(iv) Real-time PCR.

The experimental design, procedures, and data analyses followed the MIQE guidelines whenever applicable (62). Reaction mixtures (20 μl each) were set up in a MicroAmp optical 96-well reaction plate with barcode (Applied Biosystems), and each mixture contained 10 μl PowerUp SYBR green master mix (2×; Applied Biosystems), 1 μl of the diluted cDNA, and 1 μl of 5 mM forward and reverse primers. Real-time PCRs were performed with an ABI 7500 Fast real-time PCR system (Applied Biosystems). Three independent biological replicates were assayed for each strain under each experimental condition. The qPCRs for each target gene were performed in technical duplicates, with a no-template control and a no-reverse transcriptase control. The qPCR program was as follows: 50°C for 2 min, 95°C for 2 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Melting curve analysis was performed by increasing the temperature from 60°C to 90°C. The amplification efficiency of each primer was estimated with standard curves using serial dilutions of genomic DNA template and then was calculated using the slope of a linear regression model (10^−1/slope) (63). Threshold cycle (C_T) values were used for stability comparisons of candidate reference genes using BestKeeper (64). Expression levels were normalized to the geometric mean of the 16S rRNA, recA, and purC C_T values for the wild-type strain grown in glucose medium (65).