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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Penicillin-binding proteins (PBPs) are crucial antimicrobial targets involved in bacterial cell wall synthesis.
• Mycobacterial cell elongation relies on High Molecular Weight PBPs, but the role of Low Molecular Weight Class 1C PBPs, like DD-carboxypeptidases (DD-CPases), is poorly understood.
• Mycobacterium smegmatis possesses four DD-CPase homologues with homology to E. coli counterparts.

Purpose of the Study:

• To investigate the function of DD-carboxypeptidases (DD-CPases) in mycobacteria.
• To determine the role of specific DD-CPase genes, particularly MSMEG_6113 (dacB), in mycobacterial growth and cell wall synthesis.
• To explore the essentiality and function of a conserved four-gene operon in mycobacterial peptidoglycan synthesis.

Main Methods:

• Gene deletion studies were performed on Mycobacterium smegmatis to assess the impact of individual DD-CPase homologues.
• Conditional gene depletion using anhydrotetracycline-responsive repressors was employed to study essential genes.
• Spatial incorporation of new peptidoglycan was analyzed to understand cell wall synthesis localization.

Main Results:

• Individual deletion of MSMEG_1661, MSMEG_2433, or MSMEG_2432 did not affect growth, morphology, or drug susceptibility.
• Deletion of MSMEG_6113 (dacB) was only viable in a merodiploid strain expressing the homologous M. tuberculosis operon, indicating essentiality.
• Depletion of the four-gene operon (including MSMEG_6113) led to reduced bipolar peptidoglycan synthesis.

Conclusions:

• The DD-CPase MSMEG_6113 (dacB) and its associated four-gene operon are essential for mycobacterial growth.
• This conserved operon likely plays a critical role in regulating the spatial localization of peptidoglycan synthesis in mycobacteria.
• Understanding this pathway could reveal new targets for antimicrobial drug development.