Although the outer membrane contributes to the structural integrity of Gram-negative bacteria, the primary determinant of mechanical and osmotic resilience is the cell wall. This structure consists of peptidoglycan, a mesh-like polymer located in the periplasm of Gram-negative bacteria and on the surface of Gram-positive bacteria. While Gram-negatives typically possess only a single glycan layer, in Gram-positives the layer can reach up to ~100 nm in thickness. Peptidoglycan forms a continuous sacculus that encases the cytoplasmic membrane. Its glycan strands are oriented circumferentially around the cell, allowing the sacculus to act like a flexible bag that resists internal turgor pressure. This orientation, together with the rigid covalent crosslinks, prevents snapping or rupture while still allowing limited deformation. Functionally, the peptidoglycan sacculus plays a central role in maintaining cell shape, counteracting high osmotic pressures, and resisting mechanical stress. Its strength is such that catastrophic failure usually requires substantial, localized disruption. For instance, Huang et al. (2008) showed that during vancomycin treatment peptidoglycan accumulated numerous cracks without immediate lysis, with cells remaining viable until sufficient damage concentrated in one spot to form a cytoplasmic bulge. In the absence of peptidoglycan altogether, cells form circular spheroids, underlining its role in shape determination. Despite its rigidity, peptidoglycan also provides a degree of flexibility through its crosslinks, enabling cells to tolerate fluctuations in osmotic states without bursting. Importantly, the enzymes responsible for its synthesis and remodeling are targeted by β-lactam antibiotics such as penicillins, cephalosporins, and carbapenems (see [[Beta lactamases]] for more). ## **Molecular Structure** Peptidoglycan is composed of repeating disaccharide units of _N_-acetylglucosamine (_NAG_) and _N_-acetylmuramic acid (_NAM_), crosslinked by short pentapeptide side chains. - In most Gram-negative bacteria, including _E. coli_, the pentapeptide sequence is _L_-Ala–γ-_D_-Glu–meso-diaminopimelic acid (mDAP)–_D_-Ala–_D_-Ala, with an **isopeptide bond** linking _D_-Glu at position 2 to mDAP at position 3. This molecular organization underpins the circumferential architecture of glycan strands, producing the characteristic sacculus that confers both rigidity and flexibility. ## **Tethering to the OM** It is important that the less mechanically sound outermembrane stays tethered to the cell wall in order to maintain its shape and prevent cell lysis or compromised integrity. Brauns [[Lipoproteins]] play a key role in this tethering by forming covalent bonds with the PtG (the only covalent attachment of the PtG!). They form a Trimer with their lipid moiety embedded in the inner leaflet of the OM and then bind covalently to mDAP residues on PtG. The trimer that is formed is a very stable structure, which is both slow to form and slower to break, it helps maintain a good distance between the OM and cell wall. Since brauns lipoproteins is a lipoprotein and is thus tethered to the cell wall by its lipid moiety it is found predominantly in the LPS rich regions of the OM which leads me onto my next point: [[OmpA]] a highly abundant outer membrane protein has a large globular periplasmic domain that has been shown to non-covalently attach itself to the PtG this would be important in maintaining comprehensive peripheral tethering in the proteinaceous regions of the cell (Firdaus Samsudin et al., 2016) The Tol-Pal complex ([[Tol system]]) is also involved in PtG tethering and is gnerally localised to the poles or midcell during division when TolB is not bound to Pal, this is particularly important during cell division where this complex aids in the final stages of cell division and evagination. ## **Biosynthesis** ##### Stage 1 The synthesis of peptidoglycan utilises the same precursor as used in Lipopolysaccharide synthesis UDP-GlcNAc. The first step of the process of converting this fundamental building block into the thin layer of PtG is the actions of **MurAB** these proteins act to convert UDP-GlcNAc to UDP-MurNAc. **MurA** is a cytoplasmic enzyme that transfers an enolpyruvate from phosphoenolpyruvate (PEP) to UDP-GlcNAc. **MurB** another cytoplasmic protein that uses NADPH as a reducing agent to convert the enolpyruvate moiety into a D-lactate (Raina et al., 2021) producing UDP-MurNAc The pentapeptide portion of Lipid II is added by **MurC, MurD, MurE and MurF**. Interestingly this ![[MurNAc biosynthesis.jpg]] ##### Stage 2 **MraY** is a membrane anchored protein with its actions taking place in the cytoplasm, it catalyses the conversion of UDP-MurNAc-X-X-X-X-X to PP-MurNAc-X-X-X-X-X (Xs representing different amino acids) which is also known as **Lipid I**. This is through cleaving much of the UDP moiety and swapping in a phosphate. **MurG** then forms the disaccharide between a new UDP-GlcNAc and the newly synthesised Lipid I forming Lipid-PP-MurNAc-GlcNAc which is more simply referred to as **lipid II**. **MurJ** flips Lipid II across the membrane and it is added to the growing glycan chain by Peptidoglycan Glycosyltransferases. This flipping can also be done by FtsW and RodA. In the final step DD-Transpeptidases ([[penecillin binding proteins]]) will crosslink the individual glycan chains ![[esp-0010-2020_fig_004.jpg]] **references** - [Asmar, A.T. and Collet, J.-F. (2018). Lpp, the Braun lipoprotein, turns 50 major achievements and remaining issues. _FEMS Microbiology Letters_, 365(18). doi:https://doi.org/10.1093/femsle/fny199.](doi:https://doi.org/10.1093/femsle/fny199) - [Garde, S., Chodisetti, P.K. and Reddy, M. (2021). Peptidoglycan: Structure, Synthesis, and Regulation. _EcoSal Plus_, 9(2). doi:https://doi.org/10.1128/ecosalplus.esp-0010-2020.](https://journals.asm.org/doi/10.1128/ecosalplus.esp-0010-2020) - [Huang, K.C., Mukhopadhyay, R., Wen, B., Gitai, Z. and Wingreen, N.S. (2008). Cell shape and cell-wall organization in Gram-negative bacteria. _Proceedings of the National Academy of Sciences_, [online] 105(49), pp.19282–19287. doi:https://doi.org/10.1073/pnas.0805309105.](https://pmc.ncbi.nlm.nih.gov/articles/PMC2592989/) - [Firdaus Samsudin, Ortiz-Suarez, M.L., Piggot, T.J., Bond, P.J. and Khalid, S. (2016). OmpA: A Flexible Clamp for Bacterial Cell Wall Attachment. 24(12), pp.2227–2235. doi:https://doi.org/10.1016/j.str.2016.10.009.](https://www.sciencedirect.com/science/article/pii/S0969212616303173#:~:text=Summary,efforts%20to%20develop%20antibacterial%20agents.) - [Raina, D., Kumar, C., Kumar, V., Khan, I.A. and Saran, S. (2021). Potential Inhibitors Targeting Escherichia coli UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): An Overview. _Indian Journal of Microbiology_, 62(1), pp.11–22. doi:https://doi.org/10.1007/s12088-021-00988-6.](https://pmc.ncbi.nlm.nih.gov/articles/PMC8758813/ - [Silhavy, T.J., Kahne, D. and Walker, S. (2010). The bacterial cell envelope. _Cold Spring Harbor Perspectives in Biology_, [online] 2(5). doi:https://doi.org/10.1101/cshperspect.a000414.](https://pmc.ncbi.nlm.nih.gov/articles/PMC2857177/)