Virtually all bacteria possess peptidoglycan. Located exterior to the plasma membrane, it is perhaps the largest single macromolecule in bacteria and serves as their cell cytoskeleton giving it shape, rigidity while also supporting physiologically important functions. The peptidoglycan is composed of sugar backbone and peptide side-chains that are linked to each other. Historically, the chemical composition and structure of the peptidoglycan was developed using studies based on E. coli and other model organism. According to this model, the peptide side chains are exclusively crosslinked at the 4th and 3rd amino acids (4-3 crosslinks) by D,D-transpeptidases (DDTs, also known as penicillin binding proteins). However, recent studies show that peptidoglycan of mycobacteria is significantly different. Today, we know that the final step of peptidoglycan synthesis in mycobacteria (such as, Mycobacterium tuberculosis, Mycobacterium abscessus, etc) requires two enzyme sets, namely L,D-transpeptidases (LDTs) and DDTs that generate 3-3 and 4-3 linkages, respectively. For mycobacteria, LDT activity is likely more significant than DDT, as 67-80% of the linkages in their peptidoglycan are of the 3-3 type generated by the LDTs.