MSB broth and agar were used for the growth of strains under non-selective conditions. LB-0 agar was used when using selective antibiotics in transductions and transformations. Plates

were solidified with 1.5% agar. LB-0 agar or MSB broth were supplemented as needed with ampicillin (100 μg/ml) or kanamycin (20 μg/ml). Antibiotics were added to LB-0 agar after cooling to 45 degrees Celsius. Restoring msbB + genotype In order to confirm that the observed CO2 sensitivity results simply from knocking out MsbB https://www.selleckchem.com/products/Lapatinib-Ditosylate.html function, wild type msbB was expressed from the msbB promoter using plasmid pSM21 [4]. Purified plasmids were transformed into electroporation-competent cells of strains YS1 and YS873. Growth Analysis Phenotypes of strains were determined by replica plating. Master plates were made on either MSB or LB-0 agar. Replica plating was performed using a double velvet technique [4]. Replica plates were incubated for 16 hours at 37°C. To generate growth curves, 3 ml broth tubes were inoculated with single colonies and grown on a shaker overnight

at 37°C in air. Cells were diluted 1:1000 or 1:500 (β-gal strains) in LB broth. Cells were held on ice until all inoculations were completed. Triplicate cultures were then placed in a 37°C shaker with 250 rpm in air or 5% CO2. O.D.600 was measured every 60 minutes and dilutions of AR-13324 bacteria were plated onto MSB or LB agar plates to calculate the number of colony forming units (CFU) per ml. Microscopic Observation Strains 14028, 14028 zwf, YS873 and YS873 zwf were grown for 6 hours, as 3-oxoacyl-(acyl-carrier-protein) reductase described above for growth curves, at 200 RPM. The cells were then fixed for microscopy using a solution of 30 mM sodium phosphate buffer (pH 7.5) and 2.5% formaldehyde. Cell morphology was observed with a Zeiss Axiovision microscope

using differential interference contrast settings and DNA was detected via DAPI fluorescence. Fixed cells were incubated with 2 μg/ml DAPI for 10 minutes in the dark and aliquoted onto a 1% agarose pad. Mutation Frequency Determination A frozen stock of YS873 was streaked on MSB media and incubated BI 10773 clinical trial overnight at 37°C to isolate individual clones. Triplicate 3 ml of LB broth were inoculated with independent YS873 colonies. They were grown at 37°C in a shaker over night. The tubes were then placed on ice and diluted in 0.9% saline. 10-6 and 10-4 dilutions were plated in duplicates onto LB agar and incubated in air and CO2 incubators respectively overnight at 37°C to calculate the number of CFU per ml. Transduction and Transformation Salmonella P22 transductions were performed by the method of Davis et al. [30], except that LB-0 plates supplemented with the appropriate antibiotic were used. EGTA was not added to the antibiotic plates for transductions. A BioRad Gene Pulser was used for electroporation with the following settings: 2.5 kV, 1000 ohms and 25 μFD for transformation of YS1 and 1.