FIG. 1.

Protease activity and transcription of prcA-prtP in T. denticola 35405. (A) SAAPFNA activity (8) expressed in A₄₀₅/h/ml/OD₆₀₀. Error bars represent the range of values obtained with triplicate samples of a representative experiment. (B) QRT-PCR of prcA and prtP. Total RNA was extracted from cultures harvested at an OD₆₀₀ of 0.1, 0.2, and 0.3 and at stationary phase (fifth day). DNase-treated RNA samples were reversed transcribed with random hexamer primers by using the SuperScript First-Strand synthesis system for RT-PCR (Invitrogen). One microliter of the resulting first-strand cDNA was amplified by using QuantiTect SYBR Green PCR (QIAGEN) in 25 μl of reaction buffer, with 16S rRNA serving as an internal control for normalization between samples. Gene-specific primers separated by approximately 80 to 100 bp were designed with Primer Express software (Perkin-Elmer Applied Biosystems). Thermal cycling was performed in an iCycler iQ Multi-Color Real Time PCR detection system (Bio-Rad) at 95°C for 15 min, followed by 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s. Threshold values were calculated by using baseline cycles 2 to 10. Data were analyzed by using software supplied by the manufacturer. The data are shown with the gene expression level defined as 1.0 at an OD₆₀₀ of 0.1. Error bars represent standard deviations of the means of results from triplicate samples in two independent experiments.