(33) FIG. 20—shows the location of the different transposon insertions around the Clostridium acetobutylicum ATCC 824 genome. Twenty-five independent transposon insertions were sequenced. Insertions in the plus orientation are marked on the circle exterior. Insertions in the minus orientation are marked on the circle interior. Numbers indicate the precise point of insertion according to genome sequence data for C. acetobutylicum ATCC 824 (GenBank Accession Number AE001437.1)

(34) FIG. 21—shows the stability of pMTL-YZ008 and pMTL-YZ009 in the presence of IPTG. Transformed cells of Clostridium sporogenes NCIMB 10696 containing either pMTL-YZ008 or pMTL-YZ009 were plated at serial dilutions onto TYG agar with and without IPTG (1 mM) and the Colony Forming Units (CFU) estimated.

(35) FIG. 22—shows the stability of pMTL-YZ008 and pMTL-YZ009 in the presence of IPTG. Transformed cells of Clostridium botulinum ATCC 3502 containing either pMTL-YZ008 or pMTL-YZ009 were plated at serial dilutions onto TYG agar with and without IPTG (1 mM) and the Colony Forming Units (CFU) estimated.

(36) FIG. 23—shows agarose gel electrophoresis of the inverse PCR DNA fragments generated from the chromosome of 16 randomly selected putative transposon mutants in Clostridium sporogenes strain CRG3844. Genomic DNA from 16 individual transposon mutants was isolated and digested with HindIII restriction endonuclease and the resultant DNA circularised by subsequent incubation with T4 DNA ligase. Lane M, Molecular Weight marker; lanes 1 to 16, pMTL-YZ013-derived Thiamphenicol resistant transposon mutant clones 1 to 16.

CONSTRUCTION OF AN INDUCIBLE EXPRESSION CASSETTE BASED ON A LAC OPERATOR

(37) An IPTG-inducible expression system has previously been described (Heap J T et al., Journal of Microbiological Methods, 2007, 703: 452-64), and forms part of the plasmid pMTL5401Fcat as described therein. Plasmid pMTL5401Fcat was modified so that the promoter of the Clostridium pasteurianum ferredoxin included a lac operator sequence, inserted immediately downstream of the +1 transcriptional initiation site. Transcription from this promoter (termed the P.sub.fac promoter) could be repressed by the presence of the LacI protein, which was produced from a vector-borne copy of the lacI gene, which had been placed under the transcriptional control of the P.sub.ptb promoter of the Clostridium beijerinckii phosphotransbutyrylase gene (ptb). In vector pMTL5401Fcat, a promoter-less copy of the pC194 cat gene encoding chloramphenicol acetyltransferase (CAT) was placed downstream of the P.sub.fac promoter. Production of CAT was therefore induced in cells harbouring pMTL5401Fcat when IPTG was added.

(38) In order to test the ability of this inducible promoter system to control the replication of a plasmid, the control of the expression of the plasmid replication region was tested. That is replication would be dependent on the presence of IPTG (the permissive condition) and then transference of the cells to media lacking inducer (the non-permissive condition) would result in a cessation of plasmid replication and therefore plasmid loss.

(39) To test this hypothesis, it was desirable to locate the inducible system to a portable cassette which may be an inducible expression cassette comprising an inducible promoter that could be subsequently positioned 5′ to the plasmid replication region of any particular plasmid replication region. In vector pMTL5401Fcat, the ptb::lacI element and the P.sub.fac promoter were physically separated by a gene encoding the transfer function traJ and oriT sequence. As the presence of this gene and oriT region was not necessary, a strategy was devised whereby traJ was removed. Accordingly, a deletion was made between position 4058 bp and 5040 bp of pMTL5041Fcat using QuikChange II Site-Directed Mutagenesis Kit (Stratagene).

(40) Primers del (5′-CTCGATCCGGGGAATTCTCTGCAGATAATTCAGGG-3′) (SEQ ID NO: 14) and del-antisense (5′-CCCTGAATTATCTGCAGAGAATTCCCCGGATCGAG-3′) (SEQ ID NO:15) were designed, and PCR reactions were carried out according to manufacturer's instructions. PCR products were digested by enzyme DpnI to eliminate template plasmids at 37° C. for an hour, and then transformed into E. coli XL-1 Blue. Plasmids extracted from E. coli XL-1 Blue were confirmed via Sanger Sequencing. The resulting plasmid was designated pMTL-YZ006 as shown in FIG. 1 (SEQ ID NO:16).

