To measure lethality, cells were grown in LB liquid medium to mid-log phase (OD600 = 0.3 ~0.5) at 37°C with shaking. Cells were split into 1-ml aliquots in test tubes, and various concentrations

of antimicrobial agents (2 × MIC99 to 30 × MIC99) were added. After incubation for 2 hr with shaking, cells were diluted in LB liquid medium, NCT-501 which eliminated drug carryover, and 10 μl of aliquots from the dilutions were spotted in triplicate on drug-free LB agar plates. Colonies were counted after overnight incubation at 37°C. Lethality was expressed as percent of control relative to the CFU per ml at the time of drug addition. The dose that reduced CFU by 90% was taken as LD90. For screening the mutant library, kanamycin-resistant

colonies were manually replica-plated with toothpicks to a series of plates containing various concentrations of nalidixic acid and incubated overnight. Colonies exhibiting the same bacteriostatic susceptibility as the parental strain were saved for lethality measurement. Survival GM6001 purchase for each colony was measured in liquid medium after a 2-hr incubation in nalidixic acid at 20 μg/ml and 50 μg/ml as described in the previous paragraph. Colonies that exhibited decreased survival relative to the parental strain were then retested for MIC and survival as described in the previous paragraphs. Strains confirmed to have a hyperlethal phenotype were further characterized as described below. Identification of gene insertion sites Asymmetric PCR, modified from that described previously [14–16], was used to amplify E. coli genomic sequences near the ends of Tn5 that inserted into the genome. One primer, either Tn5R10 (5′ GGG ATC CCC TAC TTG TGT AT 3′) or Tn5F4568 (5′ AGA ATT CCT CCC GAG ATC TG 3′) was complementary to the sequence at an end of Tn5; the other primer contained a 6-nucleotide random before sequence followed by TGGC (Ran5-29: 5′ GTT CTA CAC GAG TCA CTG CAG NNN NNN TGG C 3′). The randomized primer binds any GCCA in the genome. However,

since PCR preferentially amplifies short fragments, combination of the two primers should amplify the sequences between one Tn5 end and the first few GCCA sequence elements. For the first 5 cycles of PCR, the BAY 11-7082 annealing temperature was high (58°C); consequently, the primer that was complementary to the sequence at the Tn5 end preferentially bound to the substrate, which caused one strand of the substrate to be asymmetrically amplified. This high-temperature annealing was followed by a cycle using low annealing temperature (30°C) to allow the randomized primer to bind the strand that had already been amplified. Then one high-temperature (58°C) and one moderate-temperature (44°C) cycle were alternated 12 times to amplify the sequence between the two primers. For all amplification cycles, the annealing time was 1 min, while the denaturation (94°C) and extension (72°C) times were 15 sec and 2 min, respectively.