Gene cloning in Clostridium difficile using Tn916 as a shuttle conjugative transposon.
A pBR322-based vector, pCI195, containing a 4.2-kb region of the conjugative transposon Tn919 was used as a vector for gene cloning in Clostridium difficile. The plasmid was found to integrate into the chromosome of a Bacillus subtilis strain that contained Tn916 delta E. Southern blot analysis of the recombinant demonstrated that pCI195 had inserted into Tn916 delta E by a recombination event. The transposon::plasmid structure could be transferred, by filter mating, from B. subtilis to C. difficile (at a frequency of 10(-8) per donor), where it entered the chromosome at a specific site. Segregation of plasmid and transposon markers was observed on transfer, although the Tn916 delta E::pCI195 was stably maintained in C. difficile. To demonstrate that pCI195 could be used for gene cloning in C. difficile, a 1.1-kb fragment of the C. difficile toxin B gene was cloned into pCI195 to generate pPPM100. Tn916 delta E::pPPM100 was transferred into a nontoxigenic C. difficile strain by filter mating, where it entered the genome at a specific site. pCI195 should be useful as a general cloning vector for C. difficile, as the transposon::plasmid structure could be transferred to different C. difficile strains. This is the first report of gene cloning in C. difficile.
Mullany
P
P
Department of Medical Microbiology, St. Bartholomew's Hospital Medical College, West Smithfield, London, United Kingdom.
Wilks
M
M
Puckey
L
L
Tabaqchali
S
S
D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D016360:Clostridioides difficile / Q000235:genetics; D003227:Conjugation, Genetic; D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015247:Deoxyribonuclease HindIII; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D015247:Deoxyribonuclease HindIII