Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration

Abstract

The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp. cremoris 901-1, was characterized. The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism. The pac region was localized on the 38.4-kb phage genome. TP901-1 belongs to the class of P335 phages (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989. Evidence is presented that the phages TP936-1 (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989) and C3-T1 (A. W. Jarvis, V. R. Parker, and M. B. Bianchin, Can. J. Microbiol. 38:398404, 1992) are very closely related to or are identical to TP901-1. The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2. Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome. Only one chromosomal attB site was found in 20 independent lysogens. The attP region of TP901-1 and the attL and attR regions were cloned and sequenced. The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA. This result was further verified by sequencing of the attB region obtained by PCR. An integration vector was constructed with the 6.5-kb EcoRl fragment from TP901-1 containing attP. This vector also functions in the plasmid-free strains MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci. In this report, we present the restriction map of phage TP901-1, including the location of the attP andpac regions. We have been able to monitor the lysogenic life cycle of the phage and demonstrate the presence of only one major attachment site. An integration vector based on TP901-1 DNA sequences was constructed, and the attP, attL, attR, and attB regions were identified, cloned, and sequenced. The integration system from TP901-1 was also shown to be functional and site specific in the laboratory strains often used for genetic studies of lactococci, namely Lactococcus lactis subsp. lactis MG1363 and LM0230. This strongly indicates that the integration system of TP901-1 may be of general use as a genetic tool with lactococci. MATERIALS AND METHODSBacteria, plasmids, and phages. Bacterial strains and plasmids used in this work are listed in Table 1. Phage DNA from phages C3-T1 (13) and XLC3 (22) Temperate phages were induced from their hosts by UV irradiation. M17 broth was inoculated with 1% (vol/vol) of an overnight culture of the lysogenic strain. The culture was at an optical density at 600 nm of 0.15, harvested at 5,000 x g for 10 min, and resuspended in 0.5 volume of NC (0.5% NaCl [wt/vol], 5 mM CaCl2). The suspension was pumped through a quartz flow cuvette placed at the bottom of a UV field (254 1069

A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1

“…The temperate bacteriophage TP901-1 integrates specifically into only one chromosomal attachment site, attB. The attB region, the attachment site of the phage (attP), and the chromosomal junctions, attL and attR, between the phage genome and the host chromosome in the lysogens were cloned and sequenced (5). In all four cases this identified the core region, where the site-specific recombination takes place, as a sequence of 5 bp, 5Ј-TCAAT-3Ј.…”

“…Transformants were selected on plates containing 2 g of erythromycin per ml. Integration was analyzed by PCR of chromosomal DNA, as previously described, with representative transformants for the presence of attL and attR and the absence of attB (5). The presence of the int gene was determined by PCR, with the primers P4Rb and Pint2.…”

Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum

“…By comparison of the nucleotide sequences of attP, attL, attR, and attB, a 17-bp common core sequence was identified. This core sequence does not show any homology to the core sequences of phages from lactic acid bacteria characterized to date (LC3 9 bp, [30]; TP901-1 5 bp [12]; adh 16 bp, [43]) or with that of other gram-positive or gram-negative bacteriophages.…”

“…Integration into a tRNA gene is a common occurrence in gram-negative phages (P22, P4, 186, HP1, 16-3, and CTX) and in actinomycete integrative plasmids (SLP1, pSAM2, pMEA100, and pSE211). In contrast, except for mycobacteriophage L5, which integrates its DNA into a tRNA gene (26), phages of gram-positive bacteria integrate either into a gene coding for a protein (L54a [24], 13 [14], LC3 [30], or TP901-1 [12]) or into an intergenic region (11 [25]). The use of the 3Ј ends of tRNA genes for the phage integration sites is a general phenomenon among various bacterial species (11,44).…”

“…There are many well-characterized examples of sitespecific recombination in gram-negative bacteriophages, and the best-studied system is that of bacteriophage (for a review, see reference 22). The integration system of phages of grampositive bacteria is less well documented, but data on sitespecific recombination are available for phages L54a, 11, and 13 of Staphylococcus aureus (14,25,55,56), for mycobacteriophage L5 (26), and for phages of lactic acid bacteria (12,30,43,51). The integration systems of integrative plasmids pSAM2, pSE211, pSE101, and SLP1 from actinomycetes appear to behave like those of temperate bacteriophages (5, 7-9).…”