Sequencing the Genome of Staphylococcal Phage 80
Abstract
The complete genome of Staphylococcus aureus phage 80 was sequenced.
The circularly permuted phage genome is 42,160 bp with an overall GC content
of 35.6%. Sixty-one potential open reading frames (ORFs) encoding proteins of
50 aa or more were identified. A predicted function was assigned to 26 of
these. Thirty other ORFs are homologous with hypothetical staphylococcal phage
proteins or putative prophage genes with unknown function. One protein is
homologous to a protein belonging to a methicillin resistant strain of S.
aureus, MRSA252, which has not previously been attributed to prophage DNA.
Four ORFs seem to belong only to phage 80, having no homologies in the GenBank
database. Phage 80 was most closely related to
fETA
in a genome comparison with other sequenced staphylococcal phages.
Introduction
Staphylococcus aureus is known to have a family of related pathogenicity islands, or PI’s, that are mobilized and replicated by specific helper phages. These PI’s are 15-20kb characterized by carriage of virulence genes, presence of flanking direct repeats, and presence of phage-like mobility genes encoding integrase, helicase, and terminase. SaPI1 and SaPI2 encode TSST-1, the toxic shock toxin, and each island is located on a specific location in the S. aureus genome. SaPI1 is located at the trp locus while SaPI2 is located at the tyrB locus. Both are excised and replicated by certain staphylococcal phages and then packaged into phage-like particles. These phage-like particles can then infect another S. aureus cell, and the SaPI DNA then integrates back into its distinct location on the genome. These infectious phage-like 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. 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. To identify mobility specific genes, comparative genome analysis must be done. In this study, we determined the DNA sequence of phage 80. Phage 80a and SaPI2 are currently being sequenced.
Materials and Methods
Phage 80 was grown in liquid culture by infecting S. aureus strain RN322 in CY broth and .06M glycerol phosphate at an multiplicity of infection (MOI) of 0.1. Once the cells had lysed, the debris was spun down and the phage was precipitated from the lysate with 10% (w/v) polyethylene glycol and .5M NaCl. The phage was further purified by CsCl density gradient centrifugation 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 using DNA-Terminator® End Repair Kit. Fragments approximately 1.5-3kb in size were purified by agarose gel electrophoresis. They were then ligated into a pSMART™-HCAmp cloning vector to create a genomic library. Plasmids were purified using QIAprep ® Spin Miniprep Kit and 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 assembled using SeqMan II and phredPhrap/Consed software. 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 sequence regions containing ambiguous bases in the assembly. ORFs greater than 50aa were identified using GeneMarkTM and TIGR’s Glimmer. Any discrepancies between the two programs were resolved by checking the sequence for candidate ribosomal binding sites. These ORFs were then compared to the database and the sequence was annotated. The genome was compared with those of other sequenced staphylococcal phages using the pairwise alignment algorithm of Wilbur and Lipman and displayed as a homology tree using the UPGMA method of Sneath and Sokal contained in the DNAMAN software package.
Results
The circularly permuted genome of phage 80 is 42,140 bp in length with a 35.6% GC content.
There were 61 predicted ORFs larger than 50aa (Fig. 1). Since the exact attachment site is not yet known, we have defined the genome start with ORF1, the int gene, which is predicted to be adjacent to attL. This facilitates alignment of the sequence with those of other staphylococcal phages in the database, most of which have been entered as prophage sequences. The predicted gene products were analyzed to find molecular weight, ribosomal binding site, closest homologue, and predicted gene function (Table 1). A homology tree was created using DNAMAN and shows that phage 80 is most closely related to fETA (Fig. 2).
Of phage 80’s 61 predicted ORF’s, functions have been proposed for 26, while 30 have homology to hypothetical Staphylococcal phage proteins or putative prophage genes. One ORF has homology to an MRSA252 protein that has not before been associated with phage and four that have no matches to any protein in the GenBank database. Phage 80 is most homologous to fETA sharing similarity in replication and morphogenesis. Lysis genes are close to that of f11 while the lysogeny genes resemble that of many different staphylococcal phages. The integrase gene is most similar to the integrase of phage 77 and 54a.
Fig. 1. Predicted ORFs larger than 50aa. ORF1 begins the genome with the int gene predicted to be adjacent to attL. Arrows indicate orientation.
