ABSTRACTM102AD is the new designation for aStreptococcus mutansphage described in 1993 as phage M102. This change was necessitated by the genome analysis of anotherS. mutansphage named M102, which revealed differences from the genome sequence reported here. Additional host range analyses confirmed thatS. mutansphage M102AD infects only a few serotype c strains. Phage M102AD adsorbed very slowly to its host, and it cannot adsorb to serotype e and f strains ofS. mutans. M102AD adsorption was blocked by c-specific antiserum. Phage M102AD also adsorbed equally well to heat-treated and trypsin-treated cells, suggesting carbohydrate receptors. Saliva and polysaccharide production did not inhibit plaque formation. The genome of this siphophage consisted of a linear, double-stranded, 30,664-bp DNA molecule, with a GC content of 39.6%. Analysis of the genome extremities indicated the presence of a 3′-overhangcossite that was 11 nucleotides long. Bioinformatic analyses identified 40 open reading frames, all in the same orientation. No lysogeny-related genes were found, indicating that phage M102AD is strictly virulent. No obvious virulence factor gene candidates were found. Twelve proteins were identified in the virion structure by mass spectrometry. Comparative genomic analysis revealed a close relationship betweenS. mutansphages M102AD and M102 as well as withStreptococcus thermophilusphages. This study also highlights the importance of conducting research with biological materials obtained from recognized microbial collections.

This is the first report of lysogenic bacteria and their inducible viruses in infected root canals.

Three phages ofStreptococcus mutans were obtained and partially characterized. The three phages, designated M102, e10, and f1, were found to be strictly lytic, with host ranges restricted to only serotype c, e, and f strains of this species, respectively. Phage sensitivity was not correlated with the presence of plasmids, at least in host strains of serotypes c and e. Each phage produced clear plaques in a number of standard media, even in the presence of sucrose, indicating that the extracellular glucan polysaccharides (mutan) produced by the hosts from this substrate do not prevent phage adsorption and growth. The phages were similar in size and morphology, having icosahedral heads and long (283-287 nm), flexible, noncontractile tails. The genome of each phage was found to consist of linear, double-stranded DNA, 31-35 kb in length, with a base composition of 37-38% G+C. Restricting phage DNAs with four enzymes produced fragment patterns unique to each phage, but common bands between M102 and e10 and between e10 and f1 were produced byBamHI. Labeled e10 and M102 DNAs hybridized strongly with all three phage DNAs, indicating that they share some common sequences. The three phages appear to be more similar than expected and probably evolved from a common ancestor.

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these speciesspecific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.Phages of oral bacteria have been studied in this laboratory for a number of years since we believe they are likely to play a significant ecological role in regulating the oral microflora. We are also interested in phage-encoded lytic enzymes as potential diagnostic tools (10,19,23) and as therapeutic agents (1,6,7,13) to control specific oral pathogens. Actinomyces naeslundii is a gram-positive, facultative anaerobe that is found in high numbers in dental plaque and is believed to be causally involved in gingivitis and root surface caries in the oral cavity, in addition to actinomycosis in other regions of the body (22). The cell wall of this organism is resistant to salivary lysozyme and most other peptidoglycan-hydrolyzing enzymes, even though it is chemically similar to many other gram-positive cell walls that are lysozyme sensitive (18). Phage Av-1, originally isolated in this laboratory (3), is a small group I phage that is lytic for several human strains of A. naeslundii (2). This implies that its genome encodes an endolysin that can degrade the cell wall of this species. Preliminary experiments in this laboratory demonstrated that phage lysates of Av-1 contained a lytic enzyme that is specific for A. naeslundii. To clone the Av-1 lysin gene in Escherichia coli, which is also resistant to the enzyme, we devised an indirect strategy in which the functional activity of a plasmid-encoded endolysin is restored via complementation of a defective holin. Since holins are not species specific, we constructed a cloning vector to isolate plasmids containing phage inserts that express lethal, holin-like activity in E. coli. By sequencing these inserts, identifying potential open reading frames (ORFs), and analyzing their deduced amino acid sequences, putative holins could be tentatively i...

A sterile-filtered, liquid medium composed of one-half-strength APT broth and 4% (wt/vol) yeast extract was found to support the production of bacteriocins by Streptococcus mutans strains BHT and GS-5. Culture supernatants, adjusted to pH 7.0 and sterilized by filtration, contained bacteriocin-like activity, which could be demonstrated by spotting dilutions onto top agar lawns seeded with Streptococcus pyogenes as the sensitive indicator and by adding dilutions to logphase indicator broth cultures. A quantitative assay was developed for BHT bacteriocin, based on its lethal effects. Bacteriocin production did not occur until after the log phase of growth had ceased and was not inducible by ultraviolet irradiation or treatment with mitomycin C. Non-bacteriocinogenic clones of strain BHT occurred spontaneously at high frequency, suggesting control by a plasmid, but this frequency was not increased by treatment with the plasmidcuring agents acridine orange and ethidium bromide.