Peptidoglycan

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.It is unclear how Gram-positive bacteria, with a thick cell wall, can release membrane vesicles. Here, Toyofuku et al. show that a prophage-encoded endolysin can generate holes in the cell wall through which cytoplasmic membrane material protrudes and is released as vesicles.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.

The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane (OM) and peptidoglycan (PG) layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking OM and PG homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in PG and incorporate d-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in Acinetobacter baumannii and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates PG precursor genes (e.g., dadA and alr), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a ∆ldtJ ∆mla double mutant, implicating the OM lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the ∆ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between PG remodeling and OM lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through PG modification but also by influencing broader regulatory and metabolic networks.IMPORTANCEAcinetobacter baumannii is a leading cause of hospital-acquired infections and is highly resistant to antibiotics. Its survival relies on the integrity of the cell envelope, composed of the peptidoglycan (PG) layer and outer membrane (OM). While LD-transpeptidases are traditionally known for reinforcing PG structure through non-canonical crosslinking, our findings reveal that the LdtJ enzyme also plays a critical role in regulating cellular metabolism and stress responses. Deletion of ldtJ results in pronounced growth defects and abnormal cell morphology-phenotypes that are fully suppressed by disrupting the OM lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between PG remodeling and OM lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria.