The nucleotide sequences of two gene clusters, cap5 and cap8, involved in the synthesis of Staphylococcus aureus type 5 and type 8 capsular polysaccharides (CPS), respectively, were determined. Each gene cluster contained 16 ORFs, which were named cap5A through cap5P for type 5 CP and cap8A through cap8P for type 8 CP. The cap5 and cap8 loci were allelic and were mapped to the Smal-G fragment in the standard SmaI map of Staph. aureus strain NCTC 8325. The predicted gene products of cap5A through cap5G and cap% through cap5P are essentially identical to those of cap8A through cap8G and cap8L through cap8P, respectively, with very few amino acid substitutions. Four ORFs located in the central region of each locus are type-specific. A comparison of the predicted amino acid sequences of cap5 and cap8 with sequences found in the databases allowed tentative assignment of functions to 15 of the 16 ORFs. The majority of the capsule genes are likely to be involved in amino sugar synthesis; the remainder are likely to be involved in sugar transfer, capsule chain-length regulation, polymerization and transport. -I

FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).

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.Staphylococcus aureus strains producing type 5 capsular polysaccharide (CP5) and CP8 account for more than 80% of clinical staphylococcal isolates (2, 3, 14, 37). These strains are referred to as microencapsulated, as they produce a small amount of CP on the cell surface (47). In comparison, rarely isolated type 1 and type 2 strains produce a large amount of CP, which results in a mucoid phenotype when these strains are grown on solid agar plates. CP1 and CP2 have been shown to be antiphagocytic virulent factors (29,30,35). However, the role of CP5 and CP8 of microencapsulated strains in virulence has been controversial (1,4,16,45,48). The controversy may stem from the fact that different systems or animal models were used by different investigators. Nevertheless, most recently, Fattom et al. (10) were able to show that the antibodies against CP5 and CP8 were protective against S. aureus infections when immunized mice were challenged intraperitoneally. A recent study also suggested that CP5 and CP8 were adhesins (43).CP8 is a trisaccharide-repeated polysaccharide with the following structure:Its structure is almost identical to that of CP5 except for the location of O acetylation and the position of the linking of the monosaccharides (12, 17, 32). Molecular characterization of the genes required for CP expression in S. aureus has not been initiated until recently. Our laboratory has reported the cloning and characterization of a cluster of 13 cap1 genes required for the biosynthesis of CP1 (22,23,29). The cloning of a type 5 capsule (cap5) gene from S. aureus Reynolds ...

This study addresses the regulation of Staphylococcus aureus type 8 capsular polysaccharide (CP8) expression by the global regulators agr and sarA. We analyzed CP8 production, cap8-specific mRNA synthesis, and blaZ reporter gene activities of the transcriptional and translational fusions in strain Becker and its agr, sarA, and agr-sarA isogenic mutants during different phases of bacterial growth. In the wild-type strain, cap8 mRNA was undetectable until the mid-logarithmic phase of growth, whereas CP8 production was undetectable until 2 h later, at the onset of stationary phase. The delay most likely reflects the time needed for completing CP8 synthesis resulting from translation of cap8 mRNA. The agr mutation caused drastic reductions in CP8 production and cap8 gene transcription, suggesting that agr is a major positive regulator of CP8 expression. The results of gene fusion studies indicated that regulation by agr is exerted at the transcriptional level. In contrast, the sarA mutation caused only a slight reduction in cap8 mRNA synthesis and reporter gene activities. By comparing CP8 production and cap8 transcription, we observed that sarA affected CP8 production both trancriptionally and posttranslationally. We showed that agr was a major activator for cap gene expression not only in type 8 strain Becker but also in strains representing the four agr groups.More than 90% of Staphylococcus aureus strains produce capsular polysaccharide (CP). Eleven serotypes of staphylococcal CP have been identified, but only CP type 1 (CP1), CP2, CP5, and CP8 have been chemically characterized. CP1 and CP2 have been shown to be antiphagocytic virulence factors. Strains producing CP1 and CP2 are heavily encapsulated; however, these strains are rarely isolated clinically. In fact, more than 80% of clinical isolates produce either CP5 or CP8 (see reference 23 for a review). Recently, CP5 has been shown to play an important role in the pathogenesis of S. aureus, most probably by allowing the organism to resist uptake and killing by phagocytes (1,27,43). Because of their prevalence, CP5 and CP8 have been used as targets for vaccine development, and specific antibodies against CP5 and CP8 have been shown to be protective against S. aureus infections (13, 24).The cap1, cap5, and cap8 gene clusters, required for the synthesis of CP1, CP5, and CP8, respectively, have been cloned and sequenced. The cap5 and cap8 operons are allelic, whereas the cap1 locus is located at a different location. Twelve of the 16 genes in the cap5 and cap8 operons have high degrees of similarity, which reflects the fact that the repeating units of CP5 and CP8 are almost identical (23). Transcriptional analyses have shown that all 16 genes of the cap8 locus are transcribed as a large transcript from a major promoter upstream of the first gene, cap8A. Although several internal promoters within the cap8 gene cluster have also been identified by genetic complementation and reporter gene fusion studies, these internal promoters are much weaker than the primary pro...

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