Bacterial Strain Construction and Validation

Bacterial strains, plasmids, and primers used in this study are listed in Tables 1 and 2. Bacteria were grown either in LB or MOPS media prepared as described [72 ,73 (link)] at 180 rotations per minute (rpm) with normal aeration or agar plates at 37°C. All mutant strains were constructed using the lambda-Red system as described in [74 (link)]. After allele substitution into the chromosome using an antibiotic resistance cassette, the constructs were genetically purified by bacteriophage phage P1 transduction, and the cassettes were removed using FLP recombinase resulting in an frt (FLP recognition target) scar. All constructs were validated by sequencing PCR products amplified from chromosomal DNA.