Escherichia coli JCL16
Random mutagenesis was performed with N'-nitro-N-nitrosoguanidine (NTG). Briefly, cultures of ppc- were grown to exponential phase in LB medium, washed twice with 0.1M citrate buffer and then concentrated two-fold by centrifugation and suspension in 0.1 M citrate buffer (pH 5.5). Samples of 2mL were exposed to NTG at a final concentration of 50 mg/ml for 30 minutes at 37°C to reach a percentage kill of approximately 50%. The cells were washed twice with 0.1 M phosphate buffer (pH 7.0) and grown in LB for one hour. The cells were then challenged by plating on glucose minimal medium and grown at 37°C for 3 days. This procedure was performed on 24 separate samples of ppc-, each of which was plated separately to ensure genetically distinct populations of mutants. One colony from each separate NTG experiment was selected, restreaked on selective medium plates to verify the phenotype and then cultured in liquid medium to obtain genomic DNA.