Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria

Abstract

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

“…These data indicated that the three new TMM reporters exhibited high specificity for members of the Corynebacterineae , with robust labeling of Msmeg and Cglut but no labeling of Bsub or E. coli (Figure A). In addition, the microscopy images in Figure B showed that treatment with compounds 4 – 6 resulted in cell‐surface fluorescence concentrated at the poles and septa of bacteria, which is consistent with a metabolic route of incorporation, the polar growth mode of Corynebacterineae , and with prior imaging studies employing O‐AlkTMM‐C7 ( 2 ) …”

“…It will be of interest to further explore the ramifications of adding large fluorophores (or other sizeable chemical cargo) to the TMM scaffold in follow‐on studies. For instance, it was recently shown that d ‐amino acid‐based reporters of peptidoglycan synthesis incorporate via different labeling routes depending on the nature of the detectable tag …”

Lsr2 is a nucleoid-associated protein that exerts pleiotropic effects on mycobacterial cellular processes

“…The Mycobacterium genus encompasses not only human pathogens that have enormous impact on global health (i.e., Mycobacterium tuberculosis, M. leprae), but also the saprophytic species, such as Mycobacterium smegmatis, which is a model organism for studies on the cell biology of tubercle bacilli. Mycobacteria possess a unique multilayered cell envelope and, unlike other rod-shaped bacteria, incorporate peptidoglycan precursors at their cell tips (Abrahams & Besra, 2018;García-Heredia et al, 2018;B. Singh et al, 2013).…”

Breaking down walls

“…This is mainly due to inherent mechanisms in M. tuberculosis that inactivate certain types of antibiotics, and to the fact that we know relatively little about how this polymer is synthesized and remodeled as the bacteria spread ( Story-Roller and Lamichhane, 2018 ). Now, in eLife, two independent groups of researchers report results that advance our understanding of these processes ( García-Heredia et al, 2018 ; Baranowski et al, 2018 ).…”

“…Sloan Siegrist of the University of Massachusetts and colleagues – including Alam García-Heredia and Amol Arunrao Pohane as joint first authors – report the results of experiments on M. tuberculosis and its close relative, M. smegmatis , where they explore peptidoglycan biology further ( García-Heredia et al, 2018 ). The team used reagents called fluorescent D-amino acids (FDAAs) to monitor the arrival and distribution of new peptidoglycan units both inside and outside the cell, as well as the remodeling of peptidoglycan outside the cell ( Figure 1 ).…”