A method to maintain introduced DNA sequences stably and safely on the bacterial chromosome: Application of prophage integration and subsequent designed excision

By application of prophage integration and subsequent intended excision; a method to maintain an introduced DNA sequence stably onto a bacterial chrom osome has been proposed. Recently-constructed integration plasmids using Ca mpbell-type prophage integration system in Lactobacillus casei strain Shiro ta and its temperate phage phi FSW was modified for this purpose and a chlo ramphenicol (Cm)-resistance gene was used as a model passenger DNA. On the integration plasmid having an erythromycin (Em)-resistance gene as a select ion 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 rec ombination 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 pheno type generated Em-sensitive and Cm-resistant derivatives under the nonselec tive conditions. Sequence analyses showed that one copy of the complete Cm- resistance gene resided at the attachment site on the host chromosome and t he other vector-derived sequences were excised probably by endogenous homol ogous recombination in the host cells to derive final integrants. The Cm-re sistant phenotype of the final integrants was stable for more than 50 gener ations under non-selective conditions. Frequency of the homologous recombin ation 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 Cam pbell-type prophage integration system and homologous recombination are pre valent among bacteria. (C) 2001 Elsevier Science B.V. All rights reserved.