RT-qPCR quantification of gene expression.

A culture from a single colony of wt UTI89 bacteria grown overnight was subcultured in LB broth at a 1:1,000 dilution under aerobic and anaerobic conditions. Aerobic cultures were grown at 37°C with vigorous agitation; samples were taken at 4 h (optical density at 600 nm [OD₆₀₀] of ∼0.5), 6 h (OD₆₀₀ of ∼0.9), and 24 h (OD₆₀₀ of >2, the overnight time point). Anaerobic cultures were grown without agitation at 37°C under a thick layer of mineral oil (catalog no. 163-2129; Bio-Rad, Singapore); both aerobic and anaerobic cultures were set up simultaneously, and samples from anaerobic cultures were aliquoted at the same time as the aerobic samples.

At each time point, a 500-μl aliquot of the respective culture was pipetted into 1 ml of RNAprotect bacterial reagent (catalog no. 1018380; Qiagen, Singapore) and then mixed thoroughly by vortexing. Individually isolated IBCs or isolated GFP-negative epithelial cells were pooled directly after isolation in a 1.5-ml Eppendorf tube and stabilized with 200 μl of RNAprotect. RNA from samples stabilized in RNAprotect was extracted by using the RNeasy minikit (catalog no. 74104; Qiagen, Singapore). DNA was removed by the addition of 4 μl of DNase I for 4 μg of the RNA extracted in a final volume of 20 μl (using RNase-free water) according to the manufacturer's protocol (Ambion DNase I [RNase free], catalog no. AM2222; Life Technologies, Singapore). For cDNA synthesis, the Superscript II kit was used, with 0.42 μl of random hexamers added to 500 ng of purified RNA in a final volume of 20 μl (using RNase-free water), according to the manufacturer's protocol (Superscript II reverse transcriptase, catalog no. 1821032; Invitrogen, Singapore).

Each quantitative PCR was performed with a total volume of 10 μl: 5 μl 2× Kapa master mix (Kapa SYBR Fast universal qPCR kit; Kapa Biosystems, Singapore), 0.2 μl each of forward and reverse primers (10 μM each), 1.5 μl cDNA, and 3.1 μl of RNase-free water. The following primers were used: AACGCGAAGAACCTTAC (V6) and CCTTTGAGTTCCCGGCC (R2) for 16S rRNA, 5′-ATGCCTTTCGTTATCGGTCTGAT-3′ and 5′-CAACGGTAAACCAGAAGCTTAAGTT-3′ for cyoB, and 5′-CGTCGATCCGGTTAAAGAA-3′ and 5′-CAGACCTTTAATGCGGGTTTCA-3′ for frdA (54). For a single biological replicate, RT-qPCRs were run in triplicate on a LightCycler 480 instrument (Roche, Singapore) with the following protocol: 95°C for 5 min followed by 40 cycles of 95°C for 30 s and 60°C for 30 s. A melting curve was run immediately afterwards with the following protocol: 95°C for 5 s, 65°C for 1 min, and a continuous acquisition step to 97°C with 20 acquisitions/°C. Each experiment was performed with at least three biological replicates.

Based on examinations of C_T values, melting temperatures, and amplification curves using serial dilutions of purified genomic DNA, maximum C_T values of 30 for the 16S rRNA gene and 34 for the cyoB and frdA genes were set as the limits of detection. All data analyzed for expression levels not only passed this C_T value threshold but also were verified to have the correct melting temperature and a reasonable amplification curve profile (based on manual examination of the baseline, curve shape, and fluorescence intensity range). Samples with detectable RNA expression for all measured genes based on these criteria were then analyzed by normalizing the cyoB and frdA expression levels to the 16S rRNA expression level. Fold changes for cyoB and frdA expression levels were then calculated (as 2^−ΔΔC_T) relative to the overnight time point for the in vitro anaerobic culture, as reported previously (54).