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Journal of Bacteriology, August 2002, p. 4177-4186, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4177-4186.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction, Characterization, and Use of Two Listeria monocytogenes Site-Specific Phage Integration Vectors

Peter Lauer,1,{dagger} Man Yin Nora Chow,1 Martin J. Loessner,1,{ddagger} Daniel A. Portnoy,1,2 and Richard Calendar1*

Department of Molecular and Cell Biology,1 School of Public Health, University of California, Berkeley, California 94720-32022

Received 28 February 2002/ Accepted 9 May 2002

Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of ~10-4 per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB' in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3' end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNAArg gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720-3202. Phone: (510) 642-5951. Fax: (510) 643-6791. E-mail: rishard{at}socrates.berkeley.edu.

{dagger} Present address: IRIS, Chiron S.p.A., 53100 Siena, Italy.

{ddagger} Present address: Institut für Mikrobiologie, FML Weihenstephan, Technische Universität München, 85830 Freising, Germany.

Journal of Bacteriology, August 2002, p. 4177-4186, Vol. 184, No. 15
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.15.4177-4186.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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