Cell Wall Damage Reveals Spatial Flexibility in Peptidoglycan Synthesis and a Nonredundant Role for RodA in Mycobacteria

Abstract

Peptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions.

The cell wall polymer initiates plasma membrane partitioning in mycobacteria

“…6A). However, as with RodA absence or moenomycin treatment (Melzer et al, 2022) there was a modest but statistically significant decrease in the polarity of nascent peptidoglycan in Δ ponA2 compared to wild-type (Fig. 6B and 6C).…”

“…We wondered whether PonA2 supports membrane homeostasis by localizing cell wall assembly. Previously we showed that the polarity of peptidoglycan synthesis decreases in the absence of RodA, a SEDS family transglycosylase (and upon treatment with moenomycin, an antibiotic that specifically inhibits transglycosylation of class A PBPs (aPBPs) such as PonA2 (Melzer et al, 2022). To more directly assay the function of PonA2, we labeled nascent peptidoglycan in wild-type and Δ ponA2 after a brief incubation in the presence of alkyne-D-alanine-D-alanine (alkDADA, also called EDA-DA (García-Heredia et al, 2018; Liechti et al, 2014)) and detected the presence of the alkyne probe via copper-catalyzed alkyne-azide cycloaddition (CuAAC) to a fluorescent azide label.…”

“…While we previously showed that spheroplasting delocalizes multiple IMD markers (García-Heredia et al, 2021), complete removal of the cell wall is likely to have pleiotropic effects on membrane physiology. More recently, we showed that limited peptidoglycan digestion by the glycoside hydrolases lysozyme and mutanolysin delocalizes the IMD-enriched protein MurG (Melzer et al, 2022). To test the specificity of this observation, we examined the distribution of a second IMD marker, GlfT2 (Hayashi et al, 2016, 2018).…”

“…We identified a factor, cell wall synthase PonA2, that establishes membrane partitioning via its transglycosylase domain. Our earlier work had suggested that peptidoglycan damage and/or removal (García-Heredia et al, 2021; Melzer et al, 2022), but not inhibition of peptidoglycan synthesis (Hayashi et al, 2018), departition the M. smegmatis membrane. Similar, seemingly-incongruous observations have been made for the roles of the cell wall and its synthesis in other bacteria (Wagner et al, 2020) and in plants (Daněk et al, 2020).…”

“…In laterally growing, rod-shaped bacteria, it is thought that the SEDS-family transglycosylase RodA lays the template for cell wall elongation while the bifunctional, transglycosylase/transpeptidase aPBPs fill in the gaps for maintenance and repair (Cho et al, 2016; Mueller et al, 2019; Murphy et al, 2021; Paradis-Bleau et al, 2010; Typas et al, 2010; Vigouroux et al, 2020). Unlike the organisms in which this model has been tested, pole-growing Mycobacteriales lack the cytoskeletal protein MreB and do not require RodA for viability or shape (Arora et al, 2018) so the division of labor is less clear (Melzer et al, 2022; Sher et al, 2021). Given that PonA2 but not RodA contributes to membrane partitioning in M. smegmatis , PonA2 may regulate the 3-dimensional density and/or heterogeneity of the polymeric cell wall network.…”