We previously cloned a 19.4-kb DNA region containing a cluster of genes affecting type 1 capsule production from Staphylococcus aureus M. Subcloning experiments showed that these capsule (cap) genes are localized in a 14.6-kb region. Sequencing analysis of the 14.6-kb fragment revealed 13 open reading frames (ORFs). Using complementation tests, we have mapped a collection of Cap-mutations in 10 of the 13 ORFs, indicating that these 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in the synthesis of the capsule was demonstrated by constructing site-specific mutations corresponding to each of the three ORFs. Using an Escherichia coli S30 in vitro transcription-translation system, we clearly identified 7 of the 13 proteins predicted from the ORFs. Homology search between the predicted proteins and those in the data bank showed very high homology (52.3% identity) between capL and vipA, moderate homology (29% identity) between capI and vipB, and limited homology (21.8% identity) between capM and vipC. The vipA, vipB, and vipC genes have been shown to be involved in the biosynthesis of SalmoneUla typhi Vi antigen, a homopolymer polysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of the components of the staphylococcal type 1 capsule. The homology between these sets of genes therefore suggests that capL, capI, and capM may be involved in the biosynthesis of amino sugar, N-acetylgalactosamino uronic acid. In addition, the search showed that CapG aligned well with the consensus sequence of a family of acetyltransferases from various prokaryotic organisms, suggesting that CapG may be an acetyltransferase. Using the isogenic Cap-and Cap' strains constructed in this study, we have confirmed that type 1 capsule is an important virulence factor in a mouse lethality test.Most strains of Staphylococcus aureus produce capsular polysaccharides. To date, 11 distinct serotypes have been reported (19,44). Type 1 or 2 capsule-producing strains produce a large amount of capsule. As a result, these strains are mucoid, phage nontypeable, and clumping factor negative and exhibit a halo around the cells under negative staining with India ink. Although these strains are antiphagocytic and virulent to mice (27,31,36), they are rarely encountered clinically. On the other hand, type 5 and 8 strains are the predominant isolates, which account for about 80% in recent isolates from all sources (2,4,14,17,19,37,44). These strains, which are referred to as microencapsulated (50), produce a small amount of capsule and do not possess the characteristics typically associated with type 1 or 2 strains. Microencapsulated type 5 and 8 strains have been shown to be antiphagocytic (18); however, this result was not supported by recent studies (1, 5, 52).S. aureus M is a type 1 capsule producer isolated from an infected human hand laceration (41). Since the report of its isolation, strain M has been subjected to intensive study as a prototype of encapsulated staphylococci (see referen...

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 ...

The lysogenization of bacteriophage +11 in Staphylococcus aureus occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the prophage were cloned, and the nucleotide sequences were determined. The attachment sites share a very short common sequence of 10 base pairs.Bacteriophage 411 is a group B phage carried as a prophage in Staphylococcus aureus NCTC 8325 (9). Genetically, it is the most characterized of all staphylococcal phages. The viral DNA is a linear, double-stranded, terminally redundant molecule of about 45 kilobases (kb) (1,6,7). In addition to the viral attachment site and the immunity region, 10 genes have been located on a circularly permuted map (3,6). During lysogeny, insertion into the chromosome occurs by site-specific recombination between the viral attachment site (attP) and the att411 site (12) on the host chromosome. To define the attachment site on the viral genome more precisely and to study the site-specific recombination mechanism, we cloned and determined the nucleotide sequences of (i) the attachment site on the phage genome (attP), (ii) the attachment site on the bacterial chromosome (attB), and (iii) the attachment sites generated at the junctions between 411 and S. aureus DNA (attR and attL).Phage 411 DNA and bulk chromosomal DNA were isolated as previously described (2). We identified the fragments containing attR, attL, and attP directly by hybridization. Chromosomal DNA from 411-lysogenized strain and 411 DNA were digested with the restriction endonuclease ClaI. The fragments were separated by agarose gel electrophoresis on adjacent gel lanes and transferred to nitrocellulose by Southern blotting (14). The blot was probed with 32P-labeled 411 DNA, and the profiles were compared. Because integration occurs between attB and attP sites and because the genome of 441 is circularly permuted, 411 gene probes should identify two unique bands in the genomic DNA of the lysogenized strain and one unique band in the genomic DNA of phage 441. Two unique junction fragments that contained attL (on a 5.1-kb fragment) and attR (on a 4.3-kb fragment) were identified. One unique fragment (8 kb) from the 411 digest that contained attP was identified. These fragments were isolated and cloned into the ClaI site of pBR322.To identify the DNA fragment containing the attB site, we digested bulk chromosomal DNA from strain 8325-4 (a nonlysogenized derivative of NCTC 8325) with Clal, electrophoresed the digest on an agarose gel, and then transferred it to nitrocellulose. A 32P-labeled DNA fragment containing either attR or attL was used for blot hybridization because such a probe was partially homologous to the DNA fragment carrying attB. A 1.4-kb ClaI fragment that con-* Corresponding author. tained attB was identified. The fragment was isolated and cloned into the ClaI site of pBR322.The primary DNA fragments isolated were subjected to further restriction mapping to identify the sm...

