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

Abstract

Autofluorescence poses an impediment to fluorescence microscopy of biological samples. In the Gram-positive, soil-dwelling bacteria of the genus Streptomyces, sources of autofluorescence have not been examined systematically to date. Here, we show that the model organism for the genus, Streptomyces venezuelae, 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 vnz15170 (fbiC) 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 fbiC 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 Streptomyces spp. and can facilitate the utilization of multi-colour imaging and fluorescence resonance energy transfer-based imaging.

Keywords: Actinomycetota; F420; Streptomyces; autofluorescence.

Fig. 1.. S. venezuelae exhibits autofluorescence in CFP and YFP channels. (a) Autofluorescence characteristics of WT S. venezuelae observed across fluorescence channels for imaging cyan (CFP), yellow (YFP) and mCherry fluorescent proteins. Fluorescence micrographs were captured with filter sets for CFP (excitation 424–448 nm; emission 460–500 nm), YFP (excitation 488–512 nm; emission 520–550 nm) and mCherry (excitation 559–585 nm; emission 600–690 nm) fluorescence channels. Representative images show the intensity distribution between the brightest and darkest pixels. Scale bar, 5 µm. (b) FbiC is a protein that consists of CofG and CofH domains and is predicted to catalyse the first steps in the F₄₂₀ biosynthesis. (c) Predicted genes involved in the F₄₂₀ synthesis pathway in S. venezuelae, including fbiC (cofGH; vnz15170), cofD (vnz13655), cofE (vnz13660) and cofC (vnz25960), based on KEGG pathway database analysis. A search for F₄₂₀ biosynthesis-related genes in S. venezuelae shows the predicted metabolic network. Genes were identified from the pathway and used to draw the depicted table.

Fig. 2.. fbiC deletion leads to the elimination of autofluorescence in CFP channels without affecting cell growth and development. (a) Spectral analysis and characterization of autofluorescence in S. venezuelae WT and fbiC mutant strains. Excitation (Ex.; emission set to 475 nm) and emission (Em.; excitation set to 420 nm) spectra are shown of cell extracts from WT S. venezuelae and fbiC mutant strain LUV320. (b) Wide-field fluorescence microscopy images of fbiC mutant captured with filters for CFP, YFP and mCherry fluorescence, respectively (representative image shows fluorescence distribution between the brightest and the darkest pixel). (c) Colony morphology and pigmentation of LUV320 and WT on MYM agar medium after 3 days of incubation at 30°C. Scale bar, 5 mm. (d) Images from live-cell imaging (Movie S1). WT and LUV320 strains were grown in a microfluidic cell perfusion system as described in the ‘Methods’ section. Phase contrast imaging of the growing cells was performed, and representative frames were extracted after sporulation. Arrows point to spore chains. Scale bar, 5 µm.

Fig. 3.. Live imaging of FtsZ-mTq2 localization during sporulation in S. venezuelae. (a) Representative fluorescence micrographs depicting the formation of FtsZ rings after the induction of sporulation. Fluorescence signal from FtsZ-mTq2 rings in the fbiC mutant strain LUV336 and WT S. venezuelae are shown below the corresponding DIC image. Cells were grown from spores to hyphae for 6 h in MYM medium in a microfluidic cell perfusion system before switching to spent MYM medium to induce sporulation. pent MYM from the WT and LUV320 strains was used, respectively. The representative micrographs were obtained at the 10 h time point of time-lapse live imaging (Movie S4). Scale bar, 10 µm. b. Close-up micrographs showing regions of FtsZ ring formation in the LUV336 and WT strain. Scale bar, 5 µm. The time points (min) of the experiment are indicated. Examples of the representative intensity profile of the Z-ring ladders at 630 min are shown to the right. The clearer peaks of FtsZ rings in the LUV320 background highlight the improved signal-to-noise ratio achieved by eliminating autofluorescence from coenzyme F₄₂₀.

Fig. 4.. Effect of fbiC deletion on visualization of mTq2-tagged DivIVA in S. venezuelae. (a) Representative fluorescence micrographs from time-lapse imaging of WT and fbiC mutant strain of S. venezuelae expressing DivIVA tagged with mTurquoise2 (DivIVA-mTq2) during vegetative growth stage (Movie S2). Scale bar, 5 µm. (b) 3D surface plot of the pixel intensity of the grayscale CFP channel from panel (a). The fluorescence data from panel (a) was plotted using a 3D surface plot in Fiji software. The x- and y-axes are each 25 µm in length, and the z-axis represents the fluorescence intensity in the range same as panel (a) (100–400). (c) Plot showing fluorescence intensity profiles DivIVA-mTq2 along the growing hyphae. The graphs show data from 20 hyphal tips in fbiC mutant strain LUV320 and 12 tips in WT background. Using Fiji software, a line of 10-pixel width was drawn across the cell tip, and the intensity profile was plotted. The peaks of the fluorescence were aligned at the maximum, and the x-axis represents the fluorescence profile from outside the cell to the inside. The y-axis is set to 0 at 10 pixels from the signal maximum. Insets show a representative line used to plot the intensity. The data was analysed in GraphPad (Prism) software.