MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli

Bohrhunter, Jessica L; Rohs, Patricia D. A.; Torres, Grasiela; Yunck, Rachel; Bernhardt, Thomas G.

doi:10.1111/mmi.14660

Cited by 25 publications

(28 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on the crystal structure, the distance from the LysM-like subdomain to the catalytic residue is approximately 50 °, but there are some additional residues between the LysM-like domain and the TM helix anchoring the protein in the membrane, which may extend the distance from the membrane to the active site. 34 Each disaccharide unit in peptidoglycan spans approximately 10 °. Overall, the dimensions of MpgA are sufficiently well-matched with what we now know about its cleavage site preferences for the proposed model to be plausible.…”

Section: Resultsmentioning

confidence: 99%

“…It is unknown whether cleavage site control is important in YceG family proteins; however, an MltG variant lacking the LysM-like subdomain was not functional in E. coli . 34 Because enzymatic activity for this MltG variant was not confirmed, it is not clear whether lack of function reflected an inability to cleave nascent peptidoglycan at all or was due to lack of wildtype cleavage site selectivity.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

Taguchi

Walker

2021

Preprint

View full text Add to dashboard Cite

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. We are only beginning to understand the roles of peptidoglycan cleavage enzymes in cell wall assembly. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end. MpgA produces much longer peptidoglycan oligomers and we show that its cleavage site selectivity is controlled by the LysM-like subdomain, which is also present in MltG. We propose that MltG's ability to complement loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

Taguchi

Walker

2021

Preprint

View full text Add to dashboard Cite

show abstract

“…MltG in E. coli have been shown to possess endolytic transglycosylase activity, i.e. breaking glycosidic bonds within a glycan strand (14,43). Structural modelling and site directed mutagenesis of the active site of the pneumococcal MltG suggest that it has the same muralytic activity (15).…”

Section: Discussionmentioning

confidence: 99%

“…Structural modelling and site directed mutagenesis of the active site of the pneumococcal MltG suggest that it has the same muralytic activity (15). It has been hypothesized that MltG in S. pneumoniae releases glycan strands made by both classes of PBPs so that they can be cross linked to new PG made by the divisome and elongasome (15,43). In light of the recent discoveries regarding the function of class A PBPs, which suggest PBP1a to be involved in maturing the newly synthesized PG by filling in gaps, we propose a different model: MltG works together with amidases to open the PG layer so that PBP2b/RodA can add new PG to the existing layer and hence elongate the dividing cell (Figure 6).…”

Section: Discussionmentioning

confidence: 99%

EloR Interacts with the Lytic Transglycosylase MltG at Midcell in Streptococcus pneumoniae R6

et al. 2021

View full text Add to dashboard Cite

The ellipsoid shape of Streptococcus pneumoniae is determined by the synchronized actions of the elongasome and the divisome, which have the task of creating a protective layer of peptidoglycan (PG) enveloping the cell membrane. The elongasome is necessary for expanding PG in the longitudinal direction whereas the divisome synthesizes the PG that divides one cell into two. Although there is still little knowledge about how these two modes of PG synthesis are coordinated, it was recently discovered that two RNA-binding proteins called EloR and KhpA are part of a novel regulatory pathway controlling elongation in S. pneumoniae. EloR and KhpA form a complex that work closely with the Ser/Thr kinase StkP to regulate cell elongation. Here, we have further explored how this regulation occur. EloR/KhpA is found at midcell, a localization fully dependent on EloR. Using a bacterial two-hybrid assay we probed EloR against several elongasome proteins and found an interaction with the lytic transglycosylase homolog MltG. By using EloR as bait in immunoprecipitation assays, MltG was pulled down confirming that they are part of the same protein complex. Fluorescent microscopy demonstrated that the Jag domain of EloR is essential for EloR’s midcell localization and its interaction with MltG. Since MltG is found at midcell independent of EloR, our results suggest that MltG is responsible for recruitment of the EloR/KhpA complex to the division zone to regulate cell elongation. Importance Bacterial cell division has been a successful target for antimicrobial agents for decades. How different pathogens regulate cell division is, however, poorly understood. To fully exploit the potential for future antibiotics targeting cell division, we need to understand the details of how the bacteria regulate and construct cell wall during this process. Here we have revealed that the newly identified EloR/KhpA complex, regulating cell elongation in S. pneumoniae, forms a complex with the essential peptidoglycan transglycosylase MltG at midcell. EloR, KhpA and MltG are conserved among many bacterial species and the EloR/KhpA/MltG regulatory pathway is most likely a common mechanism employed by many Gram-positive bacteria to coordinate cell elongation and septation.

show abstract

“…MltG associates with active PG synthetic complexes to release new strands from the cytoplasmic membrane (to which they are initially tethered via undecaprenyl pyrophosphate), presumably as they emerge from GTase activity; consequently, MltG is a strong determinant of PG strand length. [26][27][28][29] Other physiological roles assigned to LTGs include local PG editing for insertion of PGspanning protein complexes 10,12,30 and PG recycling. PG recycling, the re-incorporation of PG breakdown products into the biosynthesis cycle, starts with re-uptake of PG turnover products by the importer AmpG.…”

Section: Introductionmentioning

confidence: 99%

Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

Weaver

Álvarez

Rosch

et al. 2021

Preprint

View full text Add to dashboard Cite

The peptidoglycan cell wall is a predominant defining structure of bacteria, determining cell shape and supporting survival in diverse conditions. As a single, macromolecular sacculus enveloping the bacterial cell during growth and division, peptidoglycan is necessarily a dynamic structure that requires highly regulated synthesis of new material, remodeling, and turnover, or autolysis, of old material. Despite ubiquitous clinical exploitation of peptidoglycan synthesis as an antibiotic target, much remains unknown about how bacteria modulate synthetic and autolytic processes. Here, we couple bacterial genetics in Vibrio cholerae with compositional analysis of soluble pools of peptidoglycan turnover products to uncover a critical role for a widely misunderstood class of autolytic enzymes, the lytic transglycosylases (LTGs). We demonstrate that LTG activity is specifically required for vegetative growth. The vast majority of LTGs, however, are dispensable for growth, and defects that are ultimately lethal accumulate due to generally inadequate LTG activity, rather than the absence of specific individual enzymes. Consistent with this, we found that a heterologously expressed E. coli LTG, MltE, is capable of sustaining V. cholerae growth in the absence of endogenous LTGs. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains accumulate in the WT, and, to a higher degree, in LTG mutants, and that LTG mutants are hyper-susceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that a key function of LTGs is to prevent toxic crowding of the periplasm with synthesis-derived PG fragments. Contrary to prevailing models, our data further suggest that this process can be temporally separate from peptidoglycan synthesis.

show abstract

MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli

Cited by 25 publications

References 32 publications

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

EloR Interacts with the Lytic Transglycosylase MltG at Midcell in Streptococcus pneumoniae R6

Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

Contact Info

Product

Resources

About