Full color palette of fluorescent d-amino acids for in situ labeling of bacterial cell walls

Fluorescent d-amino acids (FDAAs) enable efficient in situ labeling of peptidoglycan in diverse bacterial species.

The reaction was quenched with cold water followed by the addition of NaHCO 3 (saturated). The resulting crude mixture was extracted with EtOAc and washed with brine, dried over sodium sulfate, and concentrated in vacuo. The resulting crude product was then separated by column chromatography (1:4 EtOAc/Hexane) to provide an off-white solid (Atto 610-P1, 6.16 grams, 24.9 mmol, 28% yield). 1
The crude reaction mixture was then concentrated in vacuo. The resulting crude mixture was diluted in brine and EtOAc. The product was extracted with EtOAc and washed with brine. The organic layer was dried over sodium sulfate and concentrated in vacuo. The resulting pink oil product (Atto 610-P2), which degrades quickly, was carried onto the next reaction without further purification (3.96 g, 15.9 mmol, 91% yield).
After an additional hour of reacting, H2SO4 (40 mL, concentrated) was carefully added and DCM was removed in vacuo. This reaction was allowed to continue for an additional 2 h. A copious amount of ethanol (~500 mL) was added followed by the addition of (nBu) 4 NIO 4 (0.8 g, 1.80 mmol). The reaction was then refluxed for 10 min. The reaction was then allowed to cool to room temperature and stir overnight. The crude mixture was then concentrated in vacuo and the product was extracted with several portions of DCM. The crude product was purified by reverse phase HPLC (10-90% ACN/H2O over 20 min) to provide a blue powdery solid TFA salt (close to 1:1 mixture of ethyl ester and methyl ester, 0.926 g, 1.84 mmol, 25%). For the ethyl ester compound: 1    An acidic solution (THF/H20/HClconc, 12:8:4.6, 24.6mL) was added to sulfonated BODIPY FL ethyl ester (153 mg, 0.36 mmol). After stirring the reaction overnight, the crude mixture was concentrated in vacuo. The product was purified by reverse-phase HPLC (10-90% acetonitrile over 10 min), and lyophilized to yield the desired product as a red solid (61.6 mg, 43% yield). 1    For TADA synthesis (Fig. S15)

3H-xanthen-3-ylidene)-N-methylmethanaminium
To a stirring solution of 5-Carboxytetramethylrhodamine TAMRA (10 mg, 0.0232 mmol) in DMF (anhydrous, 1 mL) was added CDI (4.5 mg, 0.0278 mmol, 1.2 equiv). This solution was then stirred at room temperature for 2 h. Boc-D-Dap-OH for TADA was then added (0.0278 mmol, 1.2 equiv.) and stirred continuously overnight. The reaction mixture was concentrated in vacuo and the crude residue was then stirred in TFA:DCM (10 mL of 1:1) for 2 h. The crude product was concentrated in vacuo, purified via reverse-phase HPLC (10-90% acetonitrile over 10 min), and lyophilized to yield the desired product as a red solid

SI-Methods of data acquisition
Culture growth. Strain characteristics and growth conditions are described in Table S2. Bacterial cells were streaked from -80 °C freezer stocks (LB broth with 10% DMSO) onto LB agar plates, followed by overnight growth at 37 °C for Escherichia coli and Bacillus subtilis or 30 °C for Streptomyces venezuelae and Lactococcus lactis. Single colonies from the overnight plate were transferred to liquid LB broth and incubated in an Innova  44R shaker with a shaking speed of 200 rpm. After cell cultures reached OD 600 ~ 0.5, they were diluted with culture media (10x) and grown one more round until OD 600 ~ 0.5. The cells were then used for FDAA labeling.  Image sample preparation. 24x50 mm coverslips (#1.5) were used as imaging supports for the inverted microscope system. For fluorescence imaging, the slides were rinsed with ethanol and water twice, and air-dried before use. For STORM imaging, coverslips were plasma-cleaned. Cell samples were loaded onto the coverslips, followed by laying an 8x8 mm wide, 2-mm thick PBS-agar pad on top of the cells. The coverslip-pad combination was placed onto a customized slide holder on microscopes with the pad facing upwards.
Image acquisition. Phase/fluorescence images were acquired using a Nikon Ti-E inverted microscope equipped with a 1.4NA Plan Apo 60X oil objective and Andor iXon X3 EMCCD camera. to the microscope stand. A weak cylindrical lens was introduced into the imaging path to capture three-dimensional localization information using the astigmatism imaging method (5). Shuttering of laser illumination was controlled with an acousto-optic tuneable filter (AA optoelectronics, Orsay, France) before the fibre coupler. Images were acquired with an iXon3+ 887 EMCCD (Andor Technology, South Windsor, CT, USA) camera, and synchronisation between components was achieved using µ-Manager 1.4 (6) with a microcontroller (Arduino, Almuñécar, Spain). Image processing. Unless otherwise specified, all image processing was performed in FIJI. Images were cropped, rotated, and scaled without interpolation. Contrast and brightness were adjusted. Only linear adjustments were performed. Figure  The wavelength at ~20 nm below the highest absorbance was used as the excitation wavelength for measuring excitation spectra. Excitation and emission spectra in Figs. 2 and S1 were generated using Prism. (LogD 7.4 ). The protocol was modified from previous literature (7). FDAA stock solutions were diluted with 2 mL 1x PBS to a final concentration of 0.02 mM. The absorbance at maximum  ex was recorded. The solution was then extracted with 2 mL 1- FDAA intensity was recorded in time-lapse during a continuous light exposure at the appropriate excitation wavelength ( Table 2) for ~30 s. The RAM function of NIS-Elements AR software was used for the measurement. Exponential decay curves were generated using Prism. Please note that the decay curve of FDAA intensity is dependent on imaging conditions (e.g. the power of light source).

Measurement of distribution coefficient
Thus, the photo-stability values reported here should only be used when comparing FDAAs in this study. Table 3 was accomplished using the MicrobeJ plugin in FIJI (8).

FDAA intensity quantification in
Cell morphologies and boundaries were defined using the phase channel. The fluorescence intensity of the cells (n>100) was then measured and averaged using the plugin.               Manuscript drafting by YH, JR, EK, KCH, YVB, and MSV. All authors were involved in the design of experiments in the paper.