The transition from unicellular to multicellular growth requires diversification of cellular functions within genetically identical populations. In Bacillus subtilis, biofilm formation is historically viewed as a developmental precursor to sporulation along a linear pathway. Here, we show that biofilm formation and sporulation instead diverge along a branched pathway. A subpopulation that first initiates sporulation catabolizes lipoteichoic acid through the sequential action of the enzymes ShfP (Sporulation heterogeneity factor Poison) and PhoA (alkaline phosphatase A), leading to the release of millimolar concentrations of glycerol. This glycerol impedes sporulation by disrupting cell wall synthesis and cytoplasmic pH, necessitating counteraction by another protein, ShfA (Sporulation heterogeneity factor Antidote). The extracellular glycerol, however, acts as a morphogen that directs neighboring cells to initiate biofilm formation, which we directly visualize in developing populations of cells. Thus, B. subtilis multicellularity emerges through a branched developmental program in which sporulating cells generate the cue that creates the biofilm-producing lineage via cell-cell communication through repurposing of a canonical intracellular metabolite.

Methicillin-resistant Staphylococcus aureus resists β-lactam antibiotics through the allosteric transpeptidase penicillin-binding protein 2a, which operates within staphyloxanthin-rich membrane microdomains. Statins restore susceptibility by disrupting these microdomains and impairing penicillin-binding protein 2a oligomerization, but the mechanisms enabling resistance to this resensitization remain unclear. Here we show, using evolution experiments in strains lacking a functional staphyloxanthin pathway, that mutations in gdpP, a regulator of cyclic di-adenosine monophosphate signaling, are the predominant route for restoring oxacillin resistance during membrane microdomain disruption. This adaptation is blocked by simvastatin, revealing a synthetic lethal interaction. Mechanistically, simvastatin inhibits the mevalonate pathway, depleting the essential lipid carrier undecaprenyl phosphate and exacerbating peptidoglycan precursor imbalance, an effect phenocopied by lipid carrier-targeting antibiotics such as bacitracin. Although compensatory mutations can restore resistance, they impose a fitness cost in vivo. Importantly, this vulnerability extends to Streptococcus pneumoniae, revealing a conserved strategy to overcome β-lactam resistance in Gram-positive pathogens.

SUMMARYPeptidoglycan (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.

Mycobacterium tuberculosis withstands acidic conditions to survive and replicate within macrophages. To define the genetic determinants of this adaptation, we performed a transposon screen in a lipid-rich, acidic medium that mimics the host environment and supports robust M. tuberculosis growth. This screen identified ldtB, encoding an L,D-transpeptidase, as essential for growth and survival under acid stress. Loss of LdtB decreased 3-3 peptidoglycan cross-linking, disrupted cell wall architecture, and impaired intrabacterial pH homeostasis, resulting in increased susceptibility to cell wall-active antibiotics. Notably, M. tuberculosis lacking LdtB displayed heightened sensitivity to meropenem within macrophages, suggesting that targeting this enzyme could potentiate β-lactam efficacy during infection. These findings establish LdtB as a key mediator linking peptidoglycan homeostasis to acid stress resistance and underscores the importance of in vitro culture models that recapitulate the host microenvironment for uncovering new in vivo active therapeutic targets.

