Biology subjects

Vollmer, W.

Publications and source records attributed to Vollmer, W..

At least 19 recordsLinked to original sources

bioRxiv2026

PopZ is dispensable for cell fitness but required for polar localization of ParB in Zymomonas mobilis.

Chromosome segregation is an essential process in monoploid bacteria, in which faithful inheritance of the genetic material is required for viability of daughter cell. In several alpha-proteobacteria, the polar organizing protein PopZ plays a crucial role in chromosome segregation by anchoring the chromosome partitioning machinery at a cell pole. While this process has been studied in monoploid species, it remains unclear whether this type of segregation mechanisms play a significant role in bacterial cells that carry multiple copies of chromosome. The alpha-proteobacterium Zymomonas mobilis exhibits ethanologenic physiology, and thus, is a promising chassis for biofuel production. Recent studies showed that Z. mobilis may serve as a non-model system for studying bacterial cell biology, owing to its relatively simple physiology and reduced genome size. Z. mobilis has previously been shown to be polyploid, and possesses a homolog of PopZ, a key regulator of chromosome organisation and cell polarity in alpha-proteobacteria. This raises the question of whether PopZ is involved in the organization of multiple chromosome copies. Here, we investigated the function of PopZ in Z. mobilis. Unlike in previously studied alpha-proteobacterial species, PopZ is dispensable for growth and cell morphology in Z. mobilis. However, the loss of PopZ altered the polar accumulation of chromosome segregation protein ParB, indicating an involvement in chromosome organization in Z. mobilis. These findings indicate that Z. mobilis might possess a chromosome segregation machinery that is dispensable under favourable growth conditions but might become important under certain conditions that reduce chomorome copy numbers.

microbiology↗

bioRxiv2026

Hypothetical lytic transglycosylase SleB is important for cell fitness in Zymomonas mobilis

Bacterial peptidoglycan (PG) undergoes a variety of chemical modifications. O-acetylation at the C6 hydroxyl group of N-acetylmuramic acid is a widespread PG-modification found across diverse bacterial phyla. It contributes to virulence in pathogenic bacteria because the O-acetyl group reduces the activity of the PG-degrading host defense enzyme, lysozyme. Beyond its role in host defense evasion, recent studies suggest that PG O-acetylation also regulates the activity of endogenous lytic transglycosylase (LT) autolysins. The ethanologenic alpha-proteobacterium Zymomonas mobilis O-acetylates its PG, which is associated with tolerance to environmental stresses, including salt. To better understand how PG O-acetylation contributes to stress tolerance, we investigated the predicted lytic transglycosylase SleB. Intriguingly, the sleB gene is located adjacent to the pat operon, which encodes the proteins responsible for PG O-acetylation. We showed that loss of SleB caused impaired growth and morphology, and a significant reduction of crosslinks in the PG of Z. mobilis. Furthermore, the sleB mutant was sensitive to environmental stress resembling the sensitivity of the patA mutant. Collectively, our findings unravelled an important role of SleB in PG remodelling and stress resilience.

microbiology↗

bioRxiv2026

Trichomonas vaginalis targets Lactobacillus jensenii via pseudopodia-independent phagocytosis and secreted lysozyme TvGH25

A low abundance or absence of protective lactobacilli during acute trichomoniasis is a well-known phenomenon that is associated with T. vaginalis (TV) infection. However, a crucial question that remains unanswered is whether alterations in the lactobacilli population precede TV infection or whether the parasite plays an active role in lactobacilli disappearance. Our findings showed that TV efficiently phagocytosed one of the dominant Lactobacillus species L. jensenii (LJ). Phagocytosis proceeds via a pseudopodia-independent mechanism reminiscent of sinking with a preference for viable cells. The presence of viable LJ leads to an increase in secretion of 27 TV proteins, including TvGH25 lysozyme. This enzyme cleaves peptidoglycan, a major component of the bacterial cell wall. TV overexpressing TvGH25 effectively lowers the bacterial cell count, evidencing the enzymes antimicrobial potential. These data support the notion that TV cells can suppress the Lactobacillus population through a combination of targeted secretory response and phagocytic activity, revealing novel potential targets for developing alternative therapeutic strategies against trichomoniasis. Significance StatementTrichomonas vaginalis (TV) is a sexually transmitted parasite that causes trichomoniasis and is connected to the disruption of the healthy vaginal microbiome, dominated by Lactobacillus species. However, the nature of the interactions between TV and Lactobacillus is poorly understood. In this study, we show that TV uses an unusual form of pseudopodia-independent phagocytosis to engulf L. jensenii alongside a targeted secretory response to the bacterial encounter, involving TvGH25 lysozyme. We found that TV acquired this enzyme by lateral gene transfer from bacteria and repurposed it against bacteria to degrade the major bacterial cell wall component peptidoglycan. TvGH25 thus represents an effective component of TVs antibacterial arsenal. Our findings provide new insights into the mechanistic disruption of the protective Lactobacillus microbiota.

