Construction and characterization of a vapB6::lacS disruption. (A) Multiplex linear recombination for vapB6 disruption. Two overlap extension PCR products containing lacS and either the 5′ or 3′ end of vapB6 were cotransformed into PBL2025. Recombinants arose by three crossover events (X's) between the two incoming fragments and the corresponding chromosomal allele. Primers used to verify identities of vapB6 alleles are indicated. (B) Mutant genotype. PCR amplification of the wild-type (lane 1) and lacS disrupted alleles of vapB6. (C) Thermotolerance of the vapB6 mutant. Wild-type (•) and vapB6::lacS disruption mutant (○) were cultivated in rich medium and shifted from 80°C to 95°C for the times indicated. Culturable cells were enumerated on solid medium with incubation at 80°C. (D) Arrhenius plot. The growth rate constant (k), which is related inversely to generation time, is portrayed on the y-axis. The temperature in °K is indicated on the x-axis, and the temperature in °C is indicated across the top of the figure. Strains were: wild-type (•) and vapB6::lacS disruption mutant (○). Values for the average of two independent experiments are shown.
