Laura Alvarez

SUMMARY: Peptidoglycan (PG) is a dynamic, load-bearing polymer whose crosslinking chemistry governs envelope mechanics, growth modes, and stress tolerance. For decades, PG crosslinking was viewed primarily through the lens of penicillin-binding proteins (PBPs). However, accumulating evidence over the past 2 decades has established LD-transpeptidases (LDTs) as important contributors to PG remodeling. Here, we organize the expanding LDT field into macro-domains bridging biochemistry, evolution, and ecology. We initially describe the reaction mechanisms, structural diversification, and convergent solutions and then explore the evolution across species. We highlight non-canonical LD-crosslinking chemistries, including L-Ala– meso -DAP (1–3) linkages, that broaden the design space of the sacculus. We then map functional repertoires across lineages—from reinforcement during envelope stress to outer membrane tethering, predation, specialized secretion, dormancy, and biofilms. In pathogens where LD-crosslinking is dominant or essential, carbapenems and penems remain particularly effective inhibitors of LDTs, yet family-wide diversity calls for structure-guided selectivity and ecological awareness. We also chart underexplored connections to microbiome ecology and propose LDT-derived biomarkers that report growth modes and dormancy. We integrate dispersed evidence into a complete landscape in which two-component systems and environmental cues coordinate LD pathways. Building on these threads, we propose a unifying model of LDTs as adaptive architects of PG whose acyl-enzyme intermediate and modular substrate gating endow reversibility, partner choice, and context-dependent outcomes—reinforcement, remodeling, anchoring, or controlled self-breach. Finally, we outline methods that enabled the discovery of LDTs and explore future directions. Together, these perspectives reframe LDTs from auxiliary enzymes to central designers of envelope architecture and bacterial fitness.

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species also employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

To withstand their internal turgor pressure and external threats, most bacteria have a protective peptidoglycan (PG) cell wall. The growth of this PG polymer relies on autolysins, enzymes that create space within the structure. Despite extensive research, the regulatory mechanisms governing these PG-degrading enzymes remain poorly understood. Here, we unveil a novel and widespread control mechanism of lytic transglycosylases (LTs), a type of autolysin responsible for breaking down PG glycan chains. Specifically, we show that LD-crosslinks within the PG sacculus act as an inhibitor of LT activity. Moreover, we demonstrate that this regulation controls the release of immunogenic PG fragments and provides resistance against predatory LTs of both bacterial and viral origin. Our findings address a critical gap in understanding the physiological role of the LD-crosslinking mode in PG homeostasis, highlighting how bacteria can enhance their resilience against environmental threats, including phage attacks, through a single structural PG modification.

The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDT_Go, that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans. LDT_Go-like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDT_Go can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDT_Go reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.

Most bacteria are surrounded by an essential protective mesh-like structure called peptidoglycan, made of glycan chains crosslinked through short peptides by enzymes known as transpeptidases. Of these, penicillin-binding DD-transpeptidases connect adjacent peptide stems between their 4 ^th and 3 ^rd amino acids (4,3-type), D-alanine and a meso-diaminopimelic acid (mDAP) in Gram negatives, whereas LD-transpeptidases make the 3,3-type between mDAP ³ residues. While these two processes explain the formation of crosslinks in most bacteria, recent investigations involving non-model species have brought to light novel crosslinking mechanisms that point to the existence of less-explored groups of peptidoglycan crosslinking enzymes. Here, we present the identification and characterization of a novel LD-transpeptidase found in the acetic acid bacterial Gluconobacter oxydans , named LDT _Go , which performs 1,3-type crosslinks between L-Ala ¹ and mDAP ³ . LDT _Go -like proteins are conserved among Alpha and Betaproteobacteria species that do not encode LD3,3-transpeptidases. Using a highly active ortholog, we demonstrated in vitro that this enzyme can work with non-terminal peptide bonds in the crosslinking process. This property is different from the strict specificity of typical LD- and DD-transpeptidases, which only deal with terminal peptide bonds. The high-resolution crystal structure of LDT _Go revealed significant distinctions when compared to 3,3-type LD-transpeptidases. These include a proline-rich region near the N-terminus that restricts substrate access to the active site, and an unprecedented cavity designed to accommodate both the glycan chain and the peptide stem from donor muropeptides, a feature that exhibits broad conservation among LD1,3-transpeptidases. Finally, we demonstrated the involvement of DD-crosslinking turnover in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between structurally distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans .

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCP_DRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCP_DRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

Most bacteria are encased into a load-bearing rigid framework, the cell wall (CW). The peptidoglycan (PG) layer, a network composed of glycan strands cross-linked by stem peptides, is the main component of the CW. During PG synthesis, precursors are first synthetized intracellularly, before being incorporated into the existing PG meshwork by transglycosylation (TG) and transpeptidation (TP) reactions. Covalent modifications of the PG meshwork such as amidation and acetylation participate in PG homeostasis by regulating PG-associated enzymes like PG hydrolases. Because of its essential role, PG synthesis represents a primary target for antibiotic action. Here, we investigated the effect on PG composition of antibiotics targeting intracellular and extracellular steps of PG synthesis: inhibitors of PG precursors synthesis (fosfomycin, D-cycloserine, bacitracin and tunicamycin) and TG/TP inhibitors (vancomycin and penicillin G), respectively. Our study revealed interesting correlations between crosslinking and both de-N-acetylation and amidation of the sacculus. A thorough analysis of muropeptides composition put into light an unexpected anti-correlation between the degree of PG crosslinking and accumulation of de-amidated disaccharide-tripeptide monomer subunit (M3) in the presence of TP inhibitors. We confirmed these observations by analyzing mutants of the PG synthesis pathway.

SUMMARY: Most bacteria cells are protected by a peptidoglycan cell wall. Defining the chemical structure of the peptidoglycan has been instrumental to characterize cell wall associated proteins and to illuminate the mode of action of cell wall-acting antibiotics. However, a major roadblock for a comprehensive understanding of peptidoglycan homeostasis has been the lack of methods to conduct large-scale, systematic studies. Here we have developed and applied an innovative high throughput peptidoglycan analytical pipeline to analyze the entire non-essential, arrayed mutant library of Vibrio cholerae . The unprecedented breadth of these analyses revealed that peptidoglycan homeostasis is preserved by a large percentage of the genome organized in complex networks that functionally link peptidoglycan features with genetic determinants. As an example, we discovered a novel bifunctional penicillin-binding protein in V. cholerae . Collectively, genome-wide peptidoglycan profiling provides a fast, easy, and unbiased method for systematic identification of the genetic determinants of peptidoglycan synthesis and remodeling.

Oxidative stress is caused by the production and excessive accumulation of oxygen reactive species. In bacterial cells, oxidative stress mediated by hydroxyl radicals is typically associated with DNA damage in the cytoplasm.

The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover – or autolysis – of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditional LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and, contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.