microbiology↗

bioRxiv2026

Vi-TIS: a transposon insertion sequencing framework for quantitative discrimination of viable and non viable mutants

Transposon-insertion sequencing (TIS) is a high-throughput approach that uses randomly generated transposon mutant libraries to assess bacterial gene essentiality and their contribution to fitness under defined conditions. The method integrates classical transposon mutagenesis with next-generation sequencing. However, conventional TIS workflows generally require at least one active growth step to enrich for replicating or fit mutants. The outcome of these experiments can be biased by the recovery of non-replicating cells or by the loss of slow-growing mutants. To overcome this limitation, we modified the traditional TIS workflow by incorporating propidium monoazide (PMA). PMA is membrane-impermeant and excluded from viable cells but can enter dead cells when the membrane has lost its integrity. PMA covalently crosslinks to DNA upon exposure to UV light. Such binding inhibits PCR amplification, an essential step in TIS library preparation. This property enables selective exclusion of DNA originating from dead cells and reduces the signal to noise ratio in TIS experiments. We refer to this modified approach as Viability-TIS (Vi-TIS). Applying Vi-TIS to our ultra-dense Escherichia coli K-12 BW25113 TIS library enhanced the accuracy of essential gene identification. We further exposed the library to subinhibitory concentrations of carbenicillin and found that Vi-TIS produced results consistent with those obtained using culture-based methodologies. In addition, Vi-TIS revealed previously unrecognized carbenicillin-susceptible mutants. Finally, we demonstrate that the mutations are linked to perturbations in peptidoglycan biosynthesis.

microbiology↗

bioRxiv2026

A fungal pathogen effector that shapes host plant microbiota kills bacteria through lipoteichoic acid binding and membrane disruption

Antimicrobial proteins are ancient and widespread molecules that contribute to survival across all domains of life. We previously showed that fungal plant pathogens secrete antimicrobial proteins to suppress antagonistic members of the host microbiota and thereby promote host colonization. The soil-borne plant pathogen Verticillium dahliae employs the bactericidal protein VdAve1 for this purpose. Here, we elucidate the mode of action of VdAve1 and define its mechanism as a distinct type of antimicrobial activity. Nuclear magnetic resonance (NMR) analysis revealed that VdAve1 adopts a jelly-roll barrel-like fold. Functionally, VdAve1 disrupts bacterial plasma membranes, and synthetic peptides derived from its positively charged regions retain antimicrobial activity, indicating that these regions contribute to its function. We further show that the bacterial model system Bacillus subtilis responds to VdAve1 by modifying teichoic acids, and that loss of these modifications increases bacterial sensitivity. Consistent with this, VdAve1 binds lipoteichoic acid (LTA), a major component of the Gram-positive cell wall. Together, our findings support a model in which VdAve1 binds LTA to localize at the bacterial surface, where it perturbs the plasma membrane, leading to membrane collapse and cell death. SIGNIFICANCE STATEMENTPlant-pathogenic microbes secrete effector proteins to promote host colonization. While these proteins are typically studied for their roles in host manipulation, some affect host-associated microbial communities by exhibiting antimicrobial activity. However, their modes of action remain largely unexplored. The fungal plant pathogen Verticillium dahliae secretes the antimicrobial effector VdAve1 to inhibit bacterial antagonists in the host microbiota. Here, we characterize the mode of action of VdAve1. We show that VdAve1 binds lipoteichoic acid, a major component of the cell wall of Gram-positive bacteria. This promotes VdAve1 accumulation close to the plasma membrane, and through electrostatic interactions it disrupts the plasma membrane. We further determine VdAve1 structure by NMR, revealing VdAve1 adopts a {beta}-barrel-like fold. Together, our findings show the mechanism by which a fungal effector inhibits bacterial growth and thereby how it may influence host-associated bacterial communities.

