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Journal of Bacteriology, November 1998, p. 5505-5514, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Int-B13, an Unusual Site-Specific Recombinase of the
Bacteriophage P4 Integrase Family, Is Responsible for Chromosomal
Insertion of the 105-Kilobase clc Element of
Pseudomonas sp. Strain B13
Roald
Ravatn,
Sonja
Studer,
Alexander J. B.
Zehnder, and
Jan
Roelof
van der Meer*
Swiss Federal Institute for Environmental
Science and Technology (EAWAG) and Swiss Federal Institute for
Technology (ETH), CH-8600 Dübendorf, Switzerland
Received 12 June 1998/Accepted 25 August 1998
Pseudomonas sp. strain B13 carries the
clcRABDE genes encoding chlorocatechol-degradative enzymes
on the self-transmissible 105-kb clc element. The element
integrates site and orientation specifically into the chromosomes of
various bacterial recipients, with a glycine tRNA structural gene
(glyV) as the integration site. We report here the
localization and nucleotide sequence of the integrase gene and the
activity of the integrase gene product in mediating site-specific
integration. The integrase gene (int-B13) was located near
the right end of the clc element. 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 protein (74 kDa) was observed
upon overexpression and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis separation. The N-terminal 430 aa of the predicted Int-B13 protein had substantial similarity to integrases from bacteriophages of the P4 family, but Int-B13 was much larger than P4-type integrases. The C-terminal 220 aa of Int-B13 were homologous to
an ORF flanking a gene cluster for naphthalene degradation in
Pseudomonas aeruginosa PaK1. Similar to the bacteriophages
R73 and P4, the clc 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 specificity of the integration. In
Pseudomonas sp. strain B13, a circular form 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), could be detected. Upon chromosomal
integration of the clc element into a bacterial attachment site (attB), a functional glyV was
reconstructed at the right end of the element. The integration process
could be demonstrated in RecA-deficient Escherichia coli
with two recombinant plasmids, one carrying the int-B13
gene and the attP site and the other carrying the
attB site of Pseudomonas putida F1.
*
Corresponding author. Mailing address: EAWAG,
Überlandstrasse 133, CH-8600 Dübendorf, Switzerland. Phone:
(41) 1-823-5438. Fax: (41) 1-823-5547. E-mail:
vdmeer{at}eawag.ch.

Present address: Department of Molecular Genetics and Microbiology,
UMDNJ-Robert Wood Johnson Medical School, Piscataway,
NJ
08854.
Journal of Bacteriology, November 1998, p. 5505-5514, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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