For spot tests with E. coli, saturated cultures grown in LB medium were diluted 30-fold in 1× M9 salts and transferred to M9 glucose plates (46) using a multiprong replicator device (approximately 0.1 ml of the culture per plate in total). After the spots had dried, a filter paper disc was placed on the center of each plate, and an appropriate volume of DMSO-dissolved extracts of S. epidermidis strains MO34 and 1457 (5 mg/ml) or 6-HAP, either synthesized in R. L. Gallo’s lab or synthesized by NIC, was spotted onto the paper disc. A DMSO-only control was also included. The plates were incubated overnight at 37°C and inspected the next day for zones of inhibition around the discs. The plates were then replica plated on LB plates supplemented with rifampicin (100 mg/liter), incubated overnight at 37°C, and inspected for the appearance of Rifr colonies around the discs. For quantitative mutagenesis tests with E. coli, at each concentration of MO34 extract or 6-HAP (NIC), 15 to 20 independent 1-ml M9 glucose cultures were started containing approximately 105 cells. The cultures were incubated with shaking for 24 hours at 37°C. The frequencies of d-cycloserine–resistant (Cycr) mutants occurring in the cycA gene were determined by plating 50 to 100 μl of a 10−1 dilution (for spontaneous samples) or a 10−2 dilution (for MO34- or 6-HAP–induced samples) on M9 glucose plates containing 50 μM d-cycloserine to obtain the number of Cycr colony-forming units (cfu) per milliliter, and by plating 100 μl of a 10−6 dilution on LB plates to obtain the total number of colony-forming units per milliliter. The experiments were repeated four times. To determine the exact nature of the Cycr mutations, the cycA gene was amplified from a large number of randomly chosen Cycr colonies from independent cultures in four experiments using primers cycAF1 (5′-CCCGTAAGCGTGTATTTT TG-3′) and cycAR1 (5′-CCTGGAAAGCGATGTATAACG-3′), and the PCR products were sequenced with the same primers, as described (47).

For mutagenesis experiments with yeast, S. cerevisiae strains ES15 and ES18 (ham1::LEU2) were streaked out on YPDAU-rich medium [YPD (yeast extract, peptone, dextrose) plus adenine and uracil (48)]. For each strain, six single colonies were resuspended in individual tubes with YPD medium and grown until the cultures entered the early log phase of growth (3 to 6 hours). Each culture was then split and exposed to DMSO only, 6-HAP (MP Biomedicals), or MO34- or 1457-derived extracts. After overnight incubation with shaking, cells were plated to determine canavanine-resistant mutant frequencies using plates with canavanine or minimal complete plates, as described (49).

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