INVESTIGADORES
RAYA Raul Ricardo
artículos
Título:
Site-Specific Integration of the Temperate Bacteriophage fadh into the Lactobacillus gasseni Chromosome and Molecular Characterization of the Phage (attP) and Bacterial (attB) Attachment Sites
Autor/es:
RAYA RR, FREMAUX C, DE ANTONI GL, KLAENHAMMER TR.
Revista:
JOURNAL OF BACTERIOLOGY
Editorial:
AMER SOC MICROBIOLOGY
Referencias:
Lugar: Washington, Estados Unidos; Año: 1992 vol. 174 p. 5584 - 5592
ISSN:
0021-9193
Resumen:
The temperate bacteriophage phi adh integrates its genome into the
chromosomal DNA of Lactobacillus gasseri ADH by a site-specific
recombination process. Southern hybridization analysis of BclI-digested
genomic DNA from six relysogenized derivatives of the prophage-cured
strain NCK102 displayed phage-chromosomal junction fragments identical
to those of the lysogenic parent. The phi adh attachment site sequence,
attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi
adh. This fragment was cloned and sequenced. DNA sequence analysis
revealed striking features common to the attachment sites of other
site-specific recombination systems: five direct repeats of the sequence
TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived
from the sequence of the attP-containing fragment enabled us to amplify
predicted junction fragment sequences and thus to identify attL, attR,
and attB. The core region was defined as the 16-bp sequence
TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific
insertion of phage phi adh were located in a 4.5-kb BclI fragment. This
fragment was cloned in plasmid pSA34 to generate the insertional vector
pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by
electroporation. Hybridization analysis showed that a single copy of
pTRK182 had integrated at the attB site of the NCK102
erythromycin-resistant transformants. This is the first site-specific
recombination system described in lactobacilli, as well as the first
attP-based site-specific integration vector constructed for L. gasseri
ADH.