Family of surface adhesins of dental streptococci, including P1 of was engineered. market in the oral cavity. The 185,000-serotype (35, 69) or SSP-5 of (14). DNA sequences Tubacin homologous to serotypes serotype serotype (37), and (15). In addition, Ma et al. (41) shown by Southern analysis that chromosomal DNAs from viridans and nonviridans streptococci which did not hybridize with full-length (ORF iota [9]) and within plasmid pCF10, comprising regulatory and structural genes involved in pheromone-inducible conjugation in (also includes a homologous carboxy-terminal proline-rich website believed to be involved in cell surface localization in the absence of a conserved LPXTG motif (56). In addition, the fibronectin binding proteins of and consist of proline-rich tandem repeat segments just carboxy terminal to their fibronectin binding domains which include the amino-acid sequence motif PTPPT common to the P region of P1 (29, 62, 64). Repeated proline-rich segments will also be present in the published sequences encoded by genes encoding intermedilysin (a cytolytic element of (72), immunogenic secreted protein of (44), the immunoglobulin A (IgA) Fc binding protein of group B streptococci (27), and several proteins indicated by a wide variety of bacterial varieties (10, 21, 26, 36, 52, 53, 63, 66). While the function of most microbial proline-rich repeat domains is not understood, it has been suggested that such sequences may be involved in protein-protein relationships (21, 55, 71). The function of the P region of P1 is not yet known. Nakai et al. (48) reported the P region can bind to the PAc (P1) molecule itself and proposed that this section may contribute to spontaneous self-aggregation of the molecule. Munro et al. (46) shown that a fragment of P1 (amino acid residues 816 to 1213) which includes the P region was inhibitory to adherence of to saliva-coated hydroxyapatite. Kelly et al. (32) subsequently identified a specific segment (amino acid residues 1005 to 1044) carboxy terminal to the P region as being involved in adhesion to salivary components. Other investigators have implicated amino-terminal sequences including the alanine-rich repeats (A region) in the adhesion function of P1 (11, 24, 48). A second manifestation of the interaction of P1 and related molecules with salivary components is cell-cell aggregation (13, 15). In a previous study, anti-P1 MAbs were used to inhibit adherence and aggregation mediated by the high-molecular-weight salivary agglutinin glycoprotein. MAbs specific for different epitopes demonstrated marked differences in their relative abilities to inhibit each of these processes (6), suggesting that P1 possesses adherence-specific and aggregation-specific functional domains which are not confined within discrete contiguous segments of P1. To understand the relative contribution of the Tubacin P region to the biological properties of P1, a dual strategy was undertaken. A were evaluated for reactivity with a panel of 11 anti-P1 MAbs and three polyclonal antisera. The results suggest that the P region is an integral component of conformational epitopes within the central portion of P1. The internally deleted for expression, intracellular stability, and/or translocation of the molecule to the cell surface. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. Serotype NG8 and the strains found in these tests included MC1061, SURE (Stratagene, La Jolla, Calif.), INVF (Invitrogen), TOP (Invitrogen), and M15(pREP4) (QIAGEN, Santa Clarita, Calif.). was cultivated aerobically at 37C with strenuous shaking in Luria-Bertani broth (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 1% [wt/vol] NaCl, pH 7.0) supplemented with ampicillin (50 to 100 mg/ml) or kanamycin (25 to 50 mg/ml) while appropriate. Plasmids pUC18, pCRII (Invitrogen), pMal-p (New Britain BioLabs, Inc. [NEB], Beverly, Mass.), pQE30 (QIAGEN), and pDL289 supplied by D (kindly. LeBlanc) (7) had been utilized as cloning and manifestation vectors. TABLE 1 Plasmids TSLPR and bacterial?strains Planning of plasmid and chromosomal DNAs. NG8 chromosomal DNA was ready as referred to previously (4). Plasmid DNA was purified with a revised alkaline lysis-polyethylene glycol precipitation treatment (1a) (Applied Biosystems, Inc., Foster Town, Calif.). Building and PCRs of recombinant substances. PCR amplification of (69) sequences and utilized to amplify DNA upstream from the P area, like the NG8 chromosomal DNA as the template and polymerase (Promega, Madison, Wis.), beneath the pursuing circumstances: (we) denaturation at 94C for 2 min; (ii) denaturation at 94C for 45 s, primer annealing at 53C for 1 min 30 s, and primer expansion at 72C for 3 min for 30 cycles; and (iii) primer expansion at 72C for yet another 7 min. The ensuing 2,663- and 1,982-bp gene fragments had been cloned in to the TA cloning vector pCRII (Invitrogen) with INVF as the sponsor stress. Two clones with inserts in opposing orientations with regards to the Plac promoter of pCRII had been utilized. Each purified plasmid was digested with MC1061. This plan Tubacin led to reconstitution from the vector and in-frame ligation of the internally deleted.

Family of surface adhesins of dental streptococci, including P1 of was
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