The peptidoglycan layer is a vital component of the bacterial cell wall. The existing paradigm describes the peptidoglycan network as a static structure that is cross-linked predominantly by 4-->3 transpeptide linkages. However, the nonclassical 3-->3 linkages predominate the transpeptide networking of the peptidoglycan layer of nonreplicating Mycobacterium tuberculosis. The molecular basis of these linkages and their role in the physiology of the peptidoglycan layer, virulence and susceptibility of M. tuberculosis to drugs remain undefined. Here we identify MT2594 as an L,D-transpeptidase that generates 3-->3 linkages in M. tuberculosis. We show that the loss of this protein leads to altered colony morphology, loss of virulence and increased susceptibility to amoxicillin-clavulanate during the chronic phase of infection. This suggests that 3-->3 cross-linking is vital to the physiology of the peptidoglycan layer. Although a functional homolog exists, expression of ldtMt2 is dominant throughout the growth phases of M. tuberculosis. 4-->3 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today, beta-lactams. Recently, meropenem-clavulanate was shown to be effective against drug-resistant M. tuberculosis. Our study suggests that a combination of L,D-transpeptidase and beta-lactamase inhibitors could effectively target persisting bacilli during the chronic phase of tuberculosis.