Non-surface-enhanced Raman spectroscopy using a 514.5 nm wavelength laser has been used to measure the molecular difference of conditional mutants of Mycobacterium smegmatis expressing three different alleles: wild-type wag31(Mtb), phosphoablative wag31T73(Mtb). and phosphomimetic wag31T73E(Mtb). This study demonstrates that the phosphorylation of Wag31, a key cell-division protein. causes significant differences in the quantity of amino acids associated with peptidoglycan precursor proteins and lipid 11 which are observable in the Raman spectra of these cells Raman spectra were also acquired from the isolated P60 cell envelope fraction of the cells expressing wag31T73A(Mtb) and wag31T73E(Mtb). A significant number of the molecular vibrational differences observed in the cells were also observed in the cell envelope fraction, indicating that these differences are indeed localized in the cell envelope Principal component analyses and discriminant function analyses were conducted Oil these data to demonstrate the ease of spectral classification and the reproducibility of the data (C) 2009 Elsevier Inc All rights reserved