Umeå University's logo

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.

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.

IMPORTANCE: Many 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.

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.

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.

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.

Agrobacterium tumefaciens shifts from a free-living soil bacterium to a plantinvading state upon encountering the plant root microenvironment. The acid-induced twocomponent 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.

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.

During growth and division, the bacterial cell wall is remodeled, resulting in the liberation of peptidoglycan (PG) fragments which are typically reinternalized and recycled. Recycling of PG has been studied in a few model species, but its importance and diversity are not yet well understood. Here, we review how bacteria transport and recycle the components of their PG, highlighting updates and new findings.

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.

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.