Direct fluorescence detection and volume electron microscopy reveal a role for antibiotic biosynthesis in the bacterial cell envelope

Secondary metabolites are key mediators of bacterial environmental fitness and the primary source of antibiotics and other natural products. Despite intensive study in natural product biosynthesis, the cellular biology that supports secondary metabolism is little understood. This study focuses on the linearmycin antibiotics from Streptomyces sp. Mg1. The metabolites are packaged into extracellular vesicles, which require the linearmycins for their biogenesis. This connection suggests that for some secondary metabolites, their biosynthesis is an integral function of cell organization and physiology. In this study, we employed simultaneous multi-photon fluorescence microscopy to directly detect linearmycins, enabling us to follow their biosynthesis and accumulation. We found that linearmycin biosynthesis localizes to membrane-dense regions that occur frequently where the filamentous cells form branches. Genetic disruption of linearmycin biosynthesis led to changes in the cell membrane visible using lipophilic dyes. To resolve differences between wild type and linearmycin-deficient membranes, we used serial FIB milling and scanning electron microscopy (FIB-SEM) to generate 3D volume reconstructions of S. Mg1 filaments. Using this approach, we identifed subcellular granules that require linearmycins. In the absence of linearmycins, major perturbations in the cell envelope arise, consistent with a fundamental role of the antibiotics in cell membrane integrity. This study demonstrates the combined use of advanced microscopy to reveal an intrinsic role for antibiotics in the cell biology of the producing organism.