# The combinatorial deletion of mycobacterial dd-carboxypeptidases is readily tolerated in Mycobacterium smegmatis

## TL;DR

Deleting certain enzymes in Mycobacterium smegmatis does not significantly affect its survival or response to stress.

## Contribution

The study shows that multiple deletions of dd-carboxypeptidase enzymes in M. smegmatis are well tolerated.

## Key findings

- Deleting dd-carboxypeptidase genes had minimal impact on cell viability and biofilm formation.
- Mutant strains showed normal growth under stress conditions like high salt or pH.
- No significant changes in cell wall remodeling or drug susceptibility were observed.

## Abstract

Proteins facilitating bacterial cell wall (CW) biosynthesis are crucial for survival and broadly remain the target of numerous antimicrobial agents. Herein, we focused on characterizing the physiological roles of low-molecular-weight penicillin-binding proteins (LMW PBPs), with dd-carboxypeptidase (dd-CPase) activity, in Mycobacterium smegmatis. Following various combinatorial gene deletions, cell viability, colony structure and the ability to produce biofilms remained unperturbed. Whilst small changes in cellular morphology and permeability were evident, hierarchical roles could not be ascribed to specific dd-CPase homologues. Strains exposed to lysozyme exhibited low levels of compensatory expression for the remaining homologues, but this was not evident for exposure to the CW-targeting Augmentin. When tested against a broader concentration range of various antibiotics, using MIC and spotting assays, only marginal changes in drug susceptibility were evident. Strains cultured under conditions of excess NaCl or enhanced pH levels grew normally. Given the established role of remodelling in dd-CPase enzymes of other bacteria, we further assessed whether the ability to repair lysozyme-induced CW damage was compromised. With the incorporation of the fluorescent d-amino acid peptidoglycan probe, TAMRA-d-alanine, as a proxy for remodelling, no changes in staining patterns were evident. However, the frequency of cells containing unresolved septa increased in all mutant strains, suggesting a potential role for dd-CPases in the mycobacterial cell process. In conclusion, we have demonstrated that the combinatorial deletion of non-essential mycobacterial dd-CPase homologues largely has no significant impact on mycobacterial physiology or involvement in the response to the various environmental stressors tested herein.