Homologs of the polar landmark proteins HubP and FimV are widespread among bacterial species. They all share several common features, including a periplasmic LysM-like domain, a transmembrane region, an extensive cytoplasmic domain enriched in acidic amino acids, and a C-terminal tetrapeptid-repeat (TPR) domain referred to as the FimV domain. Apart from these conserved general features, however, the proteins exhibit little homology across different bacterial genera. Here, we characterized Pseudomonas putida FimV (PpFimV) with respect to cellular processes involving FimV or HubP in other species. We found that PpFimV nonspecifically binds to peptidoglycan via its periplasmic LysM domain, which, together with an immunoglobulin-like domain, is necessary for proper polar positioning. PpFimV is required for normal flagellar-mediated swimming and the placement of the chemotaxis system. However, PpFimV is not involved in regulating the number of flagellar filaments, chromosome segregation, or type IV pilus-dependent surface motility. Thus, PpFimV has surprisingly little functional overlap with, for example, HubP from Vibrio sp. or Shewanella putrefaciens or with FimV from P. aeruginosa. PpFimV was unable to compensate for the loss of SpHubP with regard to swimming in soft agar, and vice versa. Domain swapping between SpHubP and PpFimV revealed that differences in the cytoplasmic region between the transmembrane region and the C-terminal FimV domain likely account for the proteins' distinct functions in flagella-mediated swimming. This suggests that FimV and HubP are structural homologs that have evolved to perform different, species-specific functions.IMPORTANCEMany bacterial species possess landmark proteins that organize the bacterial cell and localize specific cellular processes to the cell's polar regions by directing client proteins or protein complexes to their designated positions. FimV and its homolog HubP are landmark proteins found in many species of the gammaproteobacteria, but their roles are not well understood. Here, we demonstrate that only certain functions related to flagella-mediated motility appear to be conserved between Pseudomonas putida FimV and Shewanella putrefaciens HubP. This finding suggests a significant degree of functional diversity.

Bacterial cell growth involves expansion of the peptidoglycan cell wall. Three mutually exclusive peptidoglycan synthesis mechanisms exist in bacteria: MreB-dependent dispersed growth in many rod-shaped bacteria, polar growth involving specific proteins in actinobacteria and rhizobiales, and septal growth involving FtsZ in many cocci. Here we used imaging, mass spectrometry analysis of peptidoglycan composition, bacterial genetics and colocalization analyses to show that the actinobacterium Streptomyces venezuelae uses both canonical polar peptidoglycan synthesis and MreB-dependent dispersed peptidoglycan synthesis during rapid, exploratory growth. Transmission electron microscopy and peptidoglycan analyses showed changes in cell wall structure and composition with exploratory growth. MreB1 was essential for cell wall integrity and culture viability during exploratory growth and also localized to side walls in regions of new growth. Our results show that MreB1 is required for dynamic cell wall changes over the course of a growth cycle, contributing to a wall that is structurally distinct from that of conventionally growing streptomycetes.

The Gram-negative bacterium Pseudomonas putida bears a tuft of flagella at a single cell pole. New flagella must be assembled de novo every cell cycle to secure motility of both daughter cells. Here we show that the coordinated action of FimV, FlhF and FleN sets the location, timing and number of flagella assembled. The polar landmark proteins FimV and FlhF are independently targeted to the nascent new pole during or shortly after cell division, but FimV stabilizes FlhF association with the cell poles. FlhF determines the polar position of the flagella by targeting early flagellar components to the cell pole and preventing their nucleation at non-polar sites. FlhF also promotes efficient flagellar assembly and indirectly stimulates Class III flagellar promoter activation by promoting secretion of the anti-FliA anti-σ factor FlgM. The MinD-like ATPase FleN partitions between the cell poles and the cytoplasm. Cytoplasmic FleN regulates flagellar number by preventing excessive accumulation of FlhF at the cell poles that may otherwise lead to hyperflagellation, likely by antagonizing FleQ-dependent transcriptional activation. FimV is essential to FleN polar location. FimV and FleN temporally regulate the onset of flagellar assembly by preventing premature polar targeting of FlhF and the ensuing premature targeting of additional flagellar components. Our results shed new light on the mechanisms that ensure the timely assembly of the appropriate number of flagella at the correct polar location in polarly flagellated bacteria.

Resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus is mediated by the mecA-encoded, β-lactam-resistant transpeptidase, penicillin-binding protein 2a (PBP2a), which is capable of crosslinking peptidoglycan in the presence of β-lactam antibiotics. Here, we report that mutation of the lipoprotein signal peptidase II gene, lspA, from the lipoprotein processing pathway, significantly increased β-lactam resistance in MRSA, independent of changes in PBP2a levels or peptidoglycan composition. Exposure of MRSA to the LspA inhibitor globomycin also increased β-lactam resistance. Mutation of lgt, which encodes diacylglycerol transferase (Lgt) responsible for synthesis of the LspA substrate, did not impact β-lactam susceptibility. Furthermore, mutation of lgt in an lspA background restored β-lactam resistance to wild-type levels. These data suggest that accumulation of the LspA substrate, diacylglyceryl-lipoprotein, is associated with increased β-lactam resistance in MRSA.

Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1,000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

Peptidoglycan (PG)-modifying enzymes play a crucial role in cell wall remodeling, essential for growth and division. Cell wall degradation products are transported to the cytoplasm and recycled back in most gram-negative bacteria, and PG recycling is also linked to β-lactam resistance in many bacteria. Caulobacter crescentus is intrinsically resistant to β-lactams. Recently, it was shown that a soluble lytic transglycosylase, SdpA, is essential for β-lactam resistance. However, the precise role of SdpA in β-lactam resistance is unknown. This study investigated the PG recycling pathway and its role in antibiotic resistance in C. crescentus. Anhydromuropeptides generated by the action of lytic transglycosylases (LTs) are transported to the cytoplasm by the permease AmpG. C. crescentus encodes an ampG homolog, and deletion mutants of sdpA and ampG are sensitive to β-lactams. The ampG deletion mutant displays a significant accumulation of anhydromuropeptides in the periplasm of C. crescentus, demonstrating its essential role in PG recycling. While single knockout mutants of sdpA and ampG exhibit no growth defects, double-deletion mutants (∆sdpA∆ampG) exhibit severe growth and morphological defects. These double mutants also show enhanced sensitivity to β-lactams. Analysis of soluble muropeptides in wild-type (WT), ∆sdpA, and ∆ampG mutants revealed reduced levels of PG precursors (UDP-GlcNAc, UDP-MurNAc, and UDP-MurNAc-P5), suggesting that PG recycling products contribute toward de novo PG biosynthesis. Furthermore, supplementing the growth media with GlcNAc sugar enhanced the fitness of ∆sdpA and ∆ampG mutants under β-lactam stress. In conclusion, our study indicates that defects in PG recycling compromise cell wall biogenesis, leading to antibiotic sensitivity in C. crescentus.IMPORTANCEβ-lactam antibiotics target the peptidoglycan cell wall biosynthetic pathway in bacteria. In response to antibiotic pressures, bacteria have developed various resistance mechanisms. In many gram-negative species, cell wall degradation products are transported into the cytoplasm and induce the expression of β-lactamase enzymes. In this study, we investigated the cell wall recycling pathway and its role in antibiotic resistance in Caulobacter crescentus. Based on our data and prior studies, we propose that cell wall degradation products are utilized for the synthesis of peptidoglycan precursors in the cytoplasm. A deficiency in cell wall recycling leads to cell wall defects and increased antibiotic sensitivity in C. crescentus. These findings are crucial for understanding antibiotic resistance mechanisms in bacteria.

Bifidobacteria represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest in bifidobacteria as a live biotic therapy, our understanding of colonization, host-microbe interactions, and the health-promoting effects of bifidobacteria is limited. To address these major knowledge gaps, we used a large-scale genetic approach to create a mutant fitness compendium in Bifidobacterium breve. First, we generated a high-density randomly barcoded transposon insertion pool and used it to determine fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. Second, to enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1,462 genes. We leveraged these tools to reveal community- and diet-specific requirements for colonization and to connect the production of immunomodulatory molecules to growth benefits. These resources will catalyze future investigations of this important beneficial microbe.

