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Differential and Quantitative Analyses of mRNA Expression of Glucosyltransferases from Streptococcus mutans MT8148

¹ Departments of Pedodontics and
² Oral Microbiology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita-Osaka, 565-0871, Japan;

View larger version (42K): [in a new window]   Figure 1. Outline of and primers for real-time quantitative RT-PCR. The target for PCR was the 5' one-third region of the gtf genes shown at the top. Each gtf gene-specific primer (RT primer) corresponding to the 5' region of the catalytic domain (A) was used for the first-strand synthesis of cDNA (B). Real-time PCR was performed with the PCR primers corresponding to the hyper-variable region of each gtf gene (C). The expected sizes for each PCR product from gtfB, gtfC, and gtfD were 98 bp, 93 bp, and 83 bp, respectively (D).

View larger version (49K): [in a new window]   Figure 2. Total RNA and relative quantities of gtfB, gtfC, and gtfD mRNA in the growth phase of S. mutans MT8148. Amounts of extracted total RNA were assessed by means of a RiboGreen RNA quantitation kit with a Fluorometer. Following reverse transcription from 1 µg of total RNA, the amount of each gtf cDNA was determined by real-time PCR. Data are expressed as means and standard deviations of triplicate experiments. Statistical differences (P < 0.05, ANOVA) from early-exponential (*) and mid-exponential (#) phases are indicated.

View larger version (25K): [in a new window]   Figure 3. Southern blot analysis of real-time RT-PCR products. PCR products with/without reverse transcription (RT) reaction and recombinant plasmids carrying each gtf gene were separated by 2% agarose gel electrophoresis, then transferred onto nylon membranes. The membranes were hybridized with 32P-labeled gtfB-, gtfC-, and gtfD- specific probes, respectively. Lanes 1, RT-PCR amplicon by gtfB-specific primers; 2, RT-PCR amplicon by gtfC-specific primers; 3, RT-PCR amplicon by gtfD-specific primers; 4, PCR product without RT reaction by gtfB-specific primers; 5, PCR product without RT reaction by gtfC-specific primers; 6, PCR product without RT reaction by gtfD-specific primers; 7, pSK6 carrying gtfB; 8, pSK16 carrying gtfC; and 9, pYT104 carrying gtfD.

View larger version (22K): [in a new window]   Figure 4. Effects of pH (A) and sucrose (B) on the expression of gtf genes. S. mutans MT8148 was cultured in BHI broth with 25 mM phosphate buffer (A) with or without 2% sucrose (B) at 37°C to an optical density of 0.2 at 550 nm. Data are expressed as means and standard deviations of triplicate experiments. Statistical differences (P < 0.05, ANOVA) between pH 5 (*), pH 6(#), and pH 7 () (A), and without sucrose (*) (B) are indicated.