The Isolation And Cloning of Chromosomal Dna Specific for a Clonal Population of Staphylococcus Aureus By Subtractive Hybridisation

Subtractive hybridisation was used to screen for and identify conserved DNA sequences associated with clinical, clonal populations of Staphylococcus aureus. DNA from S. aureus strain ISP8 (a clinical isolate) was digested with TaqI and hybridised with randomly fragmented pooled DNA from 10 non-clinical (community) isolates of S. aureus. The mixture of DNA fragments was then ligated to AccI-digested and dephosphorylated pTZ19U. One recombinant plasmid (pWASA) was identified as containing specific DNA from strain ISP8; DNA sequencing revealed a 40-bp TaqI DNA fragment (WASA). PCR amplification and hybridisation analysis, with pWASA as a probe, showed that 84% of clinical isolates from a clonal line present in hospitals in major eastern Australian cities carried sequences homologous to WASA, compared with only 10% of community isolates. The isolates that hybridised were closely related by RFLP analysis to strain ISP8. the plasmid pWASA was used to identify, isolate and clone a 3.5-kb DraI fragment (DSA) from strain ISP8 total DNA. Sequence analysis of the DSA fragment identified two open reading frames (ORFs) of 2475 and 576 bp, respectively; the larger ORF1 contained a series of six tandem repeats, each consisting of 384 nucleotides. the nucleotide sequence of the repeats was 96% identical, and no significant sequence homologies to previously described protein sequences were identified. However, tandem repeats of amino acids are structural motifs characteristic of a number of gram-positive surface proteins, and are thought to play a role in generating genetic and phenotypic diversity in these and other bacteria.