Seven structural types of RPS have been identified, each composed of a somewhat different hexa- or heptasaccharide repeating unit. Each repeating unit contains a host-like motif, either beta-GalNAc1-3Gal (Gn) or beta-Gal1-3GalNAc (G) that functions as a recognition site for adhesin binding and a non-host-like antigenic region. The adhesins present on certain oral species recognize both Gn and G types of RPS whereas those present on other species are specific for one receptor type or the other. The gene clusters for these polysaccharides encode six or seven putative glycosyltransferases, the number required for synthesis of the corresponding lipid-linked hexa- or heptasaccharide repeating unit, a flippase (Wzx), a polysaccharide polymerase (Wzy) and other genes that do not directly affect polysaccharide structure. During the past year, comparative molecular studies of a major RPS group, consisting of polysaccharides that contain Glc, Rha, Galp, Galf and GalNAc, were completed. The results indicate that the diverse biological properties of these polysaccharides depend on a relatively small number of genes for allelic glycosyl or glycoslyphosphate transferases that act in different combinations. In addition to types of RPS that contain Glc and Rha, other types are known that lack these sugars but contain ribitol-5-phosphate in addition to Galp, Galf and GalNAc. Examples include the RPS of S. oralis C104 and an antigenic variant of this polysaccharide identified on S. oralis SK144. Current results from sugar analysis and NMR showed that the RPS of these S. oralis strains are identical except for the linkage between Gal and ribitol, which is alpha 1-1 in stain C104 verses alpha 1-3 in strain SK144.? ? Thirteen comparable genes were identified in the rps clusters of strains C104 and SK144. These included six genes for different transferases, wzy for a polymerase, wzx for a flippase and glf for conversion of UDP-Galp to UDP-Galf. Several of the genes for transferases were found to exhibit high homology with genes that were previously identified in the capsular polysaccharide (CPS) gene clusters of S. pneumoniae serotypes 10A and 10F. These included wcjG, which is predicted to encode a membrane-associated transferase that initiates CPS biosynthesis in S. pneumoniae. The replacement of the wcjG homologue in strain C104 with a non-polar erm cassette abolished cell surface RPS production. Plasmid-based expression of wcjG from S. pneumoniae serotype 10A in the resulting mutant restored RPS production. Surprisingly, RPS production by this mutant was also restored by expression of wbaP from Salmonella enterica; the encoded transferase (WbaP) initiates O-polysaccharide biosynthesis in this gram-negative species by linking Galp-phosphate to a carrier lipid. Thus, the ability of wbaP to complement the deletion of wcjG in S. oralis suggests that the initial step in RPS synthesis is the WcjG-mediated transfer of Galp. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the RPS cluster of strain C104 with the corresponding gene (wefN) of strain SK144. The present findings extend the characterization of ribitol-5-phosphate-containing types of RPS to the molecular level and suggest a common ancestry for these polysaccharides and S. pneumoniae serogroup 10 CPS.? ? Adhesion of bacteria to surfaces generally depends on thread-like, cell surface structures referred to as fimbriae or pili. These structures on gram-negative bacteria are composed of subunits that are held together by strong noncovalent interactions. In contrast, the fimbrial subunits of gram-positive microorganisms such as A. naeslundii, are linked covalently by the enzyme sortase, which cleaves between Thr and Gly in the cell wall sorting motif of secreted fimbrial subunits, and links the newly formed C-terminal Thr to a specific Lys in the pilin motif of the adjacent subunit. We have now identified an additional cross-link in FimP, the structural subunit of A. naeslundii type 1 fimbriae. Purified type 1 fimbriae were subjected to dilute acid hydrolysis followed by trypsin digestion. Analysis of the resulting digest by liquid chromatography-mass spectrometry revealed a number of free peptides that covered more than 80% of the FimP sequence. In addition, a triply charged ion was identified that showed two fragment ion series representing peptides from different regions of FimP. The nature of the linkage between the two FimP sequences was established following carboxypeptidase digestion and subsequent MS/MS analysis of the remaining structure. The results revealed an isopeptide bond between specific Lys and Asp residues. We predict that this bond represents an intra-subunit cross-link and have initiated site directed mutagenesis studies of fimP to test this hypothesis.
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