SEQUENCE-ANALYSIS AND MOLECULAR CHARACTERIZATION OF GENES REQUIRED FOR THE BIOSYNTHESIS OF TYPE-1 CAPSULAR POLYSACCHARIDE IN STAPHYLOCOCCUS-AUREUS

We previously cloned a 19.4-kb DNA region containing a cluster of gene s affecting type 1 capsule production from Staphylococcus aureus M. Su bcloning experiments showed that these capsule (cap) genes are localiz ed in a 14.6-kb region. Sequencing analysis of the 14.6-kb fragment re vealed 13 open reading frames (ORFs). Using complementation tests, we have mapped a collection of Cap(-) mutations in 10 of the 13 ORFs, ind icating that these 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in the synthesis of the capsu le was demonstrated by constructing site-specific mutations correspond ing to each of the three ORFs. Using an Escherichia call S30 in vitro transcription-translation system, we clearly identified 7 of the 13 pr oteins predicted from the ORFs. Homology search between the predicted proteins and those in the data bank showed very high homology (52.3% i dentity) between capL and vipA, moderate homology (29% identity) betwe en capI and vipB, and limited homology (21.8% identity) between capM a nd vipC. The vipA, vipB, and vipC genes have been shown to be involved in the biosynthesis of Salmonella typhi Vi antigen, a homopolymer pol ysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of the components of the staphylococcal type 1 capsule. The h omology between these sets of genes therefore suggests that capL, capI , and capM may be involved in the biosynthesis of amino sugar, N-acety lgalactosamino uronic acid. In addition, the search showed that CapG a ligned well with the consensus sequence of a family of acetyltransfera ses from various prokaryotic organisms, suggesting that CapG may be an acetyltransferase. Using the isogenic Cap(-) and Cap(+) strains const ructed in this study, we have confirmed that type 1 capsule is an impo rtant virulence factor in a mouse lethality test.