microbiology↗

bioRxiv2026

The phenotypic landscape of the model firmicute Bacillus subtilis

Firmicutes are gram-positive bacteria with important roles in human health, disease, and industry. However, more than a quarter of genes in the model firmicute Bacillus subtilis remain completely uncharacterized, including numerous core phylum-specific genes. Here, we design a compact pooled CRISPRi library targeting all protein-coding genes in B. subtilis and test its growth in [~]150 stress conditions. Using data from this screen as a hypothesis generator, we perform targeted experiments, revealing that the conserved essential firmicute protein YneF is part of the SRP co-translational protein secretion pathway. We also demonstrate that ECF-transporters play a previously unknown but broadly conserved role in cell wall homeostasis, perform an unbiased analysis of amino acid crossfeeding, and make additional discoveries about bacterial competition. In addition to these major contributions to our understanding of B. subtilis biology (and gram-positive firmicutes in general), this work provides a rich dataset that will nucleate future studies of uncharacterized genes and presents a framework for accessible full-genome functional genomic screens in other bacteria. SIGNIFICANCELarge-scale chemical genomics screens facilitate the characterization of genes by generating phenotypic data across a library of gene mutants. Here, using CRISPRi, we designed a compact pooled library targeting all genes in B. subtilis and screened growth of the library in [~]150 conditions. Importantly, we used this dataset to expand B. subtilis biology on levels ranging from molecular pathways to bacterial communities. We discovered a role for an essential firmicute gene in protein secretion, implicated two new players in cell wall homeostasis, and unraveled fundamental factors driving competition and cooperation in the soil. This rich dataset will serve as a hypothesis generator, expanding our set of bacterial phenotypic data and driving future experiments in B. subtilis and other gram-positive firmicutes.

microbiology↗

bioRxiv2026

PBP1A and LdtJ support cell envelope homeostasis and impact selection of Colistin-resistance in Acinetobacter baumannii

The multilayered cell envelope of Acinetobacter baumannii is an essential structure that maintains cellular integrity and protects the bacterial cell against external stresses and antibiotics. It consists of an inner membrane, a thin peptidoglycan (PG) layer and an asymmetric outer membrane (OM) enriched in lipooligosaccharide (LOS), whose lipid A moiety is the target of colistin, a last-resource antibiotic. Although lipid A is essential in most Gram-negatives, A. baumannii can survive without LOS through envelope remodeling, particularly in strains producing low levels of the bifunctional penicillin-binding protein PBP1A (encoded by mrcA) or in {Delta}mrcA mutants. Here, we identify a functional interplay between the LD-transpeptidase LdtJ, which generates 3-3 cross-links, and PBP1A, which catalyzes 4-3 transpeptidation during PG synthesis. We show that simultaneous inactivation of both enzymes severely affected growth, viability, morphology, and OM homeostasis. PG analyses revealed that the {Delta}ldtJ {Delta}mrcA mutants displays reduced overall cross-linkage and shorter glycan chains, producing a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, supporting their coordinated activity at sites of PG synthesis. Notably, {Delta}ldtJ {Delta}mrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings demonstrate that coupling between 4-3 and 3-3 transpeptidation is critical for envelope stability in A. baumannii and highlight how disrupting this coordination favors the emergence of colistin resistance. This work identifies a conserved PG remodeling vulnerability that directly links PG integrity to the evolution of antibiotic resistance, offering a new conceptual framework for destabilizing the A. baumannii envelope. ImportanceThe global rise of multidrug-resistant Acinetobacter baumannii represents an urgent clinical threat, largely driven by its extraordinary capacity to withstand cell envelope damages and escape last-resort antibiotics such as colistin. Although peptidoglycan synthesis and remodeling are known to influence outer membrane stability, how these processes are coordinated in A. baumannii has remained unclear. Here, we identify a crucial interplay between two critical envelope biogenesis and remodeling activities, 4-3 and 3-3 transpeptidations, mediated by the bifunctional PBP1A and the LD-transpeptidase LdtJ, respectively. Disrupting this coordination weakens the peptidoglycan, destabilizes the outer membrane, and increases the emergence frequency of colistin-resistant, LOS-deficient variants. These findings highlight a previously unrecognized vulnerability in the envelope homeostasis of A. baumannii, suggesting that simultaneously targeting DD and LD-transpeptidation could potentiate therapeutic strategies aimed at limiting antibiotic resistance development.

