Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof
Disclosed herein are compositions for assessing peptidoglycan biosynthesis in bacteria, for identifying bacteria, and for screening for bacterial cell wall-acting and/or cell wall-disrupting agents via modified D-amino acids and methods of use thereof. Also disclosed are live bacteria having one or more modified D-amino acids as described herein incorporated into peptidoglycan of a bacterial cell wall.
1. A method of assessing bacterial cell wall synthesis in real time, the method comprising:
providing live bacteria with a first amount of at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label selected from the group consisting of C y3B ADA, AF 647 ADA, AF 350 ADA, BADA, FADA, HADA, NADA, TADA, YADA, FDL, HDL, NDL, and TDL, and optionally a second amount of at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient for bacterial cell wall synthesis,
wherein the bacteria covalently incorporate the at least one modified amino acid and optionally the at least one additional modified amino acid into a stem peptide of peptidoglycan of the bacterial cell wall.
2. The method of claim 1 , wherein the first amount and second amount comprise a first concentration and a second concentration, respectively, wherein the first and second concentrations range from about and including 0.10 μM to about and including 1 mM.
3. The method of claim 1 , further comprising detecting a fluorescent signal emitted by the at least one modified amino acid comprising a D-amino acid covalently attached to a fluorescent label, and optionally the at least one additional modified amino acid comprising a clickable D-amino acid incorporated into the stem peptide.
4. The method of claim 1 , wherein the bacteria are Gram-positive bacteria or Gram-negative bacteria.
5. The method of claim 1 , wherein the at least one additional modified amino acid comprising a clickable D-amino acid is selected from the group consisting of EDA and ETDA.
6. The method of claim 1 , wherein the D-amino acid is selected from the group consisting of 3-amino-D-Ala, D-Ala, D-Asp, D-Cys, D-Glu and D-Lys.