(41) Following deletion of the traJ region, the lac inducible promoter cassette (P.sub.ptb::lacI element and the P.sub.fac promoter) was then PCR amplified using primers YZ4 (5′-TTTATATAGCGGCCGCGCTCACTGCCCGCTTT-3′) (SEQ ID NO:17) and YZ35 (5′-GTGCCAAGCTTGCATGCCATGGTA-3′) (SEQ ID NO: 18) (which included the emboldened restriction enzyme recognition sites 5′-GCGGCCGC-3′ and 5′-AAGCTT-3′, respectively). The amplified DNA fragment was cleaved with Not I and Hind III and the sticky-end fragment generated cloned into pMTL83251 as described in (Heap et al., Journal of Microbiological Methods, 2009, 78: 79-85) between the NotI and HindIII sites. The plasmid created was designated pMTL-YZ007 as shown in FIG. 2 (SEQ ID NO:19). A schematic diagram of the inducible expression cassette is shown in FIG. 3, and its complete nucleotide sequence corresponds to SEQ ID NO:20.

(42) In order to test that the inducible system was still functioning as expected, plasmid pMTL-YZ007 was transformed into Clostridium acetobutylicum ATCC 824 and cells anaerobically cultivated at 37° C. in 25 ml of CGM medium (Hartmanis M G N and Gatenbeck S, 1984, Applied Environmental Microbiology, 47: 1277-1283) supplemented with erythromycin (40 μg per ml). Two duplicate cultures were set up. Cells were grown to an OD.sub.600 of 0.6 at which point IPTG was added to one culture (final concentration 1 mM), whereas the duplicate culture received no inducer. Cultivation of the culture continued, and 2 ml samples were withdrawn at regular intervals followed by centrifugation and resuspension of cell pellet in 1 ml 100 mM Tris-HCl (pH 7.8). Cell lysate was achieved by sonication, and the level of expression of CAT determined according to the method of Shaw (Shaw, W. V., 1975, Methods in Enzymology, 43:737-755). The assay mixture contained 100 mM Tris-HCl (pH 7.8), 0.1 mM acetyl-coenzyme A and 0.4 mg 5,5′-dithiobis-2-nitrobenzoic acid (DTNB)/ml, and was equilibrated to 37° C. before use. Cell lysate (10 μl) and 5 mM chloramphenicol in 100% ethanol (10 μl) were added to 980 μl assay mixture in a plastic cuvette and the Absorption at 412 nm was measured for 1 min using an AnalytikJena Specord 250 spectrophotometer. Protein concentration of cell lysate was determined by Thermo Scientific NanoDrop 2000 Spectrophotometer.

(43) The level of CAT expression achieved in the two cultures is shown in FIG. 4. These data clearly show that IPTG induction of CAT production is occurring in cells carrying plasmid pMTL-YZ007.

(44) Derivation of a pCB102-Based Conditional Vector, pMTL-YZ009, Using the P.sub.fac-Based Inducible Promoter

(45) Having established the functionality of the P.sub.fac inducible promoter, the catP gene was deleted from plasmid pMTL-YZ007, in order to bring the pCB102 plasmid replication region under the transcriptional control of P.sub.fac. Two plasmids were created. In the one (pMTL-YZ008 as shown in FIG. 5, SEQ ID NO:21), pMTL-YZ007 was cleaved with BamHI and HindIII, the sticky-ends created blunt-ended by treatment with T4 polymerase, and the resultant linear fragment subjected to self-ligation. In a second plasmid (pMTL-YZ009, as shown in FIG. 6, SEQ ID NO: 22) pMTL-YZ007 was digested with BamHI and AscI, the sticky-ends created blunt-ended by treatment with T4 polymerase, and the resultant linear fragment subjected to self-ligation.

