Cat. Cmpd) including ETBR (Son et al., 2003). Overexpression of the Enterobacter cloacae sugE homolog

in E. coli generated cells with increased resistance to several QACs and ETBR (He et al., 2011). Overexpression of the Aeromonas molluscorum sugE homolog in E. coli generated resistance to ETBR, but not the QAC cetylpyridinium RG 7204 chloride (Cruz et al., 2013). Also, when the E. coli SugE protein was assembled in membrane mimics, it bound to ETBR with a Kd in the low micromolar range, which is consistent with a role in ETBR transport (Sikora & Turner, 2005). Thus, in this work, we directly tested the model that Dcm influences sugE expression and thereby affects SugE-mediated resistance to antibacterial compounds. The bacterial strains used in this study are shown in Table 1 (Baba et al., 2006; Militello et al., 2012); plasmids were a gift from Ashok VEGFR inhibitor Bhagwat (Sohail et al., 1990). The lack of 5-methylcytosine in the dcm knockout strain JW1944-2 has been previously reported (Militello et al., 2012). The absence of the sugE gene in JW5738-1 and the rpoS gene in JW5437-1 was confirmed by PCR (data not shown). Liquid bacterial cultures were grown at 37 °C at 250 r.p.m. in either Luria Broth (LB) or M9 minimal media containing 0.4% glucose (Difco). Ampicillin was added to liquid cultures containing

dcm plasmids at 25 μg mL−1. Solid cultures were grown at 37 °C in the same media containing 15 grams of agar per liter, and when necessary ampicillin was added at 50 μg mL−1. All experiments to assess the sensitivity of strains to antibacterial compounds were performed in minimal media containing glucose as many QACs precipitate in LB. Bacteria were grown in LB at 37 °C at 250 r.p.m. to early logarithmic phase (A600 nm of c. 0.45) and early stationary phase (A600 nm c. 3.0). Total RNA was isolated from

3–4 mL of bacteria cultures using the MasterPure RNA Isolation Ketotifen Kit (Epicentre). For 5-azacytidine experiments, the drug (Sigma-Aldrich) was dissolved in 1X phosphate-buffered saline (PBS), and PBS was added to untreated samples as a control. RNA quality was assessed using bioanalysis at the University of Rochester Genomics Research Center. Prior to reverse transcription, RNA was treated with RQ1 RNase-free DNase (Promega). One microgram of total RNA was used for reverse transcription using the New England BioLabs Protoscript kit with random primers. cDNA was used as a template for qPCR reactions on a Stratagene MX3000p machine. All reactions were run in triplicate or quadruplicate (technical replicates), and each experiment was performed 3–4 times (biological replicates). Data were normalized to the levels of malate dehydrogenase (mdh) using the ΔΔCt method (Livak & Schmittgen, 2001). The primer sequences are listed in Table S1.