Mycobacterium smegmatis serves as an experimental model for Mycobacterium tuberculosis , as both share key features of cell wall organization and supramolecular architecture relevant to pathogenicity. Understanding cell wall structure is essential for developing drugs to control mycobacterial growth. Here, we investigate the spectroscopic behavior of the non-canonical amino acid HADA (3-[(7-hydroxy-2-oxo-2H-1-benzopyran-3-yl)carbonylamino]-D-alanine), a fluorescent D-alanine analog used to track nascent peptidoglycan in live bacteria. HADA fluorescence enables visualization of de novo peptidoglycan synthesis at the poles and septal wall. We first examined the influence of solvent polarity on HADA’s excitation and emission spectra in water and organic solvents (chloroform, acetonitrile, ethanol and methanol). Two distinct spectral profiles were observed, independent of HADA concentration, with water spectra separated from organic solvent. Spectral phasor analysis further revealed two emission states reflecting solvent-dependent microenvironments. Building on these, in vitro studies analyzing HADA fluorescence in the cell wall of M. smegmatis revealed distinct spectral environments associated with newly synthesized versus pre-existing cell wall regions. Together, these results demonstrate that HADA emission is highly sensitive to its local environment, expanding its value beyond labeling bacterial cell wall, to study architecture and growth dynamics.
Rossello et al. (2026) studied this question.