(46) The essential difference between the two plasmids is that pMTL-YZ008 carries a transcriptional terminator (that of the ferredoxin gene of Clostridium pasteurianum) between the P.sub.fac promoter and the repH replication gene of the pCB102 plasmid replication region. This terminator was deleted in pMTL-YZ009.

(47) The two plasmids were introduced by electroporation into Clostridium acetobutylicum ATCC 824 and their ability to replicate tested in the presence or absence of the inducer IPTG in CGM media lacking any antibiotic supplementation. Unexpectedly, plasmid pMTL-YZ009 was found to only be stably maintained in the absence of IPTG as shown in FIG. 7. In the presence of IPTG (1 mM), the plasmid was rapidly lost as shown in FIG. 7, as evidenced by an almost complete loss of Colony Forming Units (CFU) on plates supplemented with erythromycin (40 μg per ml). A similar loss was not evident in cells harbouring pMTL-YZ008 (FIG. 13). It was concluded that transcriptional readthrough into the pCB102 plasmid replication region was interfering with plasmid replication/maintenance.

(48) Construction of an Inducible Expression Cassette Based on a Tet Operator

(49) In parallel, an equivalent system was constructed based on the Tet system. The system is broadly equivalent to the lac system. Thus the promoter of the tetA gene contains an operator sequence to which the TetR (equivalent to LacI) repressor protein binds. Repression is lifted through binding of tetracycline antibiotic (Tc) to TetR, which causes a conformational change, releasing it from the operator sequence. As Tc can inhibit growth, the Tc analogue anhydrotetracycline (aTc) is commonly used as a replacement. It has higher affinity for TetR compared to Tc, but has a decreased toxicity.

(50) This system has been used in a number of bacteria, including mycobacteria and staphylococcus (Corrigan, R. M. & Foster, T. J. 2009 Plasmid 61, 126-129; Ehrt, S. et al., 2005 Nucleic Acids Res 33, e21, doi:10.1093/nar/gni013). To develop an equivalent system for Clostridia, it was elected to derivatise the P.sub.fdx promoter of the Clostridium sporogenes ferredoxin gene through the incorporation of the requisite Tet operator sequence. Accordingly, we designed and had synthesised a prototype system, based loosely on the system described previously Corrigan, R. M. & Foster, T. J. 2009. Plasmid 61, 126-129. A schematic of the cassette constructed is shown in FIG. 9 (SEQ ID NO: 23). It comprises a TetR gene under the control of the P.sub.thl promoter of the Clostridium acetobutylicum thiolase gene (thl), a derivatised P.sub.fdx promoter of the Clostridium sporogenes ferredoxin gene and a TetR gene that encodes the same TetR protein as that carried by the E. coli plasmid R100 (GenBank Accession NC_002134.1), but the codons have been altered to match those generally found in Clostridium difficile.

(51) The 1249 bp synthetic sequence was introduced into the modular vector pMTL82254 (Heap et al., Journal of Microbiological Methods, 2009, 78: 79-85) as an NdeI—NotI restriction fragment between the equivalent sites of pMTL82254 to yield the plasmid pMTL-tet3nO (FIG. 10, SEQ ID NO: 24). Plasmid pMTL82254 carries a promoter-less copy of the catP gene isolated from Clostridium perfringens. Accordingly, the inducible cassette was inserted such that the P.sub.fet promoter was positioned immediately upstream of this gene. To test the functionality of the inducible system, plasmid pMTL-tet3nO was transformed into Clostridium difficile strain 630 and cells anaerobically cultivated at 37° C. in 100 ml of BHIS medium (brain heart infusion media supplemented with yeast extract [5 mg/ml, Oxoid]) supplemented with erythromycin (10 μg per ml). Two duplicate cultures were set up. Cells were grown to an OD.sub.600 of 0.6 at which point aTc was added to one culture (final concentration 316 ng per ml), whereas the duplicate culture received no inducer. Cultivation of the culture continued, and samples were withdrawn at regular intervals. At each time point, sample culture was normalized to a 10 ml equivalent of OD.sub.600 1.0, followed by centrifugation and resuspension of cell pellet in 1 ml 100 mM Tris-HCl (pH 7.8). Cell lysate was achieved by sonication, and the level of expression of CAT determined according to the method of Shaw (Shaw, W. V. 1975, Methods in Enzymology, 43:737-755.). The assay mixture contained 100 mM Tris-HCl (pH 7.8), 0.1 mM acetyl-coenzyme A and 0.4 mg 5,5′-dithiobis-2-nitrobenzoic acid (DTNB)/ml, and was equilibrated to 37° C. before use. Cell lysate (10 μl) and 5 mM chloramphenicol in 100% ethanol (10 μl) were added to 980 μl assay mixture in a plastic cuvette and the Absorption at 412 nm was measured for 1 min using an AnalytikJena Specord 250 spectrophotometer.

