Biology subjects

Yahashiri, A.

Publications and source records attributed to Yahashiri, A..

5 recordsLinked to original sources

bioRxiv2026

The lytic transglycosylase MltA participates in turnover of septal peptidoglycan in Escherichia coli

Daughter cell separation in Escherichia coli is driven primarily by two classes of peptidoglycan (PG) hydrolases that work in tandem: N-acetylmuramoyl-L-alanine amidases that strip stem peptides from the PG glycan backbone and lytic transglycosylases (LTs) that break down the PG glycan backbone. Although the relevant amidases have been known for years, which of E. colis eight LTs contribute to this process is less clear. Because the amidases process PG first, the relevant LTs must utilize peptide-free or "denuded" glycan substrates (dnGs). MltA is one of the few E. coli LTs that can break down peptide-free PG glycans in vitro, but its precise physiological roles are not known. Here we show MltA localizes to the division site in constricting E. coli cells and cells lacking MltA accumulated dnGs in septal PG. We found that MltA binds to the anhydroMurNAc ends of glycan chains, which raises the possibility that these structures are enriched in septal PG. Nevertheless, as reported previously, deletion of mltA does not impair daughter cell separation sufficiently to cause a chaining phenotype. Overall, our findings demonstrate that MltA is a physiologically relevant peptidoglycan hydrolase for cell division in E. coli. IMPORTANCEHow bacteria coordinate synthesis and cleavage of septal peptidoglycan remains poorly understood, in part because some of the relevant enzymes have yet to be identified. Here we show that the E. coli lytic transglycosylase MltA is involved in cleaving septal peptidoglycan. Besides elucidating a physiological role for MltA, our work brings the field a step closer to identifying all of the proteins involved in cell division in an important model organism.

microbiology↗

bioRxiv2024

FtsN coordinates septal peptidoglycan synthesis and degradation through self-interaction of SPOR in E. coli

The E. coli cell division protein FtsN was proposed to coordinate septal peptidoglycan (sPG) synthesis and degradation to ensure robust cell wall constriction without lethal lesions. Although the precise mechanism remains unclear, previous work highlights the importance of two FtsN domains: the E domain, which interacts with and activates the sPG synthesis complex FtsWIQLB, and the SPOR domain, which binds to denuded glycan (dnG) strands, key intermediates in sPG degradation. Here, we used single-molecule tracking of FtsN and FtsW (a proxy for the sPG synthesis complex FtsWIQLB) to investigate how FtsN coordinates the two opposing processes. We observed dynamic behaviors indicating that FtsNs SPOR domain binds to dnGs cooperatively, which both sequesters the sPG synthesis complex on dnG (termed as the dnG-track) and protects dnGs from degradation by lytic transglycosylases (LTs). The release of the SPOR domain from dnGs leads to activating the sPG synthesis complex on the sPG-track and simultaneously exposing those same dnGs to degradation. Furthermore, FtsNs SPOR domain self-interacts and facilitates the formation of a multimeric sPG synthesis complex on both tracks. The cooperative self-interaction of the SPOR domain creates a sensitive switch to regulate the partitioning of FtsN between the dnG- and sPG-tracks, thereby controlling the balance between sequestered and active populations of the sPG synthesis complex. As such, FtsN coordinates sPG synthesis and degradation in space and time.

microbiology↗

bioRxiv2024

FtsZ-mediated spatial-temporal control over septal cell wall synthesis

FtsZ, the tubulin-like GTPase, is the central organizer of the bacterial divisome, a macromolecular complex that synthesizes new septal cell wall and degrades old septal cell wall (made of septal peptidoglycan, sPG) to allow cell wall constriction and cytokinesis. In E. coli, it is well accepted that 1) FtsZ recruits all essential divisome proteins to the septum, including the core sPG synthase complex, FtsWI/QLB and its activator, FtsN; 2) FtsWI/QLB must complex with FtsN to produce sPG under the wild-type background; and 3) the Brownian ratcheting by treadmilling FtsZ polymers drives the directional movements of sPG synthase proteins along the septum circumference; and 4) FtsZ is essential for the early stage, but dispensable for the late stage of cell wall constriction. However, it remains unclear how FtsZ spatial-temporally organizes the divisome for robust bacterial cytokinesis throughout cell wall constriction process. Combining theoretical modeling with experiments in E. coli, we show that at the early stage during cell division, the Brownian ratcheting by FtsZ treadmilling acts both as a template to corral FtsWI/QLB and FtsN into close contacts for FtsWI/QLB-FtsN complex formation and as a conveyor to maximally homologize the septal distribution of sPG synthesis activities to avoid uneven cell wall constriction. When the septum constricts progressively, the FtsN septal density increases via binding to denuded sPG; consequently, the denuded PG-bound FtsN serves as the template to activate FtsWI/QLB for continued sPG synthesis, rendering FtsZ dispensable. Our work establishes an overarching framework that FtsZ spatial-temporally controls over septal cell wall constriction. SignificanceBacteria utilize FtsZ, the tubulin-like GTPase, to organize cell wall enzymes during cell division. FtsZ forms treadmilling polymers along the septum circumference and drives the directional movement of cell wall enzymes for robust cell wall constriction. How this role is achieved is unclear. We show that FtsZ treadmilling acts both as a template to corral cell wall enzymes into close contacts for priming and as a conveyor to homologize the septal distribution of cell wall synthesis activities for even septum constriction. These roles evolve at different stages of cell division and are modulated differentially by different bacteria; they likely define an overarching principle for robust cell division across the microbial world.

biophysics↗

bioRxiv2022

Comparative study of bacterial SPOR domains identifies functionally important differences in glycan binding affinity

Bacterial SPOR domains target proteins to the divisome by binding septal peptidoglycan (PG) at sites where cell wall amidases have removed stem peptides. These PG structures are referred to as denuded glycans. Although all characterized SPOR domains bind denuded glycans, whether there are differences in affinity is not known. Here we use isothermal titration calorimetry (ITC) to determine the relative PG glycan binding affinity (Kd) of four Escherichia coli SPOR domains and one Cytophaga hutchinsonii SPOR domain. We found that the Kd values ranged from approximately 1 {micro}M for E. coli DamXSPOR and C. hutchinsonii CHU2221SPOR to about 10 {micro}M for E. coli FtsNSPOR. To ask whether these differences in PG binding affinity are important for SPOR domain protein function, we constructed and characterized a set of DamX and FtsN "swap" proteins. As expected, all SPOR domain swap proteins localized to the division site, and in the case of FtsN all of the heterologous SPOR domains supported cell division. But for DamX only the high-affinity SPOR domain from CHU2221 supported normal function in cell division. In summary, different SPOR domains bind denuded PG glycans with different affinity, which appears to be very important for the function of some SPOR domain proteins (e.g., DamX) but not others (e.g., FtsN). ImportanceSPOR domain proteins are prominent components of the cell division apparatus in a wide variety of bacteria. The primary function of SPOR domains is to target proteins to the division site, which they accomplish by binding to septal peptidoglycan. But whether SPOR domains have any functions beyond septal targeting is unknown. Here we show that SPOR domains vary in their PG binding affinities and, at least in the case of the E. coli cell division protein DamX, having a high-affinity SPOR domain contributes to proper function.

microbiology↗

bioRxiv2021

FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of [~] 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the [~] 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZs treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.

microbiology↗