Biology subjects

Strahl, H.

Publications and source records attributed to Strahl, H..

16 recordsLinked to original sources

bioRxiv2026

Membrane-targeting antimicrobials trigger lysis in Bacillus subtilis by disturbing the MreB-dependent regulation of peptidoglycan hydrolases

Membrane-targeting antimicrobials are generally assumed to kill bacteria through bacteriolysis induced by the permeabilisation of the cytoplasmic membrane. This model relies on the notion that bacteriolysis is the direct cellular manifestation of the membrane-disruptive (membranolytic) activity of an antibacterial compound. However, it underappreciates the key role of peptidoglycan hydrolases in bacteriolysis. Using the Gram-positive model organism Bacillus subtilis, we demonstrate that the bacteriolytic activity of membrane-targeting antimicrobials arises from the misregulation of peptidoglycan hydrolases, regardless of whether they induce large membrane pores or trigger more subtle membrane disturbances such as depolarisation. Contrary to previous models, the autolysis of B. subtilis induced by membrane depolarising compounds does not depend on pH changes associated with the cell surface. Instead, the autolytic process is triggered by membrane depolarisation-dependent dissociation of the bacterial actin homolog MreB, a key spatial coordinator of cell wall synthesis in rod-shaped bacteria, from the cytoplasmic membrane. These findings provide valuable insights into the cellular pathways involved in autolysis, highlight the challenges in distinguishing between direct and indirect cellular effects of membrane-targeting antibiotics, and improve our understanding of antibiotic-induced bacteriolysis.

microbiology↗

bioRxiv2026

Transertion provides evidence for coupling of transcription and translation in Bacillus subtilis

In Gram-negative bacteria, co-translational insertion of membrane proteins into the plasma membrane may be coupled to ongoing transcription, a phenomenon known as transertion. Transertion results in a physical shift of the coding gene from the nucleoid towards the membrane and is one of the determinants of the shape of the nucleoid and placement of cell division in Gram-negative bacteria. In contrast, the existence of functional coupling of transcription and translation in Bacillus subtilis and potentially other Gram-positive bacteria has been questioned, suggesting that transertion may not happen. Here, by imaging vertically oriented B. subtilis cells, we show that the gene of a transmembrane protein changes its localization from inside the nucleoid to the plasma membrane upon induction of its transcription. Localization is restored to the nucleoid when induction of transcription has ceased. The shift of the gene towards the membrane is strictly dependent on transcription, its induction, translation and transmembrane nature of the coded protein. These results suggest that, at least, some principles of cellular regulation based on functional coupling of transcription and translation may be conserved between Gram-positive and Gram-negative bacteria.

molecular biology↗

bioRxiv2025

Monitoring single cell bioenergetic status and cell lysis in dense and differentiating Bacillus subtilis cultures

BackgroundThe Gram-positive bacterium Bacillus subtilis is a rapidly growing and easily manipulated microbe with a long history of exploitation for the commercial production of industrial enzymes, high-value biochemicals, antibiotics and other secondary metabolites. More recently, it has been developed as a food additive and plant probiotic . It grows on cheap substrates and remains productive during extended batch-fed growth conditions. Extensive knowledge of its genetics, biochemistry and gene regulation has facilitated the use of metabolic engineering strategies to optimise substrate utilisation and product yield. As a free-living environmental organism, B. subtilis differentiates into subpopulations with distinct biological functions (e.g. sporulation, biofilm formation, antimicrobials production, etc). However, during fermentation, cellular differentiation processes can pose a challenge for its optimal biotechnological utilization, particularly when emerging subpopulations do not contribute to product biosynthesis. Here, we present robust assays that facilitate the analysis of two previously difficult-to-study population properties of B. subtilis: (i) the energization levels of individual cells within post-exponential but actively growing cultures and (ii) the extent of cell lysis that can occur under such conditions. ResultsOur findings reveal an unappreciated level of heterogeneity in cell energization within post-exponential cultures, and a surprisingly high degree of cell lysis in seemingly healthy, actively growing populations. These data provide insights and add to our understanding of the biological complexities and single-cell heterogeneities present in superficially simple bacterial clonal cultures. They establish robust and well-validated analytical tools with which to study the associated processes and provide a foundation for further optimizing B. subtilis as an industrial production host. ConclusionsConsiderable research efforts have been aimed at increasing the productivity of B. subtilis for industrial, medical and agricultural products. However, its ability to undergo physiological and morphological differentiation processes at high cell densities ultimately limits its productivity. Our research reveals how the resulting heterogeneity impacts the population-level energy status of individual cells in the culture and the surprisingly high extent of population-level cell lysis. The ability to monitor these processes provides tools for evaluating the impact of genetic and metabolic engineering strategies to improve productivity.