microbiology↗

bioRxiv2026

Periplasmic SacB as a robust counter-selection tool for genome engineering in the polyploid bacterium Zymomonas mobilis

The alpha-proteobacterium Zymomonas mobilis exhibits exceptional ethanologenic physiology, which makes it a traditional alcoholic beverage producer and a promising chassis for biofuel production. Although genetic tools for this organism have expanded in recent years, a fundamental aspect of its chromosome organization remains to be understood. In particular, Z. mobilis has been suggested to exhibit polyploidy, but this feature is not fully confirmed because of discrepancies among studies reporting the copy number of chromosomes. Here, we tagged the chromosome-partitioning protein ParB with a fluorescent marker to visualise its cellular localisation and estimate chromosome copy number in individual cells. Imaging showed that Z. mobilis exhibits several distinctive ParB foci throughout the cytoplasm and an accumulated focus at the pole, demonstrating that a single Z. mobilis cell contains at >5 copies of the chromosome at the oriC regions. After verifying its polyploidy, we sought to establish an efficient counter-selection system, which is crucial for engineering multiple copies of the chromosome. We assessed the efficacy of levan-sucrase (SacB) toxicity in Z. mobilis. We found that, despite Z. mobilis secreting a native extra-cellular sucrase SacB, heterologous periplasmically-localised Bacillus subtilis SacB rendered Z. mobilis cells sensitive to sucrose. We successfully used this effect for counter-selection when deleting and inserting targeted DNA sequences into the Z. mobilis genome. Together, this work provides important insights and tools for advancing Z. mobilis genetics and its biotechnological applications. ImportanceZymomonas mobilis is a promising industrial bacterium with capacity to convert sugars into ethanol at nearly maximum theoretical yield. With its expanding use in industrial applications, it is crucial to clarify if individual Z. mobilis cells carry multiple copies of chromosome as this has important implications for genome engineering. Two previous studies have used quantitative PCR to address this question, but their reported chromosome copy numbers varied widely from 20 to 100. Here, we used a cell biological approach to estimate the copy number and confirmed that single Z. mobilis cell possesses multiple copies. In addition, we show that a SacB-based counter-selection works in Z. mobilis, enabling efficient and complete mutation of all chromosome copies.

microbiology↗

bioRxiv2025

Peptidoglycan recycling is critical for cell division, cell wall integrity and β-lactam resistance in Caulobacter crescentus

Most bacteria possess a peptidoglycan (PG) sacculus, which is continuously remodeled during cell growth and division. The turnover products generated in this process are typically imported into the cell and reused for PG biosynthesis. While the underlying pathways have been studied intensively in gammaproteobacteria, knowledge of their presence and physiological roles in other bacterial lineages remains limited. Here, we comprehensively investigate PG recycling in the alphaproteobacterial model organism Caulobacter crescentus. Characterizing the activities of key enzymes in vitro and in vivo, we show that this species contains a functional PG recycling pathway, including the MurU shunt. Our results reveal that PG recycling is critical for C. crescentus cell morphology and division, and is dynamically regulated to balance the flux of metabolic intermediates toward PG biosynthesis and central carbon metabolism. Importantly, defects in PG recycling strongly impair the intrinsic ampicillin resistance of C. crescentus without changing the activity of its {beta}-lactamase BlaA, likely by limiting PG precursor biosynthesis and thereby decreasing the activity of the cell wall biosynthetic machinery in the presence of residual antibiotic. These findings underscore the central role of PG recycling in bacterial fitness and suggest that inhibiting this process could provide a promising strategy to combat {beta}-lactam-resistant pathogens.

