SaPI1 Interference and Bacteriophage 80a Capsid Size Determination

Introduction

            Bacterial pathogenicity islands (PIs) are genetic elements containing nonessential virulence genes. These islands are speculated to be highly motile due to characteristic flanking direct repeats (such as IS elements) and intergrase homologues. Staphylococcus aureus contains such a region, known as SaPI1, which carries the gene encoding the toxic shock syndrome toxin (TSST-1). SaPI1 is 15.2 Kb in length and contains flanking 17 bp direct repeats. In addition to tst, the gene for TSST-1, SaPI1 has a gene for another superantigen, sek, as well as a partial sequence of a third, sel. An int homologue is present, which allows for the integration of the element into the S. aureus chromosome. Also noted is ter, which creates a protein similar to the terminases of several other phages. Other than these genes, the function of the other ORFs present in SaPI1 are unknown.

            Ordinarily SaPI1 is stable while integrated into the S. aureus chromosome, but the presence of the bacteriophage 80a will cause the excision, replication, and packaging of SaPI1 into phage-like particles. Excision of the SaPI1 genome cannot happen in the absence of phage 80a, suggesting that a phage-encoded function is needed for motility. These SaPI1 particles are smaller than those packaging 80a DNA, and the transduction frequency of SaPI1 through these elements is extremely high.

            The observed interference of SaPI1 in the lytic growth and packaging of 80a is speculated to be controlled by the PI itself. Although the mechanism for interference by SaPI1 is unknown, one model is provided by previous studies with the P4/P2 coliphages Without the presence of P2, P4 exists in E. coli as a plasmid or prophage. Superinfection with P2 allows lytic growth of P4. P4 head particles are composed of the same protein monomers as those of P2, but P4 is able to control the assemblage of these proteins. This results in a smaller head only capable of packaging the P4 genome. Because of similarities between the SaPI1-80a and P4-P2 systems, it is worth investigating the unknown ORFs of SaPI1 for a gene responsible for this packaging interference.

Methods

            Primers designed from regions just up and downstream of the ORFs of interest: orf1, orf4, orf5, will be used for PCR amplification of the readings frames. The ORFs will be cloned into the vector plasmid, pCN51 using the restriction enzymes BamHI, EcoRI, and Nde 1. The plasmid contains the ermC gene, which will allow for the selection of S. aureus colonies which have taken up the plasmid. After transfering the plasmids into S. aureus clean of SaPI1, 80a phage will be introduced.

Possible Results

            The cloning and expression of these three ORFs may or may not illustrate a change in the phage head contruction of the resultant phage particles.  If not, there are over 20 other ORF sites which can be investigated  using the same cloning methods. A

gene responsible for head assembly interference is likely to be found because of the sid  gene of P4, which has been found to produce a prodect responsible for capsid size determination. Whether or not the product of the gene in SaPI1 is similar is questionable, being that S. aureus is a Gram positive bacteria while E. coli is Gram negative.

References

Charpentier E, Anton AI, Barry P, Alfonso B, Fang Y, Novick RP. Novel cassette-based shuttle vector system for gram-positive bacteria. Appl Environ Microbiol. 2004 Oct;70(10):6076-85.

Lindqvist BH, Deho G, Calendar R., 1993. Mechanisms of genome propagation and helper exploitation by satellite phage P4. Microbiology. Rev. 57: 683-702.

Novick, RP. 2002. Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus. Plasmid 49:93-105.

Ruzin A, Lindsay J, Novick P., 2002. Molecular genetics of SaPI1 - a mobile pathogenicity island in Staphylococcus aureus. Molecular Microbiology 41(2):365-377.