During endospore formation, the mother cell and developing spore establish cell-cell signalling pathways that lead to compartment-specific transcription and key steps in morphogenesis. Endospore-forming bacteria also assemble a highly conserved essential membrane complex, called the A-Q complex, that physically connects these cells and may serve as a molecular conduit between them. While SpoIIIL was previously identified as a putative A-Q complex component in Bacillus subtilis, its exact role remains unclear. Here, we found that SpoIIIL does not function in the A-Q complex but instead acts as a forespore-specific factor required for efficient cell-cell signalling that leads to late mother cell transcription. Quantitative image analysis revealed that spoIIIL mutant spores do not exhibit hallmark phenotypes of A-Q complex mutants. Furthermore, unlike well-characterized A-Q complex proteins, SpoIIIL-GFP localizes uniformly in the forespore membrane before dispersing into the forespore cytoplasm. A synthetic sporulation screen identified a genetic relationship between spoIIIL and murAB, a paralog of murAA, required for efficient peptidoglycan precursor synthesis during sporulation. Cytological analysis indicates that the spoIIIL murAB double mutant is severely defective in the assembly of spore cortex peptidoglycan. Investigations into how SpoIIIL affects the cortex suggest it contributes to the activity of SpoIVB, a secreted forespore protease that initiates the signalling pathway required for processing of inactive pro-σK to active σK in the mother cell, which in turn up-regulates peptidoglycan precursor synthesis required for cortex formation. Accordingly, the spoIIIL mutant exhibits delayed and reduced pro-σK processing and decreased accumulation of peptidoglycan precursors. Thus, cortex assembly defects in the spoIIIL murAB double mutant results from alterations in separate pathways contributing to peptidoglycan precursor synthesis. Finally, phylogenetic analyses reveal that SpoIIIL is restricted to a subset of Bacillales species, highlighting evolutionary specialization in the signalling pathway leading to σK activation. Collectively, our findings redefine SpoIIIL as a forespore factor required for efficient cell-cell signalling that controls late mother-cell transcription.

Many bacteria form spores to endure unfavorable conditions. While Firmicutes generate endospores through cell division, sporulation in non-Firmicutes remains less understood. The Gram-negative bacterium Myxococcus xanthus undergoes sporulation through two distinct mechanisms: rapid sporulation triggered by chemical induction and slow sporulation driven by starvation, both occurring independently of cell division. Instead, these processes depend on the complete degradation of the peptidoglycan (PG) cell wall by two lytic transglycosylases (LTGs), LtgA and LtgB. Remarkably, LtgB programs the pace of PG degradation by LtgA during rapid sporulation, ensuring a controlled process that prevents abrupt PG breakdown and the formation of non-resistant pseudospores. In addition to regulation between LTGs, PG degradation is also influenced by its synthesis; cells exhibiting increased muropeptide production often circumvent sporulation. These findings not only reveal novel mechanisms of bacterial sporulation but also shed light on the regulatory network governing PG dynamics.

Murein lipoprotein (Lpp), also known as Braun's lipoprotein, stabilizes the cell wall of Escherichia coli by covalently tethering the outer membrane to the peptidoglycan (PG). Unlike E. coli, Salmonella enterica serovar Typhimurium encodes two murein lipoproteins, LppA and LppB, with LppB bearing an unusual C-terminal sequence, -RICKCOOH. Here, we investigated how LppA and LppB bind to the PG. Both lipoproteins were detected in pure PG material in a ∼ 400:1 LppA:LppB ratio with some LppB molecules forming a cysteine 78 (C78)-C78 intermolecular disulphide bridge. LppA and LppB anchor covalently to uncross-linked and cross-linked muropeptides. However, unlike LppA, which binds to 4,3- and 3,3-cross-linked muropeptides, LppB shows preferred binding to 4,3-cross-linked muropeptides. Mass spectrometry data revealed O-methylation at the terminal K79 residue in some PG-bound LppB molecules. The apparent selective anchoring of LppB to the PG and the K79 modification require the presence of the C78 residue. Anchoring of LppB to PG is mediated by the L,D-transpeptidase LdtB. A survey in more than 158,000 Salmonella genomes identified up to 31 murein Lpp variants differing in the C-terminal region that cluster in three phylogenetic groups. Most serovars of S. enterica subspecies enterica, responsible for infections in warm blooded animals, encode two or even three murein Lpp variants. Altogether, our data are consistent with subtle differences in the mode that LppB anchors to the PG and uncover an unprecedented diversity of murein lipoproteins within the Salmonella genus. The possibility that this variability evolved as strategy to evade host innate immunity, is also discussed.

