Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration.
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. 38:398-404, 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 EcoRI 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.
Christiansen
B
B
Department of Microbiology, Technical University of Denmark, Lyngby.
Johnsen
M G
MG
Stenby
E
E
Vogensen
F K
FK
Hammer
K
K
D001435:Bacteriophages / Q000235:genetics* / Q000528:radiation effects; D001483:Base Sequence; D003001:Cloning, Molecular; D004270:DNA, Circular / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D013294:Lactococcus lactis / Q000528:radiation effects; D008242:Lysogeny / Q000235:genetics*; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014466:Ultraviolet Rays; D016662:Virus Integration / Q000235:genetics*
D004270:DNA, Circular