(52) The level of CAT expression achieved in the two cultures is shown in FIG. 11. The data show that aTc induction of CAT production is occurring in cells carrying plasmid pMTL-tet3nO, and establishes that the system is functional.

(53) Derivation of a pCB102-Based Conditional Vector, pMTL-YZ011, Using the P.sub.fet-Based Inducible Promoter

(54) Having demonstrated that the P.sub.fac promoter could be used to control plasmid replication based on the pCB102 plasmid replication region, it was determined whether the P.sub.fet promoter could be similarly employed. This is important, because the P.sub.fac promoter might not be applicable to all members of the Class Clostridia. Indeed, the lac system does not function in Clostridium difficile, most likely due to failure of the IPTG inducer to enter the cell.

(55) An equivalent plasmid to pMTL-YZ008 was therefore made in which the P.sub.fac-based inducible expression cassette was replaced with the P.sub.m-based inducible expression cassette. This was accomplished by isolating the P.sub.fet promoter/P.sub.thl::tetR cassette as a 1249 bp fragment from plasmid pMTL-tet3nO following cleavage with NotI and NdeI. Plasmid pMTL-YZ008 was then cleaved with the same enzymes, excising the P.sub.fac/P.sub.ptb::lacI cassette, allowing the P.sub.fet promoter/P.sub.thl::tetR cassette to be inserted in its place. The plasmid created was designated pMTL-YZ010 (FIG. 12, SEQ ID NO: 25). In common with pMTL-YZ008, the Clostridium pasteurianum Fd terminator is positioned between the inducible promoter and the pCB102 plasmid replication region. In parallel, and equivalent vector to pMTL-YZ009 was made (plasmid pMTL-YZ011, FIG. 13, SEQ ID NO: 26) in which the same cloning strategy was used to replace the P.sub.fac/P.sub.ptb::lacI cassette with the P.sub.fet/P.sub.thl::tetR using the NotI and NdeI restriction sites. It follows that in pMTL-YZ011, there is no transcriptional terminator between the P.sub.fet promoter and the pCB102 plasmid replication region.

(56) The two plasmids were introduced by conjugation (Purdy D et al., 2002, Molecular Microbiology, 46: 439-52) into Clostridium difficile strain 630 and their ability to replicate tested in the presence or absence of the inducer aTc in BHIS medium (brain heart infusion media supplemented with yeast extract [5 mg/ml, Oxoid]). In keeping with the result with pMTL-YZ009, plasmid pMTL-YZ011 was found to only be stably maintained in the absence of aTc. In the presence of aTc (200 ng per ml), the plasmid was rapidly lost, as evidenced by an almost complete loss of CFU on plates supplemented with erythromycin (10 μg per ml). A similar loss was not evident in cells harbouring pMTL-YZ010. As demonstrated in FIG. 14, the data confirmed that transcriptional readthrough into the pCB102 plasmid replication region interferes with plasmid replication/maintenance.