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis ( Mtb ) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR ( virR mut ) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb . We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virR mut . Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

Agrobacterium tumefaciens shifts from a free-living soil bacterium to a plant-invading state upon encountering the plant root microenvironment. The acid-induced two-component sensor system ChvG-ChvI drives this shift and triggers a complex transcriptional program that promotes host invasion and survival against host immune defenses. Remarkably, ChvG-ChvI is also activated under cell wall stress conditions, suggesting that the transcriptional response may have a broader function. Here, we find that blocking cell wall synthesis either genetically or chemically leads to ChvG-ChvI activation. Mutations in key cell wall synthesis enzymes, such as penicillin-binding protein 1a and FtsW, suppress ChvG-ChvI activation in cell wall stress inducing conditions, suggesting that providing structural integrity is a primary function of the ChvG-ChvI regulon. Here, we investigated regulon components for this function. First, deletion of exoA, a gene required for production of the exopolysaccharide succinoglycan, confers resistance to multiple β-lactam antibiotics targeting different enzymes. Next, a class D β-lactamase is expressed that may contribute to the high level of β-lactam resistance in A. tumefaciens. Finally, outer membrane proteins are upregulated, suggesting that outer membrane remodeling may compensate for the accumulation of cell wall damage by providing structural integrity. Overall, we expand our understanding of mechanisms driving ChvG-ChvI activation and β-lactam resistance in a bacterial plant pathogen.

Beta-lactam antibiotics are widely used to treat bacterial infections, but their efficacy is compromised by resistance mechanisms such as the production of beta-lactamases. In Pseudomonas aeruginosa, the chromosomally encoded beta-lactamase AmpC is the primary mediator of beta-lactam resistance. ampC expression is regulated by the transcription factor AmpR, which responds to intracellular peptidoglycan (PG) fragments. Under normal conditions, AmpR binds the PG precursor (UDP-MurNAc-pentapeptide) and represses ampC expression. However, during beta-lactam treatment or in PG recycling-deficient mutants such as ampD mutants, PG degradation products (anhydromuropeptides) accumulate and activate AmpR, resulting in elevated ampC expression and beta-lactam resistance. We hypothesized that shifting the balance of PG precursors could modulate AmpR activity and suppress beta-lactamase expression, even in derepressed strains. Undecaprenyl phosphate (UndP) is a lipid carrier essential for translocating PG precursors across the bacterial inner membrane. Recent work has identified members of the DedA superfamily as UndP flippases responsible for recycling this lipid carrier. Disruption of UndP recycling leads to cytoplasmic accumulation of UDP-MurNAc-pentapeptide, the known AmpR repressor. Here, we show that deletion of dedA4, which encodes a predicted UndP flippase in P. aeruginosa, causes PG precursors accumulation and significantly reduces AmpC production and beta-lactam resistance in an ampD mutant. These findings highlight the influence of PG precursor dynamics on beta-lactamase regulation and identify DedA4 as a promising therapeutic target. Inhibiting UndP recycling offers a novel strategy to counteract beta-lactam resistance in P. aeruginosa and potentially other AmpC-producing pathogens.

For any organism, survival is enhanced by the ability to sense and respond to threats in advance. For bacteria, danger sensing among kin cells has been observed, but the presence or impacts of general danger signals are poorly understood. Here we show that different bacterial species use exogenous peptidoglycan fragments, which are released by nearby kin or non-kin cell lysis, as a general danger signal. Using microscopy and gene expression profiling of Vibrio cholerae, we find that even brief signal exposure results in a regulatory response that causes three-dimensional biofilm formation, which protects cells from a broad range of stresses, including bacteriophage predation. A diverse set of species (Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis) also respond to exogenous peptidoglycan by forming biofilms. As peptidoglycan from different Gram-negative and Gram-positive species triggered three-dimensional biofilm formation, we propose that this danger signal and danger response are conserved among bacteria. Peptidoglycan released by neighbouring kin or non-kin cell lysis induces physiological changes that protect from a range of stresses, including phage predation.

The bacterial cell wall, a sophisticated and dynamic structure predominantly composed of peptidoglycan (PG), plays a pivotal role in bacterial survival and adaptation. Bacteria actively modify their cell walls by editing PG components in response to environmental challenges. Diverse variations in peptide composition, cross-linking patterns, and glycan strand structures empower bacteria to resist antibiotics, evade host immune detection, and adapt to dynamic environments. This review comprehensively summarizes the most common modifications reported to date and their associated adaptive role and further highlights how regulation of PG synthesis and turnover provides resilience to cell lysis.

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.