microbiology↗

bioRxiv2025

Peptidoglycan remodelling improves salt resilience of Zymomonas mobilis

The alpha-proteobacterium Zymomonas mobilis is one of the most efficient microbial producers of ethanol and has the potential to be stablished as biofuel producer at industrial scale. However, a bottleneck hindering the full use of Z. mobilis in biorefinery is its sensitivity to environmental stresses, including sodium chloride (NaCl), a common component present in biomass from various sources. To address this limitation, we need to deepen our understanding of the cell envelope, the crucial barrier between bacteria and external stressors. To date, the cell envelope of Z. mobilis has remained largely uncharacterized. Here we show that the deletion of ctpA, which encodes a periplasmic protease, increases the salt resilience. Salt resilience is mediated by an increased level of the peptidoglycan endopeptidase MepM, which we identified as a substrate of CtpA through comparative proteomics. Supporting this, the overexpression of MepM in the wild-type enhanced salt resilience. We also discovered that the peptidoglycan of Z. mobilis is O-acetylated at MurNAc residues, a modification usually associated with virulence in pathogenic bacteria. Interestingly, O-acetylation was crucial for salt resilience, supporting a role in PG growth regulation under the stress condition. Overall, this study highlights the importance of investigating cell envelope biology in Z. mobilis as a foundation for engineering strains with improved resilience to environmental stress and, more general, studying the cell envelopes of non-model bacteria to expand our fundamental understanding of cell function ImportanceFossil fuels have negative impacts on the environment and will become limited in the next decades. Hence, alternative, sustainable energy sources need to be urgently established. Microbial fermentation of biomass for biofuel production presents a promising avenue. The Gram-negative alpha-proteobacterium Z. mobilis exhibits a superior capacity to convert sugars into ethanol, a clean, renewable and widely-used fuel. However, Z. mobilis has not been used as a first choice as a bio-fuel producer. The ethanol producer, bakers yeast Saccharomyces cerevisiae serves as a model species in cell biology, but we lack fundamental understanding of the cell envelope biology of Z. mobilis, which would be critical to engineer strain with increased resilience. Here, we demonstrate that knowledge about cell envelope biogenesis factors in Z. mobilis can help engineering optimised strains that grow under conditions of bio-fuel production.

microbiology↗

bioRxiv2025

Peptidoglycan DD-peptidases have distinct activities that impact fitness of Acinetobacter baumannii

The Gram-negative cell envelope is a vital interface between the bacterial cell and its environment. It acts as a selective barrier, blocking harmful agents while permitting nutrient uptake. Additionally, it enables environmental sensing and adaptive responses. Structurally, it is composed of the outer membrane, the cytoplasmic (inner) membrane, and the periplasm, which contains the peptidoglycan layer. Peptidoglycan is a conserved polymer that provides structural integrity, allowing the cell to withstand the internal turgor. It consists of glycan strands connected by short peptides, forming a mesh-like structure. In Gram-negative bacteria, the majority of the peptidoglycan subunits contain tetrapeptides. Tetrapeptides are generated through the action of DD-carboxypeptidases (DD-CPases), which cleave the terminal D-alanine from pentapeptides. Gram-negative bacteria encode multiple DD-CPases, but their precise role in maintaining cell shape and structural integrity remain poorly understood. The nosocomial pathogen Acinetobacter baumannii encodes three putative DD-CPases. To investigate the role of these enzymes, we generated single mutants, as well as double mutants in dacC, dacD, and pbpG, which encode the homologs of Escherichia coli DD-CPases PBP5, PBP6a, PBP6b, and the endopeptidase (DD-EPase) PBP7, respectively. We assessed the mutants for changes in cell morphology, growth dynamics, and stress tolerance. Additionally, we analyzed the composition of their peptidoglycan layers to determine the biochemical consequences of their inactivation. Each mutant exhibited distinct alterations in coccobacillary morphology and growth. Peptidoglycan analysis confirmed the enzymes possess DD-CPase activity, and PBP6b also demonstrated endopeptidase activity. Together, our results demonstrate that each peptidoglycan-modifying enzyme contributes uniquely to cell growth and morphology. These findings underscore their non-redundant functions and suggest their specific activities may serve as valuable targets for developing new antimicrobial therapies. ImportanceDD-peptidases, including carboxypeptidases and endopeptidases are crucial for maintaining cell envelope homeostasis, with distinct roles for each enzyme in cell wall biogenesis and structural integrity. The enzymatic characterization presented in this study not only advance our understanding of fundamental A. baumannii biology but also highlight these enzymatic activities as targets for development of innovative therapeutic strategies to combat infections caused by this multidrug-resistant microbe.

microbiology↗

bioRxiv2025

Molecular interplay between peptidoglycan integrity and outer membrane asymmetry in maintaining cell envelope homeostasis

