Structural analysis of staphylococcal bacteriophage phi 11 attachment sites

Abstract

The lysogenization of bacteriophage +11 in Staphylococcus aureus occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the prophage were cloned, and the nucleotide sequences were determined. The attachment sites share a very short common sequence of 10 base pairs.Bacteriophage 411 is a group B phage carried as a prophage in Staphylococcus aureus NCTC 8325 (9). Genetically, it is the most characterized of all staphylococcal phages. The viral DNA is a linear, double-stranded, terminally redundant molecule of about 45 kilobases (kb) (1,6,7). In addition to the viral attachment site and the immunity region, 10 genes have been located on a circularly permuted map (3,6). During lysogeny, insertion into the chromosome occurs by site-specific recombination between the viral attachment site (attP) and the att411 site (12) on the host chromosome. To define the attachment site on the viral genome more precisely and to study the site-specific recombination mechanism, we cloned and determined the nucleotide sequences of (i) the attachment site on the phage genome (attP), (ii) the attachment site on the bacterial chromosome (attB), and (iii) the attachment sites generated at the junctions between 411 and S. aureus DNA (attR and attL).Phage 411 DNA and bulk chromosomal DNA were isolated as previously described (2). We identified the fragments containing attR, attL, and attP directly by hybridization. Chromosomal DNA from 411-lysogenized strain and 411 DNA were digested with the restriction endonuclease ClaI. The fragments were separated by agarose gel electrophoresis on adjacent gel lanes and transferred to nitrocellulose by Southern blotting (14). The blot was probed with 32P-labeled 411 DNA, and the profiles were compared. Because integration occurs between attB and attP sites and because the genome of 441 is circularly permuted, 411 gene probes should identify two unique bands in the genomic DNA of the lysogenized strain and one unique band in the genomic DNA of phage 441. Two unique junction fragments that contained attL (on a 5.1-kb fragment) and attR (on a 4.3-kb fragment) were identified. One unique fragment (8 kb) from the 411 digest that contained attP was identified. These fragments were isolated and cloned into the ClaI site of pBR322.To identify the DNA fragment containing the attB site, we digested bulk chromosomal DNA from strain 8325-4 (a nonlysogenized derivative of NCTC 8325) with Clal, electrophoresed the digest on an agarose gel, and then transferred it to nitrocellulose. A 32P-labeled DNA fragment containing either attR or attL was used for blot hybridization because such a probe was partially homologous to the DNA fragment carrying attB. A 1.4-kb ClaI fragment that con-* Corresponding author. tained attB was identified. The fragment was isolated and cloned into the ClaI site of pBR322.The primary DNA fragments isolated were subjected to further restriction mapping to identify the sm...

Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites

“…Nonreplicative integration events are typical among temperate phages. The attachment site core of two temperate phages from S. aureus are the only core sequences from gram-positive bacteria that have been analyzed (18,19). They have in common a 5'-ATGGGA-3' sequence but otherwise share no extensive homology with the core sequences of phage from gram-negative bacteria.…”

“…Programmed DNA rearrangements mediated by site-specific recombination are described in several biological phenomena, including, among others, integration and excision of temperate bacteriophages (1,4,17,19,21,42) and Streptomyces spp. plasmids (5,6,23,25,37) into chromosomal DNA, DNA inversion controlling gene expression (12,36), control of plasmid amplification of the 2,um plasmid in Saccharomyces cerevisiae (9), dimer resolution and stable propagation of phage P1 (2), and resolution of cointegrates structures formed by transposons Tn3 and -yI (33).…”

Bacterial Two-component and Hetero-heptameric Pore-forming Cytolytic Toxins: Structures, Pore-forming Mechanism, and Organization of the Genes

“…In contrast, the attB for PV83-pro was the same as for 11, and these phages were integrated into the 9 bp-attB-core sequence in the intergenic region between purA and metA. 73,78,82) 3. Molecular evolution of PVL-carrying phages (1) Genomic classification of the reported staphylococcal temperate phages:…”

“…7). 73,78) Details are described bellow. (2) Genomic structure of PVL-carrying phages: (i) PVL: The complete nucleotide sequence of the PVL genome was determined for the first staplylococcal bacteriophage (Fig.…”

The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNAAlagene of Mycobacterium spp.

“…Recom binases belonging to the integrase family were found in temperate bacteriophages of both Gramnegative (Argos et at., 1986;Leong et al, 1986) and Gram-positive (Lee & Iandolo, 1986, 1988 The GenBank accession number for the sequence reported in this paper is AF030986. In mycobacteria, the only well-documented site-specific integration system is that of temperate mycobacteriophage LS.…”