Molecular characterization and transcriptional analysis of type 8 capsule genes in Staphylococcus aureus

Abstract

A 20.5-kb contiguous DNA fragment from Staphylococcus aureus Becker affecting type 8 capsule (CP8) biosynthesis was previously cloned. Sequencing analysis indicated that 16 open reading frames (ORFs) encoded within this fragment might be involved in CP8 synthesis. Using various plasmids containing DNA inserts derived from the 20.5-kb region, we showed by complementation of chemical mutants that 8 of the 16 ORFs were required for CP8 synthesis. To determine the involvement of the remaining eight ORFs, nonpolar gene-specific chromosomal mutations located in each of these ORFs were constructed. We found that three additional ORFs were also involved in the CP8 synthesis. Thus, 11 of the 16 ORFs were shown to affect CP8 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 hybridization using either the entire cap8 gene cluster or the internal fragments of individual ORFs as probes, one 17-kb cap8-specific transcript was detected. Using xylE as the reporter gene, we found that the promoter at the beginning of the cap8 operon was much stronger than any of the internal promoters. These results suggest that the cap8 genes are transcribed mainly as a single large transcript. In addition, Southern hybridization analyses showed that cap8H, cap8I, cap8J, and cap8K, located in the central region of the cap8 gene cluster, were CP8 specific.