Promoter analysis of the cap8 operon, involved in type 8 capsular polysaccharide production in Staphylococcus aureus

The production of type 8 capsular polysaccharide (CP8) in Staphylococcus au reus is regulated in response to a variety of environmental factors. The ca p8 genes required for the CP8 production in strain Becker are transcribed a s a single large transcript by a primary promoter located within a 0.45-kb region upstream of the first gene of the cap8 gene cluster. In this study, we analyzed the primary cap8 promoter region in detail. We determined the t ranscription initiation site of the primary transcript by primer extension and identified the potential promoter sequences. We found several inverted and direct repeats upstream of the promoter. Deletion analysis and site-dir ected mutagenesis showed that a 10-bp inverted repeat of one of the repeats was required for promoter activity. We showed that the distance but not th e specific sequences between the inverted repeat and the promoter was criti cal to the promoter activity. However, insertion of a DNA sequence with two or four helix turns in this intervening region had a slight effect on prom oter activity. To demonstrate the biological significance of the 10-bp inve rted repeat, we constructed a strain with a mutation in the repeat in the S . aureus Becker chromosome and showed that the repeat affected CP8 producti on mostly at the transcriptional level, By gel mobility shift assay, we dem onstrated that strain Becker produced at least one protein capable of speci fic binding to the 10-bp inverted repeat, indicating that the repeat serves as a positive regulatory protein binding site. In addition, reporter gene fusion analysis showed that the cap8 promoter activity was influenced by va rious growth media and affected most by yeast extract. Our results suggest that yeast extract may exert its profound inhibitory effect on cap8 gene ex pression through the 10-bp inverted repeat element.