Cooperativity of peptidoglycan synthases active in bacterial cell elongation

Bacteria are surrounded by a peptidoglycan (PG) cell wall that must be remodeled to allow cell growth. While many structural details and properties of PG and the individual enzymes involved are known, how the process is coordinated to maintain cell integrity and rod shape is not understood. We have developed a coarse-grained method to simulate how individual transglycosylases, transpeptidases, and endopeptidases could introduce new material into an existing unilayer PG network. We find that a simple model with no enzyme coordination fails to maintain cell wall integrity and rod shape. We then iteratively analyze failure modes and explore different mechanistic hypotheses about how each problem might be overcome by the macromolecules involved. In contrast to a current theory, which posits that long MreB filaments are needed to coordinate PG insertion sites, we find that local coordination of enzyme activities in individual complexes can be sufficient to maintain cell integrity and rod shape. We also present possible molecular explanations for the existence of monofunctional transpeptidases and glycosidases (glycoside hydrolases), trimeric peptide crosslinks, cell twisting during growth, and synthesis of new strands in pairs.coarse-grained modeling | cell wall synthesis | morphogenesis T he cytoplasmic membrane of Gram-negative rod-shaped bacteria is surrounded by a peptidoglycan (PG) sacculus that protects the cell from internal turgor pressure, and its architecture determines the cell's shape (1, 2). The sacculus is composed of long glycan strands crosslinked by peptides into a mesh-like network. The glycan repeating unit is a disaccharide of an N-acetylglucosamine and an N-acetylmuramic acid attached to a stem pentapeptide L-Ala-D-iGlu-m-A 2 pm-D-Ala-D-Ala. Crosslinks are formed between peptides on adjacent strands-most at the fourth (D-Ala) residues of the donors and the third (m-A 2 pm) residues of the acceptors. While it is now understood that, in Gram-negative cells, the glycan strands run parallel to the cell surface, how the strands are arranged in this plane is still debated. While recent atomic force microscopy images of purified sacculi were interpreted to indicate that glycan strands had random orientations (3), electron cryotomography of sacculi (4) and other evidence from both Gramnegative (1, 5) and -positive (6, 7) sacculi have, instead, pointed to a universal "circumferential" model, in which the stiff glycan strands are circumferentially around the rod and the flexible peptide crosslinks parallel to the rod's long axis.Cell growth requires that the sacculus be elongated without losing its integrity or characteristic shape. This remodeling process requires the presence of not only transglycosylases and transpeptidases, also known as penicillin-binding proteins (PBPs), to polymerize and crosslink new glycan strands into the existing network (2, 8) but also, endopeptidases to cleave covalent bonds to open space for the new material (9). How these small enzymes work together to maintain the order and...

Section: Discussionsupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape

Nguyen

Gumbart

Beeby

et al. 2015

“…2B). In addition, disrupting the activity of PBP1a, which binds PBP2 in vitro (19), either through inhibition with cefsulodin or deletion, had no effect on diffusivity (Fig. S7).…”

Section: Resultsmentioning

confidence: 99%

“…This line of reasoning is supported by the observation that growth rate is unaffected by A22 treatment (25), despite disruption of MreB spatial organization. In vitro interactions between E. coli PBP2 and PBP1a (19) and between Helicobacter pylori PBP2 and MreC (20) have been identified, and in the latter case these proteins appear to form a complex in vivo. Although perturbation of PBP1a does not affect PBP2 mobility (Fig.…”

Section: Discussionmentioning

confidence: 99%

A dynamically assembled cell wall synthesis machinery buffers cell growth

Lee

Tropini

Hsin

et al. 2014

Assembly of protein complexes is a key mechanism for achieving spatial and temporal coordination in processes involving many enzymes. Growth of rod-shaped bacteria is a well-studied example requiring such coordination; expansion of the cell wall is thought to involve coordination of the activity of synthetic enzymes with the cytoskeleton via a stable complex. Here, we use singlemolecule tracking to demonstrate that the bacterial actin homolog MreB and the essential cell wall enzyme PBP2 move on timescales orders of magnitude apart, with drastically different characteristic motions. Our observations suggest that PBP2 interacts with the rest of the synthesis machinery through a dynamic cycle of transient association. Consistent with this model, growth is robust to large fluctuations in PBP2 abundance. In contrast to stable complex formation, dynamic association of PBP2 is less dependent on the function of other components of the synthesis machinery, and buffers spatially distributed growth against fluctuations in pathway component concentrations and the presence of defective components. Dynamic association could generally represent an efficient strategy for spatiotemporal coordination of protein activities, especially when excess concentrations of system components are inhibitory to the overall process or deleterious to the cell.

“…Septal cell wall synthesis is directed by the divisome, and lateral cell wall synthesis is directed by the 'elongasome' (91). These two systems may compete with each other, because they share the same precursors (92,93) and/or coordinate with each other during both cell wall elongation and constriction (94)(95)(96)(97)(98).…”

Section: Constriction Initiation and Progress Does Not Require Z-ringmentioning

confidence: 99%

Defining the rate-limiting processes of bacterial cytokinesis

Coltharp

Buss

Plumer

et al. 2016

152

212

Bacterial cytokinesis is accomplished by the essential ‘divisome’ machinery. The most widely conserved divisome component, FtsZ, is a tubulin homolog that polymerizes into the ‘FtsZ-ring’ (‘Z-ring’). Previous in vitro studies suggest that Z-ring contraction serves as a major constrictive force generator to limit the progression of cytokinesis. Here, we applied quantitative superresolution imaging to examine whether and how Z-ring contraction limits the rate of septum closure during cytokinesis in Escherichia coli cells. Surprisingly, septum closure rate was robust to substantial changes in all Z-ring properties proposed to be coupled to force generation: FtsZ’s GTPase activity, Z-ring density, and the timing of Z-ring assembly and disassembly. Instead, the rate was limited by the activity of an essential cell wall synthesis enzyme and further modulated by a physical divisome–chromosome coupling. These results challenge a Z-ring–centric view of bacterial cytokinesis and identify cell wall synthesis and chromosome segregation as limiting processes of cytokinesis.