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During growth and propagation, a bacterial cell enlarges and subsequently divides its peptidoglycan (PG) sacculus, a continuous mesh-like layer that encases the cell membrane to confer mechanical strength and morphological robustness. The mechanism of sacculus growth, how it is regulated and how it is coordinated with other cellular processes is poorly understood. In this article, we will discuss briefly the current knowledge of how cell wall synthesis is regulated, on multiple levels, from both sides of the cytoplasmic membrane. According to the current knowledge, cytosolic scaffolding proteins connect PG synthases with cytoskeletal elements, and protein phosphorylation regulates cell wall growth in Gram-positive species. PG-active enzymes engage in multiple protein–protein interactions within PG synthesis multienzyme complexes, and some of the interactions modulate activities. PG synthesis is also regulated by central metabolism, and by PG maturation through the action of PG hydrolytic enzymes. Only now are we beginning to appreciate how these multiple levels of regulating PG synthesis enable the cell to propagate robustly with a defined cell shape under different and variable growth conditions.

We review how bacterial cell wall growth is regulated on multiple levels. We outline the assembly of synthesis complexes through protein scaffolding and how dynamic protein–protein interactions control cell wall growth. We also discuss other regulatory inputs including phosphorylation of key regulatory proteins, cell wall maturation and coordination of cell wall growth with central metabolism through sensing nutrient availability.

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Lysocin E (1) is a structurally complex 37-membered depsipeptide comprising 12 amino-acid residues with an N-methylated amide and an ester linkage. Compound 1 binds to menaquinone (MK) in the bacterial membrane to exert its potent bactericidal activity. To decipher the biologically important functionalities within this unique antibiotic, we performed a comprehensive structure-activity relationship (SAR) study by systematically changing the side-chain structures of l-Thr-1, d-Arg-2, N-Me-d-Phe-5, d-Arg-7, l-Glu-8, and d-Trp-10. First, we achieved total synthesis of the 14 new side-chain analogues of 1 by employing a solid-phase strategy. We then evaluated the MK-dependent liposomal disruption and antimicrobial activity against Staphylococcus aureus by 1 and its analogues. Correlating data between the liposome and bacteria experiments revealed that membrane lysis was mainly responsible for the antibacterial functions. Altering the cationic guanidine moiety of d-Arg-2/7 to a neutral amide, and the C7-acyl group of l-Thr-1 to the C2 or C11 counterpart decreased the antimicrobial activities four- or eight-fold. More drastically, chemical mutation of d-Trp-10 to d-Ala-10 totally abolished the bioactivities. These important findings led us to propose the biological roles of the side-chain functionalities.

What parts work best? Antibiotic lysocin E (1), a 37-membered depsipeptide, binds to menaquinone in the bacterial membrane to exert its potent bactericidal activity (see figure). Comprehensive SAR studies of 1 were performed by systematically changing the side-chain structures. The key structural features for the antimicrobial activities were established to be the cationic functionalities at d-Arg-2/7, the hydrophobic acyl group at l-Thr-1, and the indole ring at d-Trp-10.

Neisseria gonorrhoeae releases peptidoglycan (PG) fragments during infection that provoke a large inflammatory response and, in pelvic inflammatory disease, this response leads to the death and sloughing of ciliated cells of the Fallopian tube. We characterized the biochemical functions and localization of two enzymes responsible for the release of proinflammatory PG fragments. The putative lytic transglycosylases LtgA and LtgD were shown to create the 1,6-anhydromuramyl moieties, and both enzymes were able to digest a small, synthetic tetrasaccharide dipeptide PG fragment into the cognate 1,6-anhydromuramyl-containing reaction products. Degradation of tetrasaccharide PG fragments by LtgA is the first demonstration of a family 1 lytic transglycosylase exhibiting this activity. Pulse-chase experiments in gonococci demonstrated that LtgA produces a larger amount of PG fragments than LtgD, and a vast majority of these fragments are recycled. In contrast, LtgD was necessary for wild-type levels of PG precursor incorporation and produced fragments predominantly released from the cell. Additionally, super-resolution microscopy established that LtgA localizes to the septum, whereas LtgD is localized around the cell. This investigation suggests a model where LtgD produces PG monomers in such a way that these fragments are released, whereas LtgA creates fragments that are mostly taken into the cytoplasm for recycling.

Neisseria gonorrhoeae releases a significant portion of cell wall-derived peptidoglycan fragments, inducing inflammation and tissue damage in the genital tract. This work describes the functions of the two lytic transglycosylases responsible for producing the peptidoglycan fragments tracheal cytotoxin and GlcNAc-anhydro-MurNAc-tripeptide, a Nod1 agonist. Although these two enzymes can perform the same biochemical reaction, each lytic transglycosylase has different substrate specificity and cellular localization that leads to differing contributions to peptidoglycan remodeling, recycling, and release.