Transposon

pMTL-SC1

The first mariner-based transposon system developed for in vivo random mutagenesis of C. difficile. Utilises the hyperactive Himar1 variant 29 transposase under the control of the native tcdB toxin gene promoter, PtcdB. The transposase mobilises a mini-transposon containing the catP gene which confers chloramphenicol and thiamphenicol resistance Use of plasmid pMTL-SC1 was demonstrated in the epidemic BI/NAP1/027 strain R20291 where transposon insertion generated random, single insertion (98.3%) mutants [1].

pMTL-YZ14

Based on pMTL-SC1, this plasmid carries a conditional Gram-positive pCB102 replicon, whereby the presence of IPTG is the non-permissive replicative condition. The resistance marker is the macrolide-lincosamide-streptogramin B antibiotic resistance gene ermB. Replication occurs in E. coli via the pUC ColE1 origin of replication and the plasmid can be transferred to clostridial recipients using the oriT from RK2, but requires the presence of the C. difficile tcdR gene integrated in the chromosome. Initial exemplification was in Clostridium acetobutylicum and Clostridium sporogenes [2].

pMTL-CW20

pMTL-CW20 is based on the pMTL-YZ14 plasmid described above, with the following principal differences. A promoter-less codA gene from E. coli has been positioned downstream of the catP mini-transposon, such that its premature excision as a consequence of transposition leads to expression of codA by the upstream Pthl promoter. Supplementation of the host E. coli or clostridial strain with 5-FC ensures stability of the transposon as spontaneous undesired transposition results in the CodA mediated conversion of 5-FC to the toxic compound 5-FU. I-SceI recognition sites flanking the mini-transposon have also been introduced, which allow for removal of any remaining plasmid-based reads at the library sequencing stage [3]. The plasmid also carries the conditional replicon of pMTL-YZ14 [2].

pMTL-CW21 & pMTL-CW22

Details to come, but essentially as pMTL-CW20 but has swopped the PtcdB promoter with a Tet promoter, dispensing with the need for integration of tcdR in the chromosome [4].

References

[1] Cartman ST, Minton NP. A mariner-based transposon system for in vivo random mutagenesis of Clostridium difficile. Appl Environ Microbiol. 2010;76(4):1103-9.
https://doi.org/10.1128/AEM.02525-09

[2] Zhang Y, Grosse-Honebrink A, Minton NP. A Universal Mariner Transposon System for Forward Genetic Studies in the Genus Clostridium. PLoS ONE. 2015; 10(4).
https://doi.org/10.1371/journal.pone.0122411

[3] Craig Woods, Christopher M. Humphreys, Claudio Tomi-Andrino, Anne M. Henstra, Michael Köpke, Sean D. Simpson, Klaus Winzer, Nigel P. Minton. Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum. bioRxiv. 2021.05.19.444907.
https://doi.org/10.1101/2021.05.19.444907

[4] Manuscript in preparation.