Bacterial pathogenicity islands (PIs) are genetic elements containing nonessential genes, often related to virulence. These islands are speculated to be highly motile due to characteristic flanking direct repeats (such as IS elements) and intergrase homologues. Staphylococcus aureus contains numerous PIs including 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 in the S. aureus chromosome, but the presence of 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 capsid particles are composed of the same protein monomers as those of P2, but P4 is able to control the assemblage of these proteins by way of the protein Sid This results in a smaller head only capable of packaging the smaller 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.

Pathogenicity island SaPIn3, found in S. aureus strain N315, has recently been investigated for small RNAs (sRNAs) which are primarily translation regulators. Seven different sRNAs were found to be expressed in the PI and some of these expression levels varied greatly among pathogenic strains. This variation suggests their importance in the regulation of S. aureus virulence elements.

Orf1 of SaPI1 was successfully cloned into the vector plasmid pCN51 and electroporated into a SaPI1-clean strain of S. aureus. Orf2 and orf3 were well on their way to being cloned as well when this method was abandoned due to problems with pCN51 expression in S. aureus strains. Other projects in the lab using pCN51 were unable to induce adequate expression of protein using this vector, so a new plasmid will be used: pPV72. Also, instead of cloning each orf individually, a large section of SaPI1 will be investigated to accelerate the process.

Orf15 of SaPI1 is a homologue to a phage helicase, a protein which is typically expressed early in phage expression. Because early, middle, and late genes tend to be clustered together in phage, orfs 17-21 are most likely involved with early gene expression. Capsid construction happens during the late stage of phage expression, so the gene interfering is most likely to be found with late genes. Therefore, the region from orf11 to ter (small terminase subunit) will be cloned (Fig. 1). Small terminase is involved in capsid head packaging, again suggesting the presence of late expressed genes in that region of SaPI1. Primers were designed from the end of the tst gene to the end of orf15 to be sure any promoter and terminator sequences for the region are included. The PCR product will then be digested with Pst 1 and ligated into pPV72. Plasmid pPV72 is an E. coli, S. aureus shuttle vector allowing tetracycline selection in both species.

The S. aureus strain containing the vector with insert will then be infected with 80a and plated alongside a control strain without the plasmid. Significant difference in pfu (plaque-forming units) would indicated disruption in 80a capsid formation as the copies of 80a genomic DNA packaged would be reduced. If such a disruption were seen, primers would then be designed internally within the tst-orf11 region to narrow down the location of the gene responsible for this disruption. If no disruption is seen initially, then another region of SaPI1 will be explored.

Also,the entire SaPI1 sequence will be searched for regions of simularity to the sRNA sequences found in SaPIn3. These sequences were found in intergenic (noncoding regions) so areas between ORFs will be focused on primarily.

Hopefully, by using this whole region approach instead of orf by orf, I will be able to locate the region containing the gene by the end of the summer. If all goes well, I will have it down to the exact orf itself responsible for the capsid interference. The vector should not give any problems, since it is not an expression plasmid, I am using the promoters on SaPI1 itself. The only big hurdle will be getting the first PCR amplification of an almost 5 Kb region. If this proves to be unrealisitic, then I will go ahead and design the internal primers to split the region in half.

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