A method to maintain introduced DNA sequences stably and safely on the bacterial chromosome: application of prophage...

By application of prophage integration and subsequent intended excision, a method to maintain an introduced DNA sequence stably onto a bacterial chromosome has been proposed. Recently-constructed integration plasmids using Campbell-type prophage integration system in Lactobacillus casei strain Shirota and its temperate phage phi FSW was modified for this purpose and a chloramphenicol (Cm)-resistance gene was used as a model passenger DNA. On the integration plasmid having an erythromycin (Em)-resistance gene as a selection marker, N- and C-terminally-truncated Cm-resistance genes were inserted into both sides of the attP of phi FSW, within which the site-specific recombination took place with the attB of phi FSW on the recipient chromosome through the phi FSW integrase. Primary integrants of the modified plasmid (integration-excision vector) exhibiting Em-resistant and Cm-sensitive phenotype generated Em-sensitive and Cm-resistant derivatives under the nonselective conditions. Sequence analyses showed that one copy of the complete Cm-resistance gene resided at the attachment site on the host chromosome and the other vector-derived sequences were excised probably by endogenous homologous recombination in the host cells to derive final integrants. The Cm-resistant phenotype of the final integrants was stable for more than 50 generations under non-selective conditions. Frequency of the homologous recombination suggests that negative selection is also adoptable. Thus, this method using the integration-excision vector gives a stable and safe derivatives of the strain and is likely to be applicable to various bacteria, since Campbell-type prophage integration system and homologous recombination are prevalent among bacteria.