Fluorescent D-Amino Acid Labeling for Bacterial Imaging

The fluorescent d-amino acid (FDAA) NADA (4-chloro-7-nitrobenzofurazan 3-amino-d-alanine) or TADA (tetramethylrhodamine 3-amino-d-alanine) (18 (link), 51 (link)) was added to early exponential cells (OD₆₀₀ of 0.05) to a final concentration of 100 μM. Cultures were grown to an OD₆₀₀ of 0.20 and placed on ice for 2 min to halt labeling. One milliliter of culture was centrifuged at 14,000 × g for 5 min at 4°C. Cell pellets were washed once with cold medium, resuspended, and seeded into microfluidic chambers at 10°C. Flow was then initiated with prewarmed medium without peptide (control) or with the indicated concentration of peptide (treatment). Where indicated, expression of a FliM-tdTomato fusion (10 (link)) was induced with 0.1% l-Ara (Sigma-Aldrich), and cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min prior to the addition of 6-carboxyfluorescein (FAM)-labeled B22.