For screening of the 3280 strains, spot tests were conducted on agar plates using Biomek FX (Beckman Coulter) and RoToR HDA (Singer Instruments) automation systems. Strains were spotted on EMM2−N agar plates and incubated under −N conditions. Since the agar contained trace quantities of nitrogen, nitrogen starvation required slightly longer incubation times. Three independent spot tests were conducted on EMM2−N using different incubation times (14 days and 1 and 2 months) (fig. S5A). After incubation, we respotted them in quadruplicate on EMM2 plates to replenish the nitrogen source and to check colony formation. These spot tests were for the first rough screening, which could have contained false positives, so additional MC assays were conducted afterward.

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