TPW22, a lactococcal temperate phage with a site-specific integrase closely related to Streptococcus thermophilus phage integrases

Abstract

The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases.

FIG. 1

Overview of the 3.4-kb sequenced region stretching from the EcoRI site to the HindIII site positioned 3.4 kb downstream. The map includes the position of attP, putative genes drawn as arrows, four putative factor-independent terminator structures marked “term,” and a putative promoter called P_int. The clones used in integration experiments are indicated as lines. +, ability to integrate into L. lactis subsp. cremoris MG1363; −, lack of ability to integrate. The extension of the proposed integration module is indicated by a box, with the transition points shown as asterisks.

FIG. 2

Phenogram of the TPW22 integrase, integrases of closely related bacteriophages of gram-positive bacteria, and the integrase of bacteriophage λ. The GenBank accession numbers of the sequences are given in parentheses.

FIG. 3

Identification of the TPW22 phage attachment site (attP) by Southern blot hybridization. Lane 1, TPW22 phage genomic DNA digested with EcoRI. Lanes 2 to 4, EcoRI-digested chromosomal DNA of L. lactis subsp. cremoris W22, L. lactis subsp. cremoris 3107L8, and L. lactis subsp. cremoris 3107, respectively. Lane 5, TP3107 phage DNA digested with PstI. Lane 6, TPW22 phage genomic DNA digested with PstI. Lanes 7 to 8, PstI-digested chromosomal DNA of L. lactis subsp. cremoris W22 and L. lactis subsp. cremoris 3107L8. The DNA was hybridized with the labeled pAP3 insert containing attP of phage TPW22 as a probe. The migration positions and molecular sizes in kilobases of the fragments containing the left attachment site (attL), the right attachment site (attR), and the phage attachment site (attP) are marked. The autoradiograph was scanned with Hewlett-Packard DeskScan II, and labels were added in Adobe Photoshop 4.0.

FIG. 4

(A) Schematic representation of the bacterial chromosomal attachment site (attB) of L. lactis subsp. cremoris 3107 containing part of a putative gene. The deduced gene product has homology to DNA ligases. Inverted repeats are drawn as thin arrows, and the common core is indicated as a boxed region. The nucleotide positions of the sequenced part of the ORF and of the common core are indicated as numbers. (B) Aligned nucleotide sequences of the regions containing attP, attL, attR, and attB. Phage TPW22 sequences are underlined, while the L. lactis subsp. cremoris 3107 chromosomal sequences are double underlined.

FIG. 5

(A) An overview of the genome organization in the region surrounding int of the temperate lactococcal phages TPW22, r1t (GenBank accession no. U38906) (64), BK5-T (L44593) (11), Tuc2009 (L31348) (63), φLC3 (U04309 and L10286) (9, 47), and TP901-1 (Y14232) (23). ORFs or parts of ORFs with gene products showing more than 95% identity are indicated as solid horizontal arrows, and the conserved gene products are stated above. Other ORFs are presented as shaded horizontal arrows with the name stated. Vertical arrows represent conserved putative terminator structures. Arrows of the same type (except the vertical arrows of phage TPW22) indicate a high degree of homology between the putative terminator structures. The first base pair in the attachment core is 1 and is indicated as a cross. (B) Overview of homologies in the region around int of the phages TPW22 and φO1205. ORFs are indicated as shaded arrows. Homologous areas upstream and downstream of int are drawn as black lines. Homologous areas are connected with thin lines, and the identity is shown.

FIG. 6

The conserved putative factor-independent terminator structure between int and orf2 of the lactococcal phages φLC3 (GenBank accession no. L24560) (47), r1t (U38906) (64), Tuc2009 (L31348) (63), TP901-1 (X85213) (23), BK5-T (L44593) (11), and TPW22 and of the S. thermophilus phage φO1205 (U88974) (60). Residues are shaded if four or more are identical, and the consensus sequence is shown at the bottom.