INT-B13, AN UNUSUAL SITE-SPECIFIC RECOMBINASE OF THE BACTERIOPHAGE-P4INTEGRASE FAMILY, IS RESPONSIBLE FOR CHROMOSOMAL INSERTION OF THE 105-KILOBASE CLC ELEMENT OF PSEUDOMONAS SP. STRAIN B13

Pseudomonas sp. strain B13 carries the clcRABDE genes encoding chloroc atechol-degradative enzymes on the self-transmissible 105-kb clc eleme nt. The element integrates site and orientation specifically into the chromosomes of various bacterial recipients, with a glycine tRNA struc tural gene (glyV) as the integration site. We report here the localiza tion and nucleotide sequence of the integrase gene and the activity of the integrase gene product in mediating site-specific integration. Th e integrase gene (int-B13) was located near the right end of the clc e lement. It consisted of an open reading frame (ORF) of maximally 1,971 bp with a coding capacity for 657 amino acids (aa), The full-length p rotein (74 kDa) was observed upon overexpression and sodium dodecyl su lfate-polyacrylamide gel electrophoresis separation, The N-terminal 43 0 aa of the predicted Int-B13 protein had substantial similarity to in tegrases from bacteriophages of the P4 family, but Int-B13 was much la rger than P4-type integrases, The C-terminal 220 aa of Int-B13 were ho mologous to an ORF flanking a gene cluster for naphthalene degradation in Pseudomonas aeruginosa PaK1. Similar to the bacteriophages phi R73 and P4, the de element integrates into the 3' end of the target tRNA gene. This target site was characterized from four different recipient strains into which the clc element integrated, showing sequence speci ficity of the integration, In Pseudomonas sp. strain B13, a circular f orm of the clc element, which carries an 18-bp DNA sequence identical to the 3'-end portion of glyV as part of its attachment site (attP), c ould be detected. Upon chromosomal integration of the clc element into a bacterial attachment site (attB), a functional glyV was reconstruct ed at the right end of the element, The integration process could be d emonstrated in RecA-deficient Escherichia coli with two recombinant pl asmids, one carrying the int-B13 gene and the attP site and the other carrying the attB site of Pseudomonas putida F1.