Characterization of putative DD-carboxypeptidase-encoding genes in Mycobacterium smegmatis

Penicillin binding proteins (PBPs) are the target of numerous antimicrobial agents that disrupt bacterial cell wall synthesis. In mycobacteria, cell elongation occurs through insertion of nascent cell wall material in the sub-polar region, a process largely driven by High Molecular Weight PBPs. In contrast, the function of DD-carboxypeptidases (DD-CPases), which are Low Molecular Weight Class 1C PBPs, in mycobacteria remains poorly understood. Mycobacterium smegmatis encodes four putative DD-CPase homologues, which display homology to counterparts in Escherichia coli. Herein, we demonstrate that these are expressed in varying abundance during growth. Deletion of MSMEG_1661, MSMEG_2433 or MSMEG_2432, individually resulted in no defects in growth, cell morphology, drug susceptibility or spatial incorporation of new peptidoglycan. In contrast, deletion of MSMEG_6113 (dacB) was only possible in a merodiploid strain expressing the homologous M. tuberculosis operon encoding Rv3627c (dacB), Rv3626c, Rv3625c (mesJ) and Rv3624c (hpt), suggestive of essentiality. To investigate the role of this operon in mycobacterial growth, we depleted gene expression using anhydrotetracycline-responsive repressors and noted reduced bipolar peptidoglycan synthesis. These data point to a possible role for this four gene operon, which is highly conserved across all mycobacterial species, in regulating spatial localization of peptidoglycan synthesis.