The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane and peptidoglycan layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking outer membrane and peptidoglycan homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in peptidoglycan 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 peptidoglycan precursor genes (e.g., dadA, alr), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a {Delta}ldtJ {Delta}mla double mutant, implicating the outer membrane lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the {Delta}ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between peptidoglycan remodeling and outer membrane lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through peptidoglycan 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 layer and outer membrane. While LD-transpeptidases (LDTs) are traditionally known for reinforcing peptidoglycan 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 outer membrane lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between peptidoglycan remodeling and outer membrane lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria and suggest that targeting interconnected stress response pathways could offer novel strategies to undermine bacterial resilience.

microbiology↗

bioRxiv2025

An evolutionarily conserved laterally acquired toolkit enables microbiota targeting by Trichomonas

Trichomonas species are a diverse group of obligate extracellular symbionts associated with or attributed to various inflammatory diseases. They colonise mucosal surfaces across a wide range of hosts, all of which harbour a resident microbiota. Their evolutionary history likely involved multiple host transfers, including zoonotic events from columbiform birds to mammals. Using comparative transcriptomics, this study examines Trichomonas gallinae co-cultured with Escherichia coli, identifying a molecular toolkit that Trichomonas species may use to interact with bacterial members of the microbiota. Integrating transcriptomic data with comparative genomics and phylogenetics revealed a conserved repertoire of protein-coding genes likely acquired through multiple lateral gene transfers (LGT) in a columbiform-infecting ancestor. These LGT-derived genes encode muramidases, glucosaminidases, and antimicrobial peptides--enzymes and effectors capable of targeting bacterial cell walls, potentially affecting the bacterial microbiota composition across both avian and mammalian hosts. This molecular toolkit suggests that Trichomonas species can actively compete with and exploit their surrounding microbiota for nutrients, potentially contributing to the dysbiosis associated with Trichomonas infections. Their ability to target bacterial populations at mucosal surfaces provides insight into how Trichomonas species may have adapted to diverse hosts and how they could influence inflammatory mucosal diseases in birds and mammals.

molecular biology↗

bioRxiv2025

A novel peptidoglycan deacetylase modulates daughter cell separation in E. coli

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In Escherichia coli, three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC and NlpD activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, and NlpD and ActS are outer membrane lipoproteins. Here we report the identification of a novel periplasmic deacetylase called SddA that removes acetyl groups from denuded peptidoglycan glycan strands, the products of amidases. SddA is a substrate for the periplasmic protease Prc, suggesting regulation via protein degradation. The sddA gene is co-expressed with the gene encoding EnvC, linking SddA function to amidase activation. Consistent with this link, the deletion of sddA alleviates phenotypes associated with lack of amidase activation, while overexpression of sddA alleviates phenotypes related to a defective Tol-Pal system and causes cell chaining due to reduced septum peptidoglycan cleavage unless envC is co-expressed. We present a model according to which SddA modulates the activation of the septum-splitting amidases during cell division. AUTHOR SUMMARYBacteria surround their cell membrane by the essential peptidoglycan (cell wall) layer to prevent bursting open due to their turgor. During cell division, bacteria produce a septum at midcell, which must be cleaved for daughter cells to separate. Here, we report the identification of a new enzyme, SddA, that modifies a particular type of peptidoglycan material, denuded glycan chains released during the splitting of septal peptidoglycan for daughter cell separation, in the Gram-negative Escherichia coli. We propose a model in which SddA modulates a switch in the septal peptidoglycan splitting, ensuring splitting is activated from the cell membrane in the early stages of cell division and from the outer membrane in the late stages.

microbiology↗

bioRxiv2025

Disruptions in Outer Membrane-Peptidoglycan Interactions Enhance Bile Salt Resistance in O-antigen-Producing E. coli

