Sequencing the Genome of Bacteriophage 80

Introduction

Staphylococcus aureus is known to have a family of related pathogenicity islands (PI’s) that are mobilized and replicated by specific helper phages. These PI’s are characterized by genes encoding integrases and the presence of flanking direct repeats. SaPI1 and SaPI2 encode TSST-1, the toxic shock toxin, and are excised and replicated by certain staphylococcal phages and then packaged into phage-like infectious particles. These TSST-1 containing particles have smaller heads than the helper phage, associated with the smaller size of the PI genome. When the helper phages are absent the islands are stable with no detectable mobility.

SaPI1 is 15.2kb in length and has a direct repeat of 17 nucleotides. It contains a reading frame whose product is from the integrase family, but does not by itself code for excision. In the presence of phage 80a, SaPI1 can excise, replicate, and become encapsidated. 80a is not a naturally occurring staphylococcal phage. It was developed while trying to select a host range variant of another staphylococcal phage, 80. It is most likely a recombinant of 80 with two other temperate staphylococcal transducing phages, f11, and f13. Like 80a, f13 can also cause excision of SaPI1, but not replication. No excision or replication is caused by f11. It is not known whether 80 itself can induce SaPI1, because it does not grow in the strains carrying this pathogenicity island. However, phage 80 does induce excision and replication of a second TSST-1 PI, SaPI2. This PI, which is associated with isolates from menstrual strains, integrates into a different site in the S. aureus genome. The sequence of SaPI2 is not yet known, but it differs from SaPI1 in several respects. Phage 80 induces excision and replication for this island in a similar way to 80a and SaPI1.

What genes on the helper phages are responsible for excision and replication? The first step in this project will be to sequence the genome of phage 80. The genome sequences are known for f11 and f13, and sequencing of 80a is currently in progress in another lab. Once the sequence of 80 has been determined, comparative genomic analysis can be done among these four phages. This should allow identification of candidate genes for promoting SaPI excision and replication.

Progress Report

Phage 80 was grown in liquid culture by infecting S. aureus strain RN322. Once the cells had lysed, the debris was spun down and the phage was precipitated from the lysate with polyethylene glycol. The phage was further be purified by banding in cesium chloride and the DNA was then extracted. Phage DNA was physically sheared into 2-4kb fragments and purified by agarose gel electrophoresis. The ends of the fragments were repaired to convert overhangs into blunt cloning sites.  They were then ligated into the cloning vector pSMART and purified using QIA Spin Miniprep Kit.  The genome is approximately 45kb in length, 384 minipreps were made to give 25X coverage.

Goals for the Academic Year

The plasmids are being sent to NCI where they will sequence the genome using dideoxy fluorescent sequencing.  I will assemble the sequence using Phred-Phrap software.  Primers will be designed to fill any gaps.  The sequence will be annotated using TIGR's Glimmer software.

Budget

TBA

References