Abstract
Clostridium encompasses species which are relevant to human and animal disease as well as species which have industrial potential, for instance, as producers of chemicals and fuels or as tumour delivery vehicles. Genetic manipulation of these target organisms is critical for advances in these fields. DNA transfer efficiencies, however, vary between species. Low efficiencies can impede the progress of research efforts.A novel conjugal donor strain of Escherichia coli has been created which exhibits a greater than 10-fold increases in conjugation efficiency compared to the traditionally used CA434 strain in the three species tested; C. autoethanogenum DSM 10061, C. sporogenes NCIMB 10696 and C. difficile R20291. The novel strain, designated ‘sExpress’, does not methylate DNA at Dcm sites (CCWGG) which allows circumvention of cytosine-specific Type IV restriction systems.A robust protocol for conjugation is presented which routinely produces in the order of 105 transconjugants per millilitre of donor cells for C. autoethanogenum, 106 for C. sporogenes and 102 for C. difficile R20291. The novel strain created is predicted to be a superior conjugal donor in a wide range of species which possess Type IV restriction systems.
Highlights
The bacterial genus Clostridium is most often associated with disease, none more so than Clostridium difficile, the leading cause of antibiotic-associated disease and a significant burden on the finances of healthcare systems worldwide [1]
The restriction enzyme database http://rebase.neb.com [21] provides an overview of the restriction-modification (RM) systems found in the genome of a given strain
The attribution of these genes as encoding Type IV systems is due to a shared region of homology with the E. coli McrB Type IV cytosinespecific restriction enzyme
Summary
The bacterial genus Clostridium is most often associated with disease, none more so than Clostridium difficile, the leading cause of antibiotic-associated disease and a significant burden on the finances of healthcare systems worldwide [1]. The vast majority of clostridial species are entirely benign and in many instances possess properties and attributes of great benefit to mankind. There are a variety of E. coli strains which are capable of mediating the mobilisation of small, autonomous vectors to clostridial recipients during the process of conjugation. Transfer of the vector generally relies on its oriT-mediated mobilisation with the necessary transfer functions being provided in trans by genes located either on a plasmid, such as R702, co-resident in the donor E.coli host strain (eg., CA434), or in the chromosome, e.g. donor strains S17.1 and SM10 [4]. All genetic studies of C. autoethanogenum to date, and the majority of studies in C. difficile and C. sporogenes, have used the donor E. coli strain CA434 [10]
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