Eric Miller

The rapid evolution of antibiotic resistance is shrinking our stockpile of commercially available antibiotics. Therefore, new antimicrobials with novel mechanisms of action (MoAs) are desperately needed. The free fatty acid, ( Z )-13-methyltetra-4-decenoic acid (( Z )-4C-14:1), isolated from a marine sediment bacterium Olleya marilimosa , displays strong inhibition of Gram-positive pathogens with minimal cytotoxic effects to mammalian cells. Here, we applied a combination of experimental approaches to identify the mechanism by which ( Z )-4C-14:1 kills the Gram-positive bacterium Bacillus subtilis . Using quick and cost-effective bacterial cytological profiling (BCP), we established the cytological signatures of 17 antibiotics representing 6 general classes of antibiotic MoAs. We used BCP to demonstrate that while ( Z )-4C-14:1-treated B. subtilis cells display unique morphological features compared to other antibiotic classes, ( Z )-4C-14:1 mode of action shares substantial overlap with the fast-acting antibiotic colistin specifically in Gram-positive cells and daptomycin, both of which target bacterial permeability by destroying the cell membrane and causing extensive cell surface alterations. To further determine if the cell membrane of B. subtilis was the target of ( Z )-4C-14:1, we used cell membrane- and peptidoglycan-specific diagnostic stains to investigate bacterial permeability. Our results indicate that ( Z )-4C-14:1 destabilizes the cell membrane by pore formation in Gram-positive bacteria and emphasize the importance of mining the marine environment for naturally occurring antibiotics. IMPORTANCE With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, new sources of antibiotics that target pathogens of clinical importance are paramount. Here, we use bacterial cytological profiling to identify the mechanism of action of the monounsaturated fatty acid ( Z )-13-methyltetra-4-decenoic acid isolated from the marine bacterium Olleya marilimosa with antibacterial effects against Gram-positive bacteria. The fatty acid antibiotic was found to rapidly destabilize the cell membrane by pore formation and membrane aggregation in Bacillus subtilis , suggesting that this fatty acid may be a promising adjuvant used in combination to enhance antibiotic sensitivity.

Nature serves as a rich source of molecules with immense chemical diversity. Aptly named, these ‘natural products’ boast a wide variety of environmental, medicinal and industrial applications. Type II polyketides, in particular, confer substantial medicinal benefits, including antibacterial, antifungal, anticancer and anti-inflammatory properties. These molecules are produced by enzyme assemblies known as type II polyketide synthases (PKSs), which use domains such as the ketosynthase chain-length factor and acyl carrier protein to produce polyketides with varying lengths, cyclization patterns and oxidation states. In this work, we use a novel bioinformatic workflow to identify biosynthetic gene clusters (BGCs) that code for the core type II PKS enzymes. This method does not rely on annotation and thus was able to unearth previously ‘hidden’ type II PKS BGCs. This work led us to identify over 6000 putative type II PKS BGCs spanning a diverse set of microbial phyla, nearly double those found in most recent studies. Notably, many of these newly identified BGCs were found in non-actinobacteria, which are relatively underexplored as sources of type II polyketides. Results from this work lay an important foundation for future bioprospecting and engineering efforts that will enable sustainable access to diverse and structurally complex molecules with medicinally relevant properties.