Bile salts (BS) are derived from cholesterol in the liver and act as antimicrobial agents in the intestines by disrupting bacterial cell membranes and inducing oxidative stress. The gut bacterium E. coli is naturally resistant to BS, including the model strain K12 that produces a truncated LPS without O-antigen (OAg). Paradoxically, restoring a wild-type like LPS with OAg sensitises E. coli K12 to exogenous BS. In this study, we investigate this phenomenon. We show that mutations causing truncation of the LPS core oligosaccharide render these strains even more susceptible to BS, similar to the mutant strain MG1655-S{Delta}waaL defective in OAg ligase, primarily due to the accumulation of the lipid-linked intermediate UndPP-OAg. Through the characterisation of BS-resistant suppressor mutants of MG1655-S{Delta}waaL, we identify key genetic disruptions involved in resistance. Notably, we observed the highest BS resistance in strains with a weaker connection between the outer membrane (OM) and peptidoglycan (PG), including strains lacking the major OM-anchored, PG-binding proteins OmpA or Lpp, or expressing versions of these that lack PG-binding. Our data suggest that BS-induced stress in OAg-producing E. coli is due to the spatial constraints between OM and PG, and that mutations disrupting OM-PG interactions alleviate this stress, enhancing BS resistance. These findings provide new insights into a major challenge E. coli faces in the gut environment where it needs to produce OAg for stable colonisation and resists BS. E. coli can only survive BS exposure by fine-tuning the connectivity between its cell envelope layers, which highlights a potential target for modulating bacterial responses to BS in the gut. Author summaryEnteric bacteria residing in the human gut must withstand the host-derived antimicrobial agents, bile salts (BS), but the underlying resistance mechanisms are not fully elucidated. This study investigates the BS resistance mechanisms in O-antigen (OAg)-producing Escherichia coli K-12. We show that truncation of lipopolysaccharide (LPS) core oligosaccharides or restoration of OAg production increases BS sensitivity due to the accumulation of UndPP-OAg intermediates. By analysing suppressor mutants, we identify key genetic disruptions, particularly affecting the level of outer membrane-peptidoglycan (OM-PG) interactions involving OmpA and Lpp, which confer heightened BS resistance. Our findings highlight how BS-induced stress is linked to spatial constraints between the OM and PG layer, offering new insights into bacterial adaptation to BS stress. This research may provide new targets for therapeutic interventions to modulate gut microbial responses to BS.

microbiology↗

bioRxiv2024

A bacterial cell wall repair and modification system to resist host antibacterial factors

Pathogenic bacteria have acquired the ability to resist antibacterial defense mechanisms of the host. Streptococci are common in animal microbiota and include opportunistic pathogens like Group A Streptococcus (GAS) and Streptococcus pneumoniae (pneumococcus). While the conserved streptococcal S protein has been identified as a key factor in GAS virulence, its exact function is unclear. Here, we show that the pneumococcal S protein is crucial for resisting against host-derived antimicrobials by coordinating cell wall modification and repair. Specifically, we show that S proteins are septally localized through their transmembrane domain and contain an extracellular peptidoglycan (PG) binding LysM domain which is required for its function. Protein-protein and genetic interaction studies demonstrate that the pneumococcal S protein directly interacts with a PG synthase, class A penicillin binding protein PBP1a, and the PG deacetylase PgdA. Single-molecule experiments reveal that the fraction of circumferentially moving PBP1a molecules is reduced in the absence of S protein. Consistent with an impaired PBP1a function, streptococci lacking S protein exhibit increased susceptibility to cell wall targeting antibiotics and altered cell morphologies. PG analysis showed reduced N-deacetylation of glycans in the S. pneumoniae S protein mutant, indicating reduced PgdA activity. We show that pneumococci lacking the S protein cannot persist transient penicillin treatment, are more susceptible to the human antimicrobial peptide LL-37 and to lysozyme, and show decreased virulence in zebrafish and mice. Our data support a model in which S proteins regulate PBP1a activity and play a key role in coordinating PG repair and modification. This cell wall sentinel control system provides defense against host-derived and environmental antimicrobial attack.

microbiology↗

bioRxiv2024

Wall teichoic acids regulate peptidoglycan synthesis by paving cell wall microstructure

