Lysozyme treatment was for 9 h. Discussion M. tuberculosis Rv1096 protein, S. pneumoniae PgdA protein (spPdgA), L. monocytogenes PgdA (lmo0415), and L. lactis PgdA (XynD) are carbohydrate esterase MG-132 4 (CE-4) superfamily members. The CE-4 superfamily includes peptidoglycan GlcNAc deacetylases, rhizobial NodB chito-oligosaccharide
deacetylases, chitin deacetylases, acetyl xylan esterases, and xylanases . The substrates of these enzymes are polymers or basic structures that assemble PG backbone glycan strands. In this study, Rv1096 protein, over-expressed in both E. coli and M. smegmatis, was able to deacetylate M. smegmatis peptidoglycan. Therefore, M. tuberculosis Rv1096 protein is a peptidoglycan deacetylase. As shown in Figure 1, Rv1096
and three other deacetylases share sequence conservation at two catalytic histidine residues (H-326 and H-330) . The metal ligand sites, Lorlatinib concentration including Asp (D-275), Arg (A-295), Asp (D-391) and His (H-417) residues, which were identified in the S. pneumonia PgdA protein [5, 10, 28], are all present in the Rv1096 protein. These highly conserved sequences in Rv1096 suggest that it may have metallo-dependence. Indeed, our results show that the enzymatic activity of Rv1096 increased after supplementation with divalent cations, especially Co2+. Taken together, our results suggest that Rv1096 may use similar catalytic mechanisms as the S. pneumoniae PgdA protein to deacetylate PG. It has been reported that PG deacetylase contributes to lysozyme resistance in some bacterial species, such as Bacillus cereus , S. pneumonia  , L. monocytogenes  and Shigella flexneri . Generally, pdgA mutants are more sensitive to lysozyme degradation in the stationary phase. Similarly, M. smegmatis over-expressing Rv1096 protein showed remarkable resistance to lysozyme at the end of log phase growth. In the present study, the viability
of M. smegmatis/Rv1096 was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment, indicating that PG deacetylation by the Rv1096 deacetylase had increased lysozyme resistance. The morphological changes observed between wild-type M. smegmatis and M. smegmatis/Rv1096 provides strong evidence that Rv1096 activity helped to preserve the integrity of the cell wall during Methane monooxygenase lysozyme treatment. Wild-type M. smegmatis lost its acid-fastness because of the increased cell wall permeability caused by lysozyme treatment. SEM ACY-1215 nmr observations showed that wild-type M. smegmatis had a wrinkled cell surface with outward spilling of its cell contents, while M. smegmatis/Rv1096 maintained its cell wall integrity and acid fastness. Therefore, it is likely that the functionality of the Rv1096 protein of M. smegmatis/Rv1096 contributed to its cell wall integrity. In fact, PG N-deacetylase has been shown to be a virulence factor in several bacteria including S. pneumonia , S. iniae  , L. monocytogenes  and H. pylori . For example, the S.