M63 media

M63 media (VWR Life Science) solution was made by first diluting 1 liter of presterilized M63 5× (BioWORLD, GeneLinx International Inc.) stock solution using autoclaved Millipore water. Filter-sterilized magnesium sulfate anhydrous (MgSO₄, Fisher Scientific) water solution, of volume 1 mL and molarity of 1 M, was added to the diluted media solution following standard protocol. Sodium arsenate stock solution (RICCA Chemical Company, 100 mM) was first filter-sterilized and then diluted with sterilized DI water to reach concentrations of 0.1 mM and 0.1 µM and stored under 4 °C. Potassium dichromate (Fisher Scientific) solution was made by first dissolving sodium dichromate crystal into sterilized DI water to reach concentrations of 17 mM, and then, the solution was filter-sterilized and diluted with sterilized DI water again to reach concentrations of 0.34 mM and 0.34 µM and stored at 4 °C. Prior to exposure to bacterial cultures, working solutions were placed at room temperature for 30 min to equilibrate to ambient temperature and then titrated to the culture to target exposure concentration. Anhydrous dextrose (glucose, Fisher Scientific), 1 g, was dissolved in 10 mL DI water and filter-sterilized to form 10% (w/v) glucose stock solution, which was added into the media solution later to provide energy source for bacteria.

To determine whether mutations in crbS, crbR, or acsA have an effect on acetate metabolism in non-O1/non-O139 strains of V. cholerae, strains carrying these mutations, as well as deletions in REC domains of crbS and crbR, were grown on M63 media (VWR) with 15mM supplemental sodium acetate (Sigma). Single colonies were grown on fresh LB plates overnight at 37°C, inoculated into LBM media, and grown overnight with shaking at 200 rpm at 37°C. The bacteria were spun down at 8000 g for 3 minutes, the supernatant was removed, and bacteria were resuspended in M63 media with 15mM sodium acetate to a final OD of 0.010 in 125 mL Erlenmeyer flasks. The bacteria were grown with shaking at 200 rpm at 37°C, and the optical density was measured at 600 nm on a spectrophotometer.