Antibacterial Synnepyrroles from Human-Associated Nocardiopsis sp. Show Protonophore Activity and Disrupt the Bacterial Cytoplasmic Membrane.

Actinobacteria have traditionally been an important source of bioactive natural products, although many genera remain poorly explored. Here, we report a group of distinctive pyrrole-containing natural products, named synnepyrroles, from Nocardiopsis synnemataformans. Detailed structural characterization by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy combined with isotope-labeling experiments revealed their molecular structures and biosynthetic precursors acetate, propionate, aspartate, and (for branched analogues) valine. The biosynthetic data points toward an unusual pathway for pyrrole formation via condensation of aspartate with diverse fatty acids that give rise to a unique pyrrole-3,4-dicarboxylate core and variable linear or terminally branched alkyl side chains. In addition, the bioactivity and mode of action of synnepyrrole A were characterized in Bacillus subtilis. Orienting assessment of the phenotype of synnepyrrole A-treated bacteria by high-resolution microscopy suggested the cytoplasmic membrane as the target structure. Further characterization of the membrane effects demonstrated dissipation of the membrane potential and intracellular acidification indicative of protonophore activity. At slightly higher concentrations, synnepyrrole A compromised the barrier function of the cytoplasmic membrane, allowing the passage of otherwise membrane-impermeable dye molecules.

Physical, genetic and functional interactions between the eisosome protein Pil1 and the MBOAT O-acyltransferase Gup1.

The Saccharomyces cerevisiae MBOAT O-acyltransferase Gup1 is involved in many processes, including cell wall and membrane composition and integrity, and acetic acid-induced cell death. Gup1 was previously shown to interact physically with the mitochondrial membrane VDAC (Voltage-Dependent Anion Channel) protein Por1 and the ammonium transceptor Mep2. By co-immunoprecipitation, the eisosome core component Pil1 was identified as a novel physical interaction partner of Gup1. The expression of PIL1 and Pil1 protein levels were found to be unaffected by GUP1 deletion. In ∆gup1 cells, Pil1 was distributed in dots (likely representing eisosomes) in the membrane, identically to wt cells. However, ∆gup1 cells presented 50% less Pil1-GFP dots/eisosomes, suggesting that Gup1 is important for eisosome formation. The two proteins also interact genetically in the maintenance of cell wall integrity, and during arsenite and acetic acid exposure. We show that Δgup1 Δpil1 cells take up more arsenite than wt and are extremely sensitive to arsenite and to acetic acid treatments. The latter causes a severe apoptotic wt-like cell death phenotype, epistatically reverting the ∆gup1 necrotic type of death. Gup1 and Pil1 are thus physically, genetically and functionally connected.