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
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.
Methods
Phage 80 will be grown in liquid culture by infecting S. aureus strain RN322. Once the cells have lysed, the debris will be spun down and the phage will be precipitated from the lysate with polyethylene glycol. The phage will further be purified by banding in cesium chloride and the DNA will be extracted. Phage DNA will be physically sheared into 2-4kb fragments and purified by agarose gel electrophoresis. These fragments will be then be ligated into the cloning vector pSMART to create a genomic library. Individual plasmids from 4 96-well plates will be sequenced, using pSMART sequencing primers SL1 and SL2 and a commercial sequencing kit (such as the ABI PRISM® BigDye™ Terminators Cycle Sequencing Kit) that uses fluorescent dideoxynucleotides. Sequencing reactions will be analyzed on an automated sequencer. The sequence will then be assembled and annotated.
There may be some trouble getting the cloning to work with high efficiency, although this should be minimized by use of the pSMART kit which contains vector that is predigested and dephosphorylated. It is possible that when we assemble the individual sequences we will not obtain a single contig because there are regions of the genome that are difficult to clone. In this case, we will have to design phage-specific primers to amplify and sequence directly from phage genomic DNA in order to close the gaps. Prediction of most of the genes should be relatively straightforward, since many will have homologues in the other sequenced phages. The precise time frame of this project is not known, although it is expected to run through the year.
References
Iandolo J, Worrell V, Groicher K, Qian Y, Tian R, Kenton S, Dorman A, Ji H, Lin S, Loh P, Qi S, Zhu H, Roe BA. 2002. Comparative analysis of the genomes of the temperate bacteriophages φ11, φ12, and φ13 of Staphylococcus aureus 8325. Gene 289(1-2):109-18.
Ruzin A, Lindsay J, Novick P. Molecular genetics of SaPI1 - a mobile pathogenicity island in Staphylococcus aureus. Molecular Microbiology 41(2):365-377.