| 2237 Characterization of Genetic Transformation in Streptococcus oralis 34 | ||
|
A.P. BURLESON1, D.S. BLEHERT2, and P.E. KOLENBRANDER2, 1Medical University of South Carolina, Mount Pleasant, USA, 2NIH/NIDCR, Bethesda, MD, USA Objectives: Streptococci constitute 60-90% of the bacteria that initially colonize the tooth surface following a professional cleaning. Of these known early colonizers, several strains of mitis-group streptococci, including Streptococcus gordonii, Streptococcus sanguis, and Streptococcus oralis, are naturally transformable and attain a state of competence during which the cells take up DNA. In this study we characterized competence development in Streptococcus oralis 34, and optimized transformation conditions by transforming wild-type S. oralis 34 with homologous genomic DNA isolated from a spontaneous streptomycin-resistant mutant. Additionally, transformations were conducted with the broad-host range plasmids pCM18 and pDL276. Methods: For a typical transformation, an anaerobic overnight culture of S. oralis 34 in Todd Hewitt broth supplemented with 5% heat-inactivated horse serum (TH+) was subcultured at a 1:20 dilution and incubated in air at 37°C, without shaking, for 2 hours. A 25 µl sample of the subculture was then added to 450 ml TH+ broth, DNA was added, and cells were plated hourly, for up to seven hours, on solid medium containing the appropriate antibiotics. Results: When transforming S. oralis 34 with homologous genomic DNA, the greatest transformation efficiencies were observed following four to six hours of incubation with 5 µg of DNA. Plasmid transformation of S. oralis 34 was not as efficient as transformation with genomic DNA. However, transformation with pCM18, a plasmid containing a constitutively expressed green fluorescent protein (GFP) gene, yielded fluorescent transformants, demonstrating that GFP can be used as a marker in S. oralis 34. Conclusions: The ability to transform S. oralis 34 will facilitate the molecular genetic analysis of this strain, and the expression of GFP in S. oralis 34 may serve as a useful species-specific marker for in vitro biofilm studies. | ||
| Seq #204 - Gram-positive Cocci: Molecular Biology II 11:00 AM-12:15 PM, Friday, 8 March 2002 San Diego Convention Center Exhibit Hall C | ||
|
Back to the Microbiology / Immunology and Infection Control Program
| ||