Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria

Abstract

1 2 D-amino acid probes label cell wall peptidoglycan at both the poles and sidewall of pole-growing 3 mycobacteria. Since peptidoglycan assembly along the cell periphery could provide a rapid, 4 growth-independent means by which to edit the cell wall, we sought to clarify the precise 5 metabolic fates of these probes. D-amino acid monopeptides were incorporated into 6 peptidoglycan by L,D-transpeptidase remodeling enzymes to varying extents. Dipeptides were 7 incorporated into cytoplasmic precursors. While dipeptide-marked peptidoglycan synthesis at 8 the poles was associated with cell elongation, synthesis along the periphery was highly 9 responsive to cell wall damage. Our observations suggest a post-expansion role for 1 0 peptidoglycan assembly along the mycobacterial sidewall and provide a conceptual framework 1 1 for understanding cell wall robustness in the face of polar growth.

Trehalose recycling promotes energy-efficient mycomembrane reorganization in nutrient-limited mycobacteria

“…We hypothesized that the AGM synthesis that we observe under carbon deprivation ( Figure 1C ) is a cell-wide response, similar to peptidoglycan repair. Quantitative fluorescence microscopy revealed that O-AlkTMM labeling of M. smegmatis growing in carbon-replete medium comprised polar gradients ( Figure 1F ) as expected (Foley et al, 2016; Garcia-Heredia et al, 2018). However, in slow or non-growing, carbon-deprived M. smegmatis , O-AlkTMM-labeled species were more evenly distributed around the periphery of the cell.…”

“…We previously showed that the fluorescent D-amino acid HADA and alkDala incorporate into M. smegmatis peptidoglycan via both cytoplasmic and L,D-transpeptidase enzymes (Garcia-Heredia et al, 2018). HADA and alkDala labeling roughly correlated with mycobacterial growth rate under different amounts of glucose or acetate ( Figures 1C, S1B, S1C ).…”

“…Mycobacteria growing in nutrient-replete medium construct their cell envelope in gradients that emanate from the poles and continue along the sidewall (Foley et al, 2016; Garcia-Heredia et al, 2018). While polar peptidoglycan synthesis promotes cell elongation, sidewall synthesis occurs in response to cell wall damage.…”

“…Our data collectively indicate that the net result of such reactions is decreased TDM and spatial rearrangement of AGM. We previously showed that synthesis of peptidoglycan along the non-expanding sidewall of M. smegmatis is enhanced in response to cell wall damage (Garcia-Heredia et al, 2018). AGM synthesis under carbon starvation also occurs along the cell periphery ( Figure 1F ), further supporting the notion that mycobacteria can edit their cell surface in a growth-independent fashion.…”

Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins inMycobacterium smegmatis

“…Mycobacteria must retain this complex cell envelope as they grow and extend their rod-shaped cell. New components for cell envelope extension are added at the subpolar regions (6–12). The intracellular membrane domain (IMD) is distinct from the conventional plasma membrane, and is found enriched in the subpolar regions of actively growing cells (13, 14).…”