| Summary: | Bibliography: pages 105-115. === A cloning system, using metronidazole as a screening tool and E. coli FI9 as a selection host, was previously established to clone C. acetobutylicum P262 electron transport genes which may play a role in solvent metabolism in this bacterium. In theory, metronidazole would be reduced under anaerobic conditions to a cytotoxic intermediate by C. acetobutylicum P262 electron transport genes or reductive enzymes cloned into a recombinant plasmid. This intermediate would kill the host E. coli FI9. One C. acetobutylicum P262 clone, pMETIOB, was found to render the E. coli strain FI9 sensitive to metronidazole, under anaerobic conditions. A number of subclones of the 2.56kb C. acetobutylicum P262 insert DNA were generated in Bluescript pKS and pSK. A range of exonuclease III deletions of this C. acetobutylicum P262 insert DNA were also generated which were shown to lose the metronidazole sensitivity phenotype on progressive deletion of the insert DNA. In vitro and in vivo protein transcription/translation experiments failed to reveal a protein product that was related to the metronidazole sensitivity phenotype. DNA hybridization confrrmed that the insert DNA of pMETIOB hybridized to C. acetobutylicum P262 chromosomal DNA, but not to E. coli chromosomal DNA. The nucleotide sequence of a 933-bp fragment of the C. acetobutylicum P262 insert DNA was determined.
|
|---|