microbiology↗

bioRxiv2024

The last resort antibiotic daptomycin exhibits two independent antibacterial mechanisms of action

Daptomycin is a lipopeptide antibiotic, commonly used as last resort treatment against multidrug resistant Gram-positive pathogens. Despite its clinical success, the mechanism through which daptomycin exerts its antibacterial properties has remained controversial. Much of the debate is focused around daptomycins ability to depolarise the cytoplasmic membrane, potential formation of large membrane pores, and its more recently discovered capability to inhibit cell wall synthesis and disturb membrane lipid domain organisation through interactions with cell wall precursor lipids. Here we show that, rather than representing different facets of a single underlying activity, daptomycin exhibits two independent antibacterial mechanisms of action: (i) targeting of cell wall precursor lipids that leads to cell wall synthesis inhibition and (ii) membrane depolarisation that does not rely on interactions with cell wall precursor lipids. This dual mechanism of action provides an explanation for the frequently disagreeing findings obtained through in vivo and in vitro studies and explains why resistance development towards daptomycin is slow and multifactorial. In a broader context, this demonstrates that dual mechanism antibiotics can be clinically successful and exhibit favourable characteristics in terms of real-world, clinically relevant resistance development.

microbiology↗

bioRxiv2024

Cyclical MinD membrane affinity differences are not necessary for MinD gradient formation in Bacillus subtilis

Proteins can diffuse micrometers in seconds, yet bacterial cells are able to maintain stable protein gradients. The best studied bacterial protein gradient is the Min system of Escherichia coli. In rod-shaped bacteria the MinCD proteins prevent formation of minicells by inhibiting FtsZ polymerization close to the cell poles. In E. coli these proteins oscillate between cell poles within a minute, resulting in an increased MinCD concentration at the poles. This oscillation is caused by the interaction between MinD and the protein MinE, which form an ATP-driven reaction-diffusion system, whereby the ATPase MinD cycles between a monomeric cytosolic and a dimeric membrane attached states. Bacillus subtilis also has MinCD, but lacks MinE. In this case MinCD form a static gradient that requires the transmembrane protein MinJ, located at cell poles and cell division sites. A recent reaction-diffusion model was successful in recreating the MinD gradient in B. subtilis, assuming that MinD cycles between cytosol and membrane, like in E. coli. Here we show that the monomeric and dimeric states of B. subtilis MinD have comparable membrane affinities, that MinD interacts with MinJ as a dimer, and that MinJ is not required for membrane localization of MinD. Based on these new findings we tested different models, using kinetic Monte Carlo simulations, and found that a difference in diffusion rate between the monomer and dimer, rather than a difference in membrane affinity, is important for B. subtilis MinCD gradient formation.

microbiology↗

bioRxiv2024

Mechanism of phage sensing and abortion by toxin-antitoxin-chaperone systems

Toxin-antitoxins (TAs) are prokaryotic two-gene systems comprised of a toxin neutralised by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilises the antitoxin by recognising its chaperone addiction (ChAD) element. TACs have been shown to mediate antiphage defence, but the mechanisms of viral sensing and restriction are unexplored. We identify and characterise two Escherichia coli antiphage TAC systems containing HigBA and CmdTA TA units, HigBAC and CmdTAC. The HigBAC is triggered through recognition of the gpV major tail protein of phage {lambda}. Both the ChAD and gpV are recognised by the HigC chaperone through analogous aromatic molecular patterns, explaining the mechanism of activation. We show that the CmdT ADP-ribosyltransferase toxin modifies mRNA to shut down protein synthesis. We establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with the HigC chaperone phage sensor. HighlightsE. coli HigBAC and CmdTAC are translation-targeting phage immunity TAC systems HigC chaperone recognises phage {lambda} major tail protein to trigger HigBAC toxicity CmdT ADP-ribosyltransferase toxin abrogates translation through modification of mRNA HigC combined with CmdTA yields hybrid broad-spectrum antiphage defence system

microbiology↗

bioRxiv2023

Lipoteichoic acid biosynthesis by Staphylococcus aureus is controlled by the MspA protein through competitive interference.