The cell wall is a polymeric exoskeleton that defines the size and shape of bacteria; it is composed of peptidoglycan and, in Gram-positive bacteria, wall teichoic acid polymers. Two systems synthesize peptidoglycan in rod-shaped bacteria, which are found pervasively across bacterial clades: the multi-protein Rod complexes synthesize anisotropic peptidoglycan, which is required for rod shape because it reinforces the cell wall along its circumference1-3, whereas the non-essential enzyme PBP1 synthesizes isotropic peptidoglycan1,4 (Fig. 1A). In Gram-positive bacteria, rod shape also requires wall teichoic acids5 for unknown reasons. Here, we show that wall teichoic acids promote rod shape by preventing the formation of nanoscopic pores in the Bacillus subtilis cell wall, which lead to amorphous growth by activating PBP1 and inhibiting Rod complexes. Depleting wall teichoic acids resulted in pores within minutes, coinciding with a rapid increase in PBP1-mediated synthesis. PBP1s ability to sustain teichoic acid-less growth depended on its intrinsically disordered domain. In contrast to previous steady-state measurements6,7, we found that wall teichoic acid depletion caused the transient arrest of Rod complexes prior to the onset of amorphous growth. Finally, one of the two synthetically lethal cell wall hydrolases in B. subtilis8, LytE, became essential during wall teichoic acid depletion, meaning that PBP1 and LytE execute a novel, amorphous mode of growth. Collectively, our results identify the cell wall, via its molecular-scale structure, as a non-canonical auto-regulator of its own synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/610702v3_fig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@154996forg.highwire.dtl.DTLVardef@12561c3org.highwire.dtl.DTLVardef@1356cacorg.highwire.dtl.DTLVardef@719d22_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1:C_FLOATNO Inhibiting wall teichoic acid synthesis decreases Rod complex activity prior to cell shape loss.(A) Schematic of Gram-positive cell wall synthesis. (B) Cell length growth rate [Figure 1] for wild-type cells during 0.5 g/mL tunicamycin treatment. Blue line shows smoothed population median, error bars show standard deviation. Data shown for 2,048 discrete cell tracks from 3 biological replicates. Inset: micrographs taken (i) before and (ii) after 45 min tunicamycin treatment. Scale bars 5 m. (C) Time course for the percentage of processive Mbl filaments during 0.5 g/mL tunicamycin treatment. Error bars show 95% confidence intervals across biological replicates (bootstrap analysis). Analysis performed on 62,901 discrete filament tracks from 4 biological replicates. Inset shows representative fluorescent kymographs of Mbl motion across the cell waist in (i) LB and (ii) after 30 min tunicamycin treatment. Red dotted line follows a processive Mbl filament. Scale bars 1 m. (D) Mean fluorescent puncta per unit cell length for wild-type, {Delta}ponA and ponATP-cells during tunicamycin treatment, labeled with fluorescent D-amino acids. Error bars show 95% confidence intervals. Analysis performed over 2,017 segmentations from 4 biological replicates (wild-type), 857 segmentations from 3 biological replicates ({Delta}ponA) and 257 cells from 2 biological replicates (ponATP-). All timepoints show statistical significance for difference in mean between cell types (Students T-test, P<0.01). Dots show individual replicates. Measurements at 50 min tunicamycin treatment used a lower exposure time so are plotted separately. Inset shows micrographs of (i,iii,v) wild-type and (ii,iv,vi) {Delta}ponA cells at (i,ii) 0 min, (iii,iv) 30 min, and (v,vi) 50 min of treatment. Micrographs are identically saturated across cell types. Scale bars 5 m. (E) Percentage of peptidoglycan subunits in monomers, measured by HPLC (Methods). Two biological replicates per strain. Mean values calculated across biological replicates. Error bars show 95% confidence intervals. Individual datapoints shown in red. Statistical significance calculated using Students T-test, P<0.05. C_FIG

microbiology↗

bioRxiv2024

Surface versus volume synthesis governs growth-dependent efficacy of a β-lactam antibiotic

The efficacy of {beta}-lactam antibiotics depends strongly on bacterial growth rate. This can lead to poor correlation between in vivo action and in vitro assays, hindering effective prescribing - yet the mechanisms underlying growth-rate dependent {beta}-lactam action remain unclear. Here, we investigate growth-rate dependent action of mecillinam, a {beta}-lactam that targets the elongation-mediating PBP2 peptidoglycan transpeptidase enzyme, on Escherichia coli cells. We show that mecillinam alters the balance between the rates of cell surface area and volume synthesis in a growth-rate dependent manner. Under mecillinam treatment, cell volume increases exponentially at a rate [fi]xed by the growth medium, but the cells ability to produce new surface area is compromised by the antibiotic. On rich medium, this imbalance leads to lysis, but on poor medium, slow-growing cells reach a new balance between surface area and volume synthesis, allowing sustained growth even at concentrations of mecillinam far above the EUCAST MIC value. A mathematical model based on surface area vs volume synthesis can quantitatively explain growth-medium dependent differences in mecillinam killing, as well as rescue from killing when cell morphology is perturbed in a microfluidic device. {beta}-lactam antibiotic action is mechanistically complex, yet our work suggests that simple conceptual principles can help understand the interplay between molecular mechanism and cell physiology, potentially contributing to more effective use of these drugs.

microbiology↗