We characterized the minimum length of the DNA sequence of the attachment sites involved in the integrative recombination of staphylococcal bacteriophage L54a. A DNA fragment carrying the functional viral attachment site (aiP) or the bacterial attachment site (altO) was sequentially trimmed, redoned, and tested for integrative recombination in vivo. The size of the functional aitd site was at least 228 base pairs (bp) but no more than 235 bp. The left endpoint of the aIdP site was located to between positions -142 and -140, whereas the right endpoint was located to between positions +86 and +93 with respect to the center of the core sequence. The atOB site was located to within a 27-bp sequence, from position -15 to + 12, which included the 18-bp core sequence.Staphylococcal bacteriophage L54a is a temperate phage that was originally discovered as one of the two prophages in Staphylococcus aureus PS54 (4). Its genome size is about 45 kilobases (kb), and the genome is circularly permuted (unpublished data). During lysogeny, bacteriophage L54a inserts its genome into the host chromosome. Integrative recombination occurs between the specific viral attachment site (attP) on the bacteriophage genome and the attL54a site (attB) on the host chromosome. As a result of integration, two additional attachment sites, attL and attR, are generated on the left and the right sides of the prophage genome, respectively. In excision, attL and attR recombine and regenerate attB and attP. The attB site is located at the 3' end of the lipase (geh) structural gene. As a consequence, lysogens of L54a are lipase negative because of insertional inactivation of the lipase structural ;ene (11,12). Curing of the prophage restores the lipase activity of the host. We have previously cloned and sequenced the DNA fragments containing the four attachment sites (13). All four att sites share an identical 18-base-pair (bp) core sequence. Apparently, recombination requires the core sequence. However, the question as to how much of the DNA sequence outside the core sequence is required for the function of the attachment sites attP and attB remains unanswered. Here we report the minimum sizes of the attB and attP sites. MATERIALS AND METHODSPhage and bacterial strains. The isolation and propagation of staphylococcal bacteriophage L54a were done as described previously (11). S. aureus RN4220 (10), a restrictiondeficient variant of S. aureus 8325-4, was used as the recipient in protoplast transformations. Protoplast transformation was performed by the procedure of Chang and Cohen (2). Staphylococcal bacteriophage transductions were performed at a multiplicity of infection of 0.1, as described previously (25). Escherichia coli LE392 was used for transformation with cloned DNA and for the preparation of plasmid DNA.Chenmcals and media. Media used for routine cultivation and detection of lipase activity have been described previously (11 Recombinant DNA methods. General DNA methods were carried out as described by Maniatis et al. (15). Rapid small-scale D...