(57) Use of the Conditional Vector, pMTL-YZ013, for Transposon Delivery

(58) The inducer-mediated loss of plasmids such as pMTL-YZ009 and pMTL-YZ011 from Clostridial cells could potentially allow the conditional delivery of a transposon element. To test this possibility, a derivative of the mariner transposon vector pMTL-SC1 (Cartman S T and Minton N P, 2010, Applied Environmental Microbiology, 76: 1103-1109) was constructed. To achieve this, a conditional replicon cassette was constructed, essentially by locating the P.sub.fac/P.sub.ptb::lacI cassette plus the pCB102 plasmid replication region to a portable AscI-FseI fragment suitable for incorporation into the pMTL80000 modular format (Heap J. T. et al, 2009, Journal of Microbiological Methods, 78: 79-85). To achieve this, the NotI site of pMTL-YZ008 was changed to an AscI site using QuikChange II Site-Directed Mutagenesis Kit (Stratagene), to yield the plasmid pMTL-YZ012 (FIG. 15, SEQ ID NO: 27). Briefly, primers NotI/AscI, SEQ ID NO: 28 (5′-aacagctatgaccggcgcgccgctcactgcccgc-3′) and NotI/AscI antisense, SEQ ID NO:29 (5′-gcgggcagtgagcggcgcgccggtcatagctgtt-3′) were designed, and PCR reactions were carried out according to manufacturer's instructions. PCR products were digested by enzyme DpnI to eliminate template plasmids at 37° C. for an hour, and then transformed into E. coli XL-1 Blue. Plasmids extracted from E. coli XL-1 Blue were confirmed via Sanger Sequencing. Thereafter, a 3543 bp AscI-FseI fragment was isolated from pMTL-YZ012 and inserted between the equivalent sites of pMTL-SC1. This essentially replaced the pBP1-based replicon of pMTL-SC1 with the new conditional replicon cassette. The plasmid generated was designated pMTL-YZ013 (FIG. 16, SEQ ID NO: 30).

(59) The mariner transposon system carried by pMTL-SC1 was specifically adapted to function in the pathogen C. difficile. Thus, the transposase gene is expressed by the promoter of the Toxin B (tcdB) gene. One benefit of the use of this promoter is that it does not function in the donor E. coli host, as it is only recognised by a specific C. difficile sigma factor. However, for the transposon to work in a clostridial host other than C. difficile, the TcdR sigma factor needs to be present. In order to achieve this a strain of Clostridium acetobutylicum ATCC 824 was generated in which a promoter-less copy of the tcdR gene of Clostridium difficile 630 was inserted into the genome immediately downstream of the pyrE gene using Allele-Couple Exchange (ACE) Technology. This was accomplished using the described procedures (Heap et al, Nucleic Acids Research, 2012 40(8): e59) and the vector pMTL-ME6c (FIG. 17, SEQ ID NO: 31). The strain generated was designated Clostridium acetobutylicum CRG3011 (FIG. 18).

(60) Plasmid pMTL-YZ013 was transformed into strain CRG3011, and the transformed cells plated on CGM agar (Hartmanis M G N and Gatenbeck S, 1984, Applied Environmental Microbiology, 47: 1277-1283) containing erythromycin (40 μg per ml). Cells were harvested and plated on CGM agar containing thiamphenicol (15 μg per ml) and IPTG (1 mM). In total, 80% of colonies were thiamphenicol resistant and erythromycin sensitive, indicative of successful insertion of the catP mini-transposon into chromosome and plasmid loss.

(61) To establish whether transposition had occurred, inverse PCR was performed according to the procedure of Cartman and Minton (Cartman S T and Minton N P, 2010, Applied Environmental Microbiology, 76: 1103-1109). Genomic DNA was isolated from individual transposon mutants and digested overnight with HindIII at a concentration of 200 ng/μl. The HindIII restriction endonuclease was heat inactivated (65° C. for 30 min), and DNA was diluted to a concentration of 5 ng/μl in a reaction with T4 DNA ligase to favor self-ligation (and thus circularization) of restriction fragments. Ligation reaction mixtures were incubated at ambient temperature for 1 h, and then the T4 ligase was heat inactivated (65° C. for 30 min). Inverse PCRs were carried out in 50-μl volumes using the KOD Hot Start DNA polymerase Master Mix kit (Novagen), with 100 ng of ligated DNA and primers catP-INV-F1 (5′-TAAATCATTTTTAGCAGATTATGAAAGTGATACGCAACGGTATGG-3′) (SEQ ID NO:32) and catP-INV-R1 (5′-TATTGTATAGCTTGGTATCATCTCATCATATATCCCCAATTCACC-3′) (SEQ ID NO: 33), which face out from the transposon-based catP sequence. Inverse PCR products were run out on a 0.8% (wt/vol) agarose gel as shown in FIG. 19), purified with the QIAquick gel purification kit (Qiagen), and sequenced using the primer catP-INV-R2 (5′-TATTTGTGTGATATCCACTTTAACGGTCATGCTGTAGGTACAAGG-3′) (SEQ ID NO: 34). To identify the genomic location of transposon insertions, sequence data were analyzed using GENtle open source software. The data revealed that each transposon insertion had taken place at a different position around the genome as illustrated in FIG. 20.

