Deletion of fbiC in Streptomyces venezuelae removes autofluorescence in the excitation-emission range of cyan fluorescent protein

Autofluorescence poses an impediment to fluorescence microscopy of biological samples. In the Gram-positive, soil-dwelling bacteria of the genus <i>Streptomyces</i>, sources of autofluorescence have not been examined systematically to date. Here, we show that the model organism for the genus, <i>Streptomyces venezuelae</i>, shows autofluorescence in two of the commonly used fluorescence channels for visualizing cyan and green/yellow fluorescent proteins. We identify the source of autofluorescence in the cyan fluorescence channel as redox cofactor factor 420 (F₄₂₀) and target its synthesis to remove it. By deleting the <i>vnz15170</i> (<i>fbiC</i>) gene, which is a key biosynthetic gene for the production of F₄₂₀, we were able to create an autofluorescence-free strain in the cyan range of fluorescence excitation-emission. We demonstrate the usefulness of this strain by imaging the mTurquoise-tagged polar growth-related protein DivIVA and the cell division-related protein FtsZ in the <i>fbiC</i> deletion background. Using live-cell imaging to follow the dynamics of DivIVA and FtsZ, we demonstrate an improved signal-to-noise ratio in the mutant strain. We show that this strain can be a suitable tool for visualizing the localization of proteins in <i>Streptomyces</i> spp. and can facilitate the utilization of multi-colour imaging and fluorescence resonance energy transfer-based imaging.