TARGETED INTEGRATION OF GENES INTO THE CLOSTRIDIUM-ACETOBUTYLICUM CHROMOSOME

A number of non-replicative, integrational plasmids have been introduced into the chromosome of the NCIMB 8052 strain of Clostridium acetobutylicum. Integration occurred via homologous recombination, apparently by a Campbell-like mechanism. For plasmids containing internal fragments of gutD or spo0A, insertion was mutagenic leading, respectively, to an inability to use sorbitol as sole source of carbon for energy and growth or an inability to form endospores. One insertion was used to determine the map location of a randomly cloned DNA fragment and another underwent amplification in the bacterial chromosome. These results suggest that integrational plasmids will be very useful tools for genetic analysis in Clostridium acetobutylicum. To our knowledge, this is the first demonstration of gene transfer involving homologous recombination with the chromosome of any species of Clostridium.