MOLECULAR CHARACTERIZATION AND TRANSCRIPTIONAL ANALYSIS OF TYPE-8 CAPSULE GENES IN STAPHYLOCOCCUS-AUREUS

A 20.5-kb contiguous DNA fragment from Staphylococcus aureus Becker af fecting type 8 capsule (CP8) biosynthesis was previously cloned. Seque ncing analysis indicated that 16 open reading frames (ORFs) encoded wi thin this fragment might be involved in CP8 synthesis. Using various p lasmids containing DNA inserts derived from the 20.5-kb region, we sho wed by complementation of chemical mutants that 8 of the 16 ORFs were required for CP8 synthesis. To determine the involvement of the remain ing eight ORFs, nonpolar gene-specific chromosomal mutations located i n each of these ORFs mere constructed. We found that three additional ORFs were also involved in the CP8 synthesis. Thus, 11 of the 16 ORFs were shown to affect CPS synthesis. Complementation analyses of these 11 type 8 capsule (cap8) genes affecting CP8 production showed several promoters within the cap8 gene cluster. However, by Northern hybridiz ation using tither the entire cap8 gene cluster or the internal fragme nts of individual ORFs as probes, one 17-kb cap8-specific transcript w as detected. Using xylE as the reporter gene, we found that the promot er at the beginning of the cap8 operon was much stronger than any of t he internal promoters. These results suggest that the cap8 genes are t ranscribed mainly as a single large transcript. In addition, Southern hybridization analyses showed that cap8H, cap8I, cap8J, and cap8K, loc ated in the central region of the cap8 gene cluster, were CP8 specific .