FIG. 2.

Effects of different sugars on the induction of the fuculose kinase (fcsK) gene measured by quantitative, real-time SybrGreen RT-PCR. S. pneumoniae R6 was grown statically at 37°C in AGCH-YE medium and supplemented with different test sugars. Total RNA was extracted from late-logarithmic-phase-grown S. pneumoniae cells by using the Bio 101 FastRNA kit (Vista, Calif.) following glass bead cell disruption and a hot phenol lysis step (5). DNase-treated RNA was reverse transcribed to cDNA with a First Strand synthesis kit (Invitrogen). Relative levels of bacterial transcripts in each sample were quantified by PCR following SybrGreen dye incorporation (SybrGreen PCR core reagent kit; Applied Biosystems, Perkin-Elmer), and products were detected in real time with the 7700 sequence detection system (Applied Biosystems) as described previously (22, 35, 37). Template primers used in the PCRs are available on request. The quantity of cDNA estimated was normalized to a housekeeping gene, era. Changes in steady-state levels of fcsK mRNA in each sample were expressed relative to the uninduced control (0.2% glucose).