Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea

Abstract

pSE211 from Saccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination between attP and attB, the plasmid and chromosomal attachment sites. Integration depends on the presence of int, an open reading frame (ORF) that lies adjacent to attP and encodes the putative integrase. Immediately upstream of int lies xis (formerly called orf2) which encodes a basic protein that is thought to exhibit DNA binding. xis and int were cloned in various combinations in pUC18 and expressed constitutively in Escherichia coli from the lac promoter. attP and attB were cloned in Streptomyces or E. coli plasmids containing kanamycin resistance (KmR) or chloramphenicol resistance (CmR) markers. Stable KmR CmR cointegrates formed by attP x attB or attP x attP recombination (integration) were obtained in E. coli hosts that expressed int. Co-integrates were not found in hosts expressing int + xis. Excision (intraplasmid att site recombination) was examined by constructing plasmids carrying attL and attR or two attP sites separating CmR from KmR and by following segregation of the markers in various hosts. Both attL x attR and attP x attP excision depended on both xis and int in E. coli. pSE211 att site integration and excision were not affected by a deletion in himA, the gene encoding a subunit of integration host factor.