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

Abstract

Secondary metabolites support the environmental fitness of a broad taxonomic diversity of bacteria and fungi, yet the cellular biology that supports secondary metabolism is little understood. This study focuses on the linearmycin antibiotics from Streptomyces sp. Mg1. These membrane-disruptive metabolites are packaged into extracellular vesicles, which in turn 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 fluorescence localizes to membrane-dense regions that also coincide with localization with a fluorescent fusion protein, LnyI-Ypet, that is essential for biosynthesis. 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 identified granular subcellular compartments that require linearmycins. Disruption of linearmycin synthesis causes, in addition to disappearance of the compartments, visible distortions in the cell envelope, suggesting an integral role for the metabolites in membrane dynamics of the producer bacteria. We propose that the subcellular compartments coalesce near hyphal cell division and branch points and are regions for linearmycin biosynthesis. This study demonstrates the combined use of advanced microscopy to reveal an intrinsic role for antibiotics in the cell biology of the producing organism.