Transcriptional analysis of type 1 capsule genes in Staphylococcus aureus.
Serotype 1 capsule of Staphylococcus aureus plays an important role in staphylococcal infections. A 14.6 kb chromosomal DNA fragment containing 13 cap1 genes responsible for the synthesis of type 1 capsule in S. aureus strain M has been previously cloned and sequenced in our laboratory. These genes are closely linked and are transcribed in one orientation. To study the transcription of these genes, we employed genetic complementation using a set of plasmids with various deletions in the 14.6 kb region to complement mutants mapped in each of the 13 genes. We found that there were six transcriptional units. By Northern hybridization using the entire gene cluster as a probe, we identified a large 14 kb band and several smaller bands ranging from 0.3 to 6 kb. The 14 kb band was also identified by various individual gene probes, suggesting that all 13 genes are transcribed into a large transcript. The 14 kb transcript and the smaller bands were not detected when a 533 bp fragment containing the potential promoter region upstream of the first gene (cap1A) was deleted from the chromosome, suggesting that the small transcripts are the degradation (processing) products of the 14 kb transcript. The activities of the promoters identified by genetic complementation tests were quantitatively measured by transcriptional fusions using xylE as the reporter gene. The result showed that the promoter preceding the first cap1 gene was 45-198-fold higher than the downstream promoters. The result from gene-fusion studies is in agreement with the Northern analysis in that only the transcript transcribed from the first promoter was detected. Taken together, these results suggest that the cap1 genes are organized as a large operon with at least five weak internal promoters, and the long polycistronic transcript is processed (degraded) into several smaller transcripts. The transcription initiation site of the primary transcript was mapped by S1 nuclease mapping and the major promoter was defined by deletion analysis of the promoter-xylE fusion. In addition, we showed that the internal promoters, despite their weak activities, were sufficiently effective at expressing their downstream genes required for producing capsule, compared to the wild-type strain.
Ouyang
S
S
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Centre, Kansas City 66160, USA.
Lee
C Y
CY
D016667:Bacterial Capsules / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D005798:Genes, Bacterial / Q000235:genetics*; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013211:Staphylococcus aureus / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins