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Recent studies have renewed interest in β-lactam antibiotics as a potential treatment for Mycobacterium tuberculosis infection. To explore the opportunities and limitations of this approach, we sought to better understand potential resistance mechanisms to β-lactam antibiotics in M. tuberculosis. We identified mutations in the penicillin-binding protein (PBP) ponA2 that were able to confer some degree of resistance to the cephalosporin subclass of β-lactams. Surprisingly, deletion of ponA2 also confers resistance, demonstrating that β-lactam resistance can spontaneously arise from PBP loss of function. We show that ponA2 mutants resistant to the cephalosporin subclass of β-lactams in fact show increased susceptibility to meropenem, a carbapenem that is known to target l,d-transpeptidases, thereby suggesting that in the absence of PonA2, an alternative mode of peptidoglycan synthesis likely becomes essential. Consistent with this hypothesis, a negative genetic selection identified the l,d-transpeptidase ldtMt2 as essential in the absence of ponA2. The mechanism of β-lactam resistance we outline is consistent with emerging models of β-lactam killing, while the investigation of ponA2 downstream and synthetic lethal genes sheds light on the mechanism of cell wall biosynthesis and the interaction between conventional PBPs and l,d-transpeptidases.