The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.

The complete nucleotide sequence was determined for the Streptococcus downei (previously Streptococcus sobrinus) MFe28 gtfS gene which specifies a glucosyltransferase (GTF-S) producing water-soluble glucan. A single open reading frame which encodes a mature protein with a molecular weight of 147,408 (1,328 amino acids) and a putative signal peptide 36 or 37 amino acids in length was detected. GTF-S shares extensive sequence similarity with GTF-I (g#f) from S. downei and GTF-I (gtJB) and GTF-SI (g#U) from Streptococcus mutans. GTF-S contains a highly conserved enzymatic domain and C-terminal repeated sequences which appear to be involved in glucan binding. Comparison of the deduced GTF-S protein sequence with other sequenced GTF genes of mutans streptococci revealed that these C-terminal repeats occurred in all cases, although the patterns of repeated sequences varied with respect to each other and to the glucan-binding protein of S. mutans. GTF-S contains four C-terminal repeat sequences ranging from 49 to 51 amino acids in length and a partial repeat of 13 amino acids. Nuclear magnetic resonance analysis of the glucan produced by GTF-S revealed that the product consisted of more than 90% a-1,6-linked glucosyl residues.

A model for the protracted (30-day) colonization of smooth surfaces by Streptococcus gordonii that incorporates the nutrient flux that occurs in the oral cavity was developed. This model was used to characterize the biphasic expansion of the adherent bacterial population, which corresponded with the emergence of higherorder architectures characteristic of biofilms. Biofilm formation by S. gordonii was observed to be influenced by the presence of simple sugars including sucrose, glucose, and fructose. Real-time PCR was used to quantify changes in expression of S. gordonii genes known or thought to be involved in biofilm formation. Morphological changes were accompanied by a significant shift in gene expression patterns. The majority of S. gordonii genes examined were observed to be downregulated in the biofilm phase. Genes found to be upregulated in the biofilm state were observed to encode products related to environmental sensing and signaling.Tooth surfaces are persistently colonized by a complex but highly organized biota termed dental plaque, a microbial biofilm with the capacity to adapt to, and to endure, cyclic variation in nutrient availability as well as harsh mechanical and biological forces targeted at its containment and removal. Streptococcus gordonii is among the pioneering species to colonize a tooth surface (29,30,34,39). Binding of these organisms to the tooth enamel creates a template for the subsequent attachment of other bacteria in establishment of the complex oral biofilm (20, 22). As succeeding layers of different bacterial species attach to the plaque, new binding templates and nutritional microenvironments are formed, which may ultimately favor the attachment and residence of periodontal pathogens (6). The net effect is the establishment of an ordered community of heterogeneous microbial species, with each member playing a role in maintaining the vitality and structure of plaque. A key element in the formation and stability of the plaque biofilm, therefore, is the persistent colonization of the smooth surface of the tooth at the base of this complex community.Several studies have now demonstrated that cells existing in the biofilm state have phenotypic characteristics distinct from those of their planktonic counterparts, with significant changes in the patterns of gene expression (9, 45). This differential expression appears to be governed by communication between bacteria of the same or other species, in addition to cues emanating from the host and the environment (23). Specific intercellular communication mediated by N-(3-oxododecanoyl)-L-homoserine lactone has been shown to be central to the differentiation of the biofilm architecture by Pseudomonas aeruginosa (11).Little is known of the physiologic changes that accompany persistent colonization of the tooth surface by pioneering species in the formation of the dental plaque biofilm. It was therefore of interest to develop a model of protracted smoothsurface colonization with cyclic variation in nutrient availability as a first approx...

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The gtfI and gtfS genes of Streptococcus downei were investigated to determine the contribution of the respective enzymes to glucan production in the presence and absence of other glucosyltransferases. Extracts of Escherichia coli expressing cloned gtfS produced a short linear dextran from sucrose which could act as a primer for insoluble glucan synthesis when mixed with extracts of a strain expressing recombinant gtfI. To elucidate the contribution of gtfS to glucan production by S. downei, a mutant was constructed by insertionally inactivating gtfS. S. downei (gtfS mutant) colonies exhibited a marked phenotypic change on sucrose-containing media and a decreased ability to adhere to glass and produced no detectable water-insoluble glucan. These experiments confirm that expression of gtfS is essential for normal insoluble glucan synthesis by S. downei.