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Research Paper |
Department of Oral Biology, State University of New York, 3435 Main St, Buffalo NY 14214, USA1
Author for correspondence: Howard K. Kuramitsu. Tel: +1 716 829 2068. Fax: +1 716 829 3942. e-mail: kuramits{at}buffalo.edu
The glucosyltransferases of Streptococcus mutans are recognized as important virulence factors for this cariogenic bacterium. To study the expression of the gtfB gene of S. mutans in biofilms, a gtfB promoter (PgtfB)–green fluorescent protein (GFP) reporter system was developed. A Streptococcus–Escherichia coli shuttle vector harbouring a PgtfB::gfp cassette was introduced into S. mutans GS-5, and the expression of GFP by the transformed S. mutans cells was confirmed by fluorescence microscopy. Furthermore, confocal laser scanning microscopy was carried out on biofilms attached to polystyrene plates; enhanced gtfB expression was observed in various microcolonies across these biofilms. To further test the hypothesis that gtfB expression is upregulated in biofilms, flow cytometry analysis was done on planktonic and biofilm cells; this analysis showed an approximately five-fold increase in gtfB expression in the biofilm cells relative to the planktonic cells. Real-time (TaqMan) PCR analysis confirmed that gtfB expression in the biofilm cells was enhanced relative to the planktonic cells. Previously, it has been suggested that the S. mutans gtfC gene might be co-transcribed with gtfB. Therefore, RT-PCR analysis was performed on gtfB-expressing S. mutans; this analysis demonstrated that gtfC was co-transcribed with gtfB. These results indicated that GFP expression can be utilized to examine gene regulation in S. mutans biofilm formation.
Keywords: glucosyltransferase (GTF)
Abbreviations: CDM, chemically defined medium; CLSM, confocal laser scanning microscopy; Ct, threshold cycle; gfp and GFP, green fluorescent protein; gtf and GTF, glucosyltransferase; PgftB, gftB promoter
a Present address: Department of Preventive Dentistry, Kyushu University Faculty of Dental Science, Fukuoka 812-8582, Japan.
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