(62) In parallel, CRG3011 cells were also transformed with unadulterated pMTL-SC1, and selected transformants selected on rich media containing erythromycin (40 μg per ml). Cells were harvested and plated on CGM agar containing thiamphenicol (15 μg per ml). A total of 24 thiamphenicol resistant colonies were picked and re-streaked 3 times, only 2 of them became erythromycin sensitive, indicative of plasmid loss. These results suggested that the plasmid replication region of pMTL-SC1 is very stable in C. acetobutylicum host, which is not ideal for transposon mutagenesis.

(63) Demonstration of Conditional Vector, pMTL-YZ009, in Clostridium sporogenes

(64) To test the conditionality of plasmid pMTL-YZ009 (FIG. 6, SEQ ID NO: 22), plasmids pMTL-YZ008 (FIG. 5, SEQ ID NO: 21) and pMTL-YZ009 were introduced by electroporation into Clostridium sporogenes NCIMB 10696. Their ability to replicate tested in the presence or absence of the inducer IPTG in TYG media lacking any antibiotic supplementation. As expected, plasmid pMTL-YZ009 was found to only be stably maintained in the absence of IPTG as shown in FIG. 21. In the presence of IPTG (1 mM), the plasmid was rapidly lost as shown in FIG. 21, as evidenced by an almost complete loss of Colony Forming Units (CFU) on plates supplemented with erythromycin (20 μg per ml). A similar loss was not evident in cells harbouring pMTL-YZ008 (FIG. 21). It was concluded that transcriptional readthrough into the pCB102 plasmid replication region was interfering with plasmid replication/maintenance.

(65) Demonstration of Conditional Vector, pMTL-YZ009, in Clostridium botulinum

(66) To test the conditionality of plasmid pMTL-YZ009 (FIG. 6, SEQ ID NO: 22), plasmids pMTL-YZ008 (FIG. 5, SEQ ID NO: 21) and pMTL-YZ009 were introduced by electroporation into Clostridium botulinum ATCC 3502. Their ability to replicate tested in the presence or absence of the inducer IPTG in TYG media lacking any antibiotic supplementation. As expected, plasmid pMTL-YZ009 was found to only be stably maintained in the absence of IPTG as shown in FIG. 22. In the presence of IPTG (1 mM), the plasmid was rapidly lost as shown in FIG. 22, as evidenced by an almost complete loss of Colony Forming Units (CFU) on plates supplemented with erythromycin (20 μg per ml). A similar loss was not evident in cells harbouring pMTL-YZ008 (FIG. 22). It was concluded that transcriptional readthrough into the pCB102 plasmid replication region was interfering with plasmid replication/maintenance.

(67) Use of the Expression System for Expression of the Transposase Himar1 C9 in Clostridium sporogenes

(68) Transposon mutagenesis using the mariner transposon-based transposon vector pMTL-YZ013 (FIG. 16, SEQ ID NO: 30), is reliant on TcdR-mediated expression of the mariner transposase. Accordingly, the introduction of this vector into the Clostridium sporogenes strain CRG3817 should result in transposition of the mini-transposon carrying the catP gene and conditional plasmid loss.

(69) To determine whether transposition would occur in strain CGR3817, plasmid pMTL-YZ013 was transformed into Clostridium sporogenes strain CRG3817 and transformants selected on TYG plates containing 40 μg/ml erythromycin. Plates carrying greater than 10 isolated, transformant colonies were then incubated at 37° C. for 48 hours. All of the colony growth was scraped from the plate using a sterile loop and the cells resuspended in TYG media containing +20% Glycerol. The cell suspension was then plated at serial dilutions onto TYG agar plates containing 15 μg/ml thiamphenicol and 1 mM IPTG. A total of 100 colonies were then patch plated onto TYG plates containing 15 μg/ml thiamphenicol and TYG plates containing 40 μg/ml erythromycin as a simple test to ascertain whether the plasmid pMTL-YZ013 was still present. All 100 colonies render sensitivity to erythromycin and resistance to thiamphenicol, indicating that under the conditions employed, the plasmids had all been lost and transposition occurred from the population.

