Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors

Abstract

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 approximately 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 tRNA(Arg) 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.

FIG. 1.

(A) Plasmid map of the pPL1 integration vector. Chloramphenicol resistance genes and E. coli origin of replication are shown in grey, the RP4 origin of transfer is shown in white, and the U153 integrase gene and L. monocytogenes p60 promoter are shown in black. The multiple cloning site (MCS) is shown at the bottom of the plasmid, with unique restriction sites noted below in a box. pPL24 and pPL25 inserts are shown schematically below the multiple cloning site and were cloned as described in Materials and Methods. Final sizes of the plasmid constructs and the restriction sites used in cloning are noted for each of the inserts. (B) Plasmid map of the integration vector pPL2. The color scheme and genes are the same as in panel A except for the PSA integrase and PSA attPP′ sites, as noted. The multiple cloning site with 13 unique restriction sites is shown below the plasmid.

FIG. 2.

Genomic organization of the attachment sites within the comK gene (A and B) and the tRNA^Arg gene (C and D). (A) Nonlysogenic L. monocytogenes strain with an intact comK gene. Primers PL60 and PL61 amplify across the bacterial attachment site comK-attBB′. (B) Lysogenic L. monocytogenes strain (with approximately 40 kb of phage DNA inserted into the comK gene) or integrated strain (with pPL1 construct inserted into the comK gene). Primers PL14 and PL61 amplify across the hybrid attachment site comK-attPB′. (C) L. monocytogenes serotype 4b strain nonlysogenic at tRNA^Arg-attBB′. Primers NC16 and NC17 amplify across the bacterial attachment site tRNA^Arg-attBB′ in serotype 4b strains. Asterisk indicates that primers NC16 and NC17 are substituted with PL102 and PL103 to amplify across the bacterial attachment site tRNA^Arg-attBB′ in serotype 1/2 strains. (D) Lysogenic L. monocytogenes strain with approximately 40 kb of phage DNA or 6 kb of pPL2 vector DNA inserted at the 3′ end of the tRNA^Arg gene. Primers NC16 and PL95 amplify across the hybrid attachment site tRNA^Arg-attBP′ in both serotype 4b and 1/2 strains.

FIG. 3.

Expression and functional complementation of ActA in SLCC-5764. (A) Coomassie blue-stained SDS-PAGE of SLCC-5764-derived strains grown to late log phase. ActA is indicated by an arrow. Lane 1, molecular size markers; lane 2, DP-L3780; lane 3, DP-L4083; lane 4, DP-L4086; lane 5, SLCC-5764; lane 6, DP-L4082; lane 7, DP-L4084; lane 8, DP-L4085; lane 9, DP-L4087. Strains are described in Table 1. (B) Actin tail formation and movement of DP-L4087 in Xenopus cell extracts. The top panel is a phase image; the bottom panel is a fluorescent image of the same field.

FIG. 4.

(A) Cloverleaf diagram of tRNA^Arg used as the PSA attachment site. The arginine anticodon is circled. The region with sequence identity between the tRNA gene and the PSA attPP′ is outlined. The boundaries of the tRNA^Arg gene and Cove score (82.37) were predicted with tRNAscan-SE (32). (B) Alignment of the tRNA^Arg-attBB′ region of L. monocytogenes WSLC 1042 (top line) and the attPP′ region of PSA downstream of the integrase gene. The 74-nucleotide tRNA^Arg gene of L. monocytogenes is boxed, and the 17-bp overlapping region of sequence identity (core integration site) is shaded grey. The tRNA^Arg gene anticodon is shown in bold and underlined. Identical nucleotide residues are indicated by a colon. The numbers located at the left indicate the nucleotide position in the DNA sequences of the WSLC 1042 attachment site (AJ314913) and PSA genome (AJ312240).