Isolation of potential new SPase inhibitors producing strains by soft agar overlay assay method.

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The arylomycins are a class of compounds that target bacterial signal peptidases (SPases). In an effort to discover new compounds in this class and other small molecule SPase inhibitors, a rapid screening method was developed and applied to bacteria cultured from marine sediment samples. The assay consisted of a soft agar overlay using an engineered E. coli strain MG1655 ΔlepB::kan + pTetBHR2‐LepB designated as CUA‐604. Sixty marine sediment samples from Fiji and 200 from the Channel Islands were processed for the cultivation of marine bacteria. The primary isolation plates were overlaid with strain CUA‐604 and following 24–48 h, colonies showing zones of inhibition were removed and purified using standard microbiological methods. Six strains from Fiji and six from the Channel Islands were confirmed as inhibiting the growth of CUA‐604. It was determined that pH and iron limitation were not the cause of inhibition. The most active strain was identified as a Bacillus sp. To isolate the active compound, multiple overlay plates were prepared and the agar in the zones of inhibition removed and extracted with a variety of organic solvents. The crude butanol, methanol, and acetone extracts were active at a concentration of 10 mg/mL. The results of the bioassay‐guided isolation of the active constituents will be presented.