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 non-classical 3→3 linkages predominate the transpeptide networking of the peptidoglycan layer of non-replicating M. tuberculosis 1,2. The molecular basis of these linkages, their significance to the physiology of the peptidoglycan layer, virulence and susceptibility of M. tuberculosis to drugs remain undefined. Here, we identify MT2594 (Rv2518c) as an L,D-transpeptidase that generates 3→3 linkages in M. tuberculosis. We show that the loss of this gene 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 MT2594 is dominant throughout the growth phases of M. tuberculosis. The 4→3 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today, namely β-lactams. Recently, meropenem–clavulanate was shown to be effective against drug resistant M. tuberculosis 3. Our study suggests that a combination of an L,D-transpeptidase and a β-lactamase inhibitors could effectively target persisters during chronic phase of TB.