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 phages, 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 direct terminal repeats 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 of the SaPIs? 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.
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 purified by banding in cesium chloride and the DNA was then extracted. Phage DNA was physically sheared into 2-4kb fragments using a HydroShearTM
instrument and the ends of the fragments were repaired to convert overhangs into blunt cloning sites. Fragments approximately 1.5-3kb in size were purified by agarose gel electrophoresis. They were then ligated into a pSMART cloning vector to create a genomic library. Individual plasmids from 4 96-well plates were sequenced and analyzed at NCI, using pSMART sequencing primers SL1 and SL2 and fluorescent dideoxynucleotides (such as the ABI PRISM® BigDye™ Terminators Cycle Sequencing Kit) and analyzed on an automated sequencer.
The sequence was then assembled using SeqMan II software and phredPhrap/Consed to confirm results. Before the genome was fully sequenced there were several areas of the genome with only one strand coverage. Primers were designed for PCR and sequencing reactions to increase the coverage. Primers were also designed to resequence regions containing ambiguous bases in the assembly. Once sequenced fully, ORFs greater than 50 aa were identified using GeneMarkTM. The sequence will be analyzed for ORFs another time using TIGR’s Glimmer software. These ORFs will then be compared to the database and the sequence will be annotated.
The primary goal of this summer will be to complete the assembly and annotation of the 80 genome sequence and prepare it for submission to GenBank. Phage genome sequences are generally deposited either as virion DNA or as prophage DNA. The genome sequence we have obtained is circular, indicating that the virion DNA is circularly permuted, so that there are no discrete ends. The other staphylococcal phages in the database were sequenced as prophages. For comparative purposes, therefore, it makes the most sense to enter the 80 sequence as prophage DNA as well. This requires identification of the phage attachment site and flanking bacterial DNA. This sequence identification will be accomplished by using Inverse PCR, where the primers developed walk the opposite direction identifying the flanking sequence to a known piece of DNA. Genomic DNA from a strain lysogenic for phage 80 will be digested with a restriction endonuclease, re-ligated to form circles, and amplified with primers from the genes that are predicted to be near the ends of prophage DNA. The phage-bacterial DNA junctions will be sequenced from these amplicons. Once the prophage ends have been identified, the sequence will be prepared for entry into GenBank.
When phage 80a is sequenced, these two genomes will be compared to those of f11 and f13 to identify candidate genes involved in pathogenicity island mobilization.
References
A.L. Delcher, D. Harmon, S. Kasif, O. White, and S.L. Salzberg. Improved microbial gene identification with GLIMMER (306K, PDF format) Nucleic Acids Research, 27:23, 4636-4641. http://www.tigr.org/~salzberg/glimmer2.pdf
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.
Lindsay JA, Ruzin A, Ross HF, Kurepina N, Novick RP. 1998. The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Molecular Microbiology 29(2):527-543.
Novick, RP. 2002. Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus. Plasmid 49:93-105.
Phred-Phrap Version: 4.0 Copyright (C) 2002-2006 by Deborah A. Nickerson, Scott Taylor, Natali Kolker and Jim Sloan University of Washington
Ruzin A, Lindsay J, Novick P. Molecular genetics of SaPI1 - a mobile pathogenicity island in Staphylococcus aureus. Molecular Microbiology 41(2):365-377.
S. Salzberg, A. Delcher, S. Kasif, and O. White. http://www.tigr.org/software/glimmer/glimmer-nar.pdf (73K, PDF format) Nucleic Acids Research 26:2 (1998), 544-548.