(70) To establish whether transposition had occurred, inverse PCR was performed according to the procedure of Cartman and Minton (Cartman S T and Minton N P Applied Environmental Microbiology 2010, 76:1103-9). Genomic DNA was isolated from 16 individual thiamphenicol resistant clones and digested overnight with HindIII at a concentration of 200 ng/μl. The HindIII restriction endonuclease was heat inactivated (65° C. for 30 min), and DNA was diluted to a concentration of 5 ng/μl in a reaction with T4 DNA ligase to favor self-ligation (and thus circularization) of restriction fragments. Ligation reaction mixtures were incubated at ambient temperature for 1 h, and then the T4 ligase was heat inactivated (65° C. for 30 min). Inverse PCRs were carried out in 50-μl volumes using the KOD Hot Start DNA polymerase Master Mix kit (Novagen), with 100 ng of ligated DNA and primers catP-INV-F1, SEQ ID NO: 32 (5′-TAAATCATTTTTAGCAGATTATGAAAGTGATACGCAACGGTATGG-3′) and catP-INV-R1, SEQ ID NO: 33 (5′-TATTGTATAGCTTGGTATCATCTCATCATATATCCCCAATTCACC-3′), which face out from the transposon-based catP sequence. Inverse PCR products were run out on a 0.8% (wt/vol) agarose gel (FIG. 23), purified with the QIAquick gel purification kit (Qiagen), and sequenced using primer catP-INV-R2 SEQ ID NO: 34 TATTTGTGTGATATCCACTTTAACGGTCATGCTGTAGGTACAAGG-3′). To identify the genomic location of transposon insertions, sequence data were analyzed using GENtle.

(71) These data revealed that in all of the colonies tested, the transposon had inserted into 18 different locations within the Clostridium sporogenes genome (Table 1.) Two of the 16 clones possess double insertions. These data provide proof of principle that the presence of tcdR in the genome of CRG3817 allows transposition of the mariner transposon in Clostridium sporogenes.

(72) TABLE-US-00009 TABLE 1 Sequence analysis of the Inverse PCR products from eight randomly selected thiamphenicol resistant colonies carrying pMTL-YZ013. Position of Insertion in C. sporogenes Colony NCIMB 10696 Gene Number genome affected Gene function  1 2440051 (reverse CS1546 pyridine nucleotide-disulfide strand) oxidoreductase family protein  2 1827633 (forward CS1723 DEAD/DEAH box helicase family strand) protein  3 2319672 (forward CS2157 KWG leptospira repeat protein strand)  4 2960953 (forward CS2817 heparinase II/III-like family protein strand)  5 3527420 (forward CS3350 putative CoA-substrate-specific strand) enzyme activase domain protein  6 1682076 (forward CS1592 HAD ATPase, P-type, IC family strand) protein  7 prfC found in botulinum not in sporogenes  8 1454638 (forward CS1379 4Fe-4S binding domain protein strand)  9 842007 (reverse CS3053 putative membrane protein strand) 10 897474 (reverse CS3008 HNH endonuclease family protein strand) 11 1028948 (forward CS0978 conserved hypothetical protein and strand) and putative membrane protein 0979 12a 3297392 (reverse CS0721 conserved hypothetical protein strand) 12b 3065003 (forward CS2912 polysaccharide deacetylase family strand) protein 13 879201 (forward CS0821 2-amino-4-hydroxy-6- strand) hydroxymethyldihydropteridine pyrophosphokinase 14 3443288 (forward CS3275 dihydrodipicolinate reductase, strand) family protein 15 2768400 (forward CS1233 L-serine dehydratase, iron-sulfur- strand) and dependent, beta subunit and L- 1234 serine dehydratase, iron-sulfur- dependent, alpha subunit 16a 3213733 (reverse CS0806 branched-chain amino acid strand) transport system II carrier protein 16b 1643917 (forward CS1552 ftsX-like permease family protein strand)