(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity, which was expanded to 53% when conservative amino acid changes were included. When the streptococcal protein was compared with an AAD(3')(9) protein of E. coli, the degrees of identity were 27 and 47%, on the basis of actual amino acids and conservative replacements, respectively. The cloning and nucleotide base sequence analyses of the spectinomycin AAD(9) determinant from E. faecalis that results in high-level Spr when transferred to S.sanguis or E. coli are presented.

The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coliStreptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM␤1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(؊). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289⌬202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289⌬202 were mobilizable by pAM␤1 into S. sobrinus, with frequencies of 3 ؋ 10 ؊6 and 1 ؋ 10 ؊7 transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EII Suc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289⌬202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM␤1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in double crossovers was confirmed by phosphotransferase system assays. This is the first report of allelic replacement in S. sobrinus.Streptococcus mutans and Streptococcus sobrinus, members of the mutans streptococci, are principal agents of human dental caries (32). While S. mutans is detected with greater frequency in the oral cavity than S. sobrinus (22), fresh isolates of S. sobrinus have been more cariogenic than freshly isolated S. mutans (12).Both the presence of acidogenic bacteria on the tooth surface and a source of fermentable carbohydrate are essential for caries formation (36, 52). Sucrose is the most common dietary sugar, and its consumption has been positively correlated with caries incidence (47, 51). The metabolism of sucrose by S. mutans and S. sobrinus is complicated, as there are several routes by which this carbohydrate can be processed (reviewed in reference 24). Less than 10% of available sucrose is converted to extracellular polysaccharides through the action of glucosyltransferases and, in the case of S. mutans, fructosyltransferases. The majority of available car...

The complete nucleotide sequences of Streptococcus sobrinus 6715 scrA and scrB, which encode sucrosespecific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-phosphate hydrolase, respectively, have been determined. These two genes were transcribed divergently, and the initiation codons of the two open reading frames were 192 bp apart. The transcriptional initiation sites were determined by primer extension analysis, and the putative promoter regions of these two genes overlapped partially. The gene encoding enzyme IIScr, scrA, contained 1,896 nucleotides, and the molecular mass of the predicted protein was 66,529 Da. The hydropathy plot of the predicted amino acid sequence indicated that enzyme I1Scr was a relatively hydrophobic protein. The gene encoding sucrose-6-phosphate hydrolase, scrB, contained 1,437 nucleotides. The molecular mass of the predicted protein was 54,501 Da, and the encoded enzyme was hydrophilic. The predicted amino acid sequences of the two open reading frames exhibited approximately 45 and 70% identity with those encoded by scrA and scrB, respectively, from Streptococcus mutans GS5. Homology also was observed between the N-terminal region of the S. sobrinus 6715 enzyme IISr and other enzyme Ils specific for the glucopyranoside molecule, all of which generate glucopyranoside-6phosphate during translocation and phosphorylation of the respective substrates. The sequence of the C-terminal domain of the S. sobrinus 6715 enzyme IIscr shared significant homology with enzyme IIIGIc from Escherichia coli and SalmoneUa typhimurium and with the C-terminal domain of enzyme IlBg' from E. coli, indicating that the two functional domains, enzyme IIScr and enzyme IIIScr, were covalently linked as a single polypeptide in S. sobrinus 6715. The deduced amino acid sequence of the gene product of S. sobrinus scrB shared strong homology with sucrase from Bacilus subtilis, Klebsiella pneumoniae, and Vibrio alginolyticus, suggesting conservation based on the physiological roles of these proteins. * Corresponding author.ing EIIs"1 scrA, and sucrose-6-phosphate hydrolase, scrB,