Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli

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: 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

“…S1), one of the simplest models to explain the LpoB-bypass phenotype of the PBP1b* variants is that the proteins have gained the ability to synthesize PG in the absence of LpoB. To investigate this possibility, we monitored PG synthesis in etherpermeabilized cells (EPCs) by supplying them with UDP-MurNAcpep 5 and radiolabeled UDP-[ 14 C]GlcNAc (9). EPCs use the soluble precursors to produce lipid-II for PG synthesis by the PBPs.…”

Section: Resultsmentioning

confidence: 99%

“…Precursor synthesis begins in the cytoplasm with the production of the activated sugar molecules uridine diphosphate (UDP)-N-acetylmuramic acid pentapeptide (UDP-MurNAc-pep 5 ) and UDP-N-acetylglucosamine (UDPGlcNAc). In the second, membrane-associated phase, UDPMurNAc-pep 5 is converted to the precursor lipid-I by MraY, which transfers phospho-MurNAc-pep to the lipid carrier undecaprenol-phosphate (Und-P).…”

mentioning

confidence: 99%

Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

Markovski

Bohrhunter

Lupoli

et al. 2016

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.T he peptidoglycan (PG) layer forms a protective shell that surrounds the cytoplasmic membrane of bacteria to prevent osmotic rupture and provide cells with their characteristic shape (1). This complex macromolecule is composed of glycan strands crosslinked to one another by attached peptide chains to form the exoskeletal matrix. Because of its essentiality and uniqueness to bacteria, the PG layer is an important therapeutic target. Many antibiotics in our current arsenal block PG assembly, with penicillin and related beta-lactam drugs being the most wellknown and widely used. These molecules target major PG synthase enzymes called penicillin-binding proteins (PBPs) (1).The PBPs function in the final stage of the three-part pathway for PG biogenesis (1). Precursor synthesis begins in the cytoplasm with the production of the activated sugar molecules uridine diphosphate (UDP)-N-acetylmuramic acid pentapeptide (UDP-MurNAc-pep 5 ) and UDP-N-acetylglucosamine (UDPGlcNAc). In the second, membrane-associated phase, UDPMurNAc-pep 5 is converted to the precursor lipid-I by MraY, which transfers phospho-MurNAc-pep to the lipid carrier undecaprenol-phosphate (Und-P). Lipid-II is formed by MurG via the addition of GlcNAc to lipid-I from UDP-GlcNAc. This final precursor contains the basic monomeric unit of PG, the disaccharide-pentapeptide. After its production, lipid-II must be flipped by MurJ (2, 3) ...

“…Efforts to elucidate its function are under way. PBP1B interacts with the essential, IM-anchored cell division proteins PBP3 and FtsN (8,10). FtsN also stimulates the activity of PBP1B, presumably by stabilizing its dimeric form, which is more active in in vitro assays (22).…”

Section: Discussionmentioning

confidence: 99%

“…Soluble versions of LpoB with or without the oligohistidine-tag [His-LpoB(sol) and LpoB(sol)] and a His-tagged version of the UB2H domain of PBP1B (His-UB2H) were prepared as described in SI Appendix, Supplemental Materials and Methods. Antisera against PBP1B and LpoB (Eurogentec) were purified over an antigen column as described previously (8). VIM-4 β-lactamase was a gift from Adeline Derouaux (Centre for Bacterial Cell Biology, Newcastle University, Newcastle upon Tyne, United Kingdom).…”

Section: Methodsmentioning

confidence: 99%

Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B

Egan¹,

Jean

Koumoutsi

et al. 2014

Self Cite

134

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer. Growing and dividing cells expand their PG layer by using membrane-anchored PG synthases, which are guided by dynamic cytoskeletal elements. In Escherichia coli, growth of the mainly single-layered PG is also regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required for the activation of penicillin-binding protein (PBP) 1B, which is a major, bifunctional PG synthase with glycan chain polymerizing (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. Here, we report the structure of LpoB, determined by NMR spectroscopy, showing an N-terminal, 54-aalong flexible stretch followed by a globular domain with similarity to the N-terminal domain of the prevalent periplasmic protein TolB. We have identified the interaction interface between the globular domain of LpoB and the noncatalytic UvrB domain 2 homolog domain of PBP1B and modeled the complex. Amino acid exchanges within this interface weaken the PBP1B-LpoB interaction, decrease the PBP1B stimulation in vitro, and impair its function in vivo. On the contrary, the N-terminal flexible stretch of LpoB is required to stimulate PBP1B in vivo, but is dispensable in vitro. This supports a model in which LpoB spans the periplasm to interact with PBP1B and stimulate PG synthesis. P eptidoglycan (PG) is an essential component of the bacterial cell envelope, required for cell shape and stability. It is composed of glycan chains that are connected by short peptides, and forms a net-like, elastic structure, called the sacculus, which encases the cytoplasmic/inner membrane (IM) (1). In Gramnegative bacteria, such as Escherichia coli, the sacculus is mainly single-layered and is firmly attached to the outer membrane (OM) by abundant OM proteins. Some of the most effective antibiotic agents, such as the β-lactams and glycopeptides, inhibit PG biosynthesis, resulting in cell lysis.Bacteria enlarge their sacculus by polymerizing new PG from lipid II precursor at the outer face of the IM and incorporating the newly made material into the existing PG layer. At the same time, a significant amount of old material is released. For synthesis and hydrolysis to be coupled, the corresponding enzymes have to be tightly regulated and coordinate their actions (2). How does this happen? The current view is that PG synthases [penicillin-binding proteins (PBPs)] and hydrolases form membrane-anchored multienzyme complexes, which are driven by cytoskeletal elements. More recently, it was established that dedicated regulators tightly control the activities of PG synthases and hydrolases and/or couple it to other cell envelope processes (3-6).PG synthesis requires glycosyltransferases (GTases) to polymerize the glycan chains and transpeptidases (TPases) to form peptide cross-links. Most bacteria carry several PG synthases, which can perform one or both enzymatic reactions. In E. coli, the bifunctional GTase/TPases PBP1A and PBP1B provide the main PG...