Staphylococcus aureus produces a plethora of virulence factors critical to its ability to establish an infection and cause disease. We have previously characterised a small membrane protein, MspA, which has pleiotropic affects on virulence and contributes to S. aureus pathogenicity in vivo. Here we report that mspA inactivation triggers overaccumulation of the essential cell wall component lipoteichoic acid (LTA) which, in turn, decreases autolytic activity and leads to increased cell size due to a delay in cell separation. We show that MspA directly interacts with the LTA synthesis enzymes UgtP, LtaA and LtaS, and competitively interferes with the association between LtaA and LtaS. While complementation of the mspA mutant with wild-type mspA reduces the amount of LTA, expression of a mutated version of MspA that does not interfere with the interactions between LtaA and LtaS does not. We suggest that MspA contributes to maintaining a physiological level of LTA in the cell wall by interacting with the LTA synthetic enzymes. In conclusion, this study uncovers the critical role of the MspA protein in regulating cell envelope biosynthesis and pathogenicity. IMPORTANCEThe S. aureus cell envelope, comprising of the cytoplasmic membrane, a thick peptidoglycan layer and the anionic polymers lipoteichoic acid and wall teichoic acids, is fundamental for bacterial growth and division, as well as being the main interface between the pathogen and the host. It has become increasingly apparent that the synthesis and turnover of cell envelope components also affect the virulence of S. aureus. In this study, we show that MspA, a novel effector of S. aureus virulence, contributes to the maintenance of normal levels of lipoteichoic acid in the cell wall, with implications on cell cycle and size. These findings further our understanding of the connections between envelope synthesis and pathogenicity and suggests MspA as a novel a target for therapeutic intervention.

microbiology↗

bioRxiv2023

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Our data, thus, demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

microbiology↗

bioRxiv2022

Shield co-opts an RmuC domain to mediate phage defence across Pseudomonas species.

Competitive bacteria-bacteriophage interactions have resulted in the evolution of a plethora of bacterial defense systems preventing phage propagation. In recent years, computational and bioinformatic approaches have underpinned the discovery of numerous novel bacterial defense systems. Anti-phage systems are frequently encoded together in genomic loci termed defense islands. Here we report the identification and characterisation of a novel anti-phage system, which we have termed Shield, that forms part of the Pseudomonas defensive arsenal. The Shield system comprises a membrane-bound protein, ShdA, harboring an RmuC domain. Heterologous production of ShdA alone is sufficient to mediate bacterial immunity against a panel of phages. We show that ShdA homologues can degrade phage DNA in vitro and, when expressed in a heterologous host, can alter the organisation of chromosomal DNA to a nucleoid structure. Further analysis reveals that Shield can be divided into four subtypes, three of which contain additional components that in some cases can modulate the activity of ShdA and/or provide additional lines of phage defence. Collectively, our results identify a new player within the Pseudomonas bacterial immunity arsenal that displays a novel mechanism of protection, and reveals a surprising role of RmuC domains in phage defence. SIGNIFICANCEThe evolutionary pressure exerted by bacteriophages has driven bacteria to acquire numerous defense systems. Recent studies have highlighted the extraordinary diversity of these systems, uncovering exciting links between bacterial and eukaryotic immunity. Here we describe a novel anti-phage system, named Shield, found within Pseudomonas species. We identify several Shield subtypes, all harboring the same core component, and describe its mode of action. The growing instance of multidrug-resistant bacterial infections urgently requires the development of alternative treatments. Phage therapy is a particularly pertinent approach to treat multi-drug resistant Pseudomonas aeruginosa strains causing severe lung infection in cystic fibrosis patients. A detailed understanding of bacterial immunity and phage counter-strategies is an essential step to underpin the rational design of phage therapy to fight disease.

microbiology↗

bioRxiv2022

A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes

Transmembrane potential is one of the main bioenergetic parameters of bacterial cells, and is directly involved in energising key cellular processes such as transport, ATP synthesis, and motility. The most common approach to measure membrane potential levels is through use of voltage-sensitive fluorescent dyes. Such dyes either accumulate or are excluded from the cell in a voltage-dependent manner, which can be followed by means of fluorescence microscopy, flow cytometry, or fluorometry. Since the cells ability to maintain transmembrane potential relies upon low membrane ion conductivity, voltage-sensitive dyes are also highly sensitive reporters for the activity of membrane-targeting antibacterials. However, the presence of an additional membrane layer in Gram-negative (diderm) bacteria significantly complicates their use. In this manuscript, we provide guidance on how membrane potential and its changes can be reliably monitored in Gram-negatives using the voltage-sensitive dye DiSC3(5). We also discuss the confounding effects caused by the presence of the outer membrane, or by measurements performed in buffers rather than growth medium. We hope that the discussed methods and protocols provide an easily accessible basis for the use of voltage-sensitive dyes in Gram-negative organisms, and raise awareness of potential experimental pitfalls associated with their use.

microbiology↗

bioRxiv2021

Panacea: a hyperpromiscuous antitoxin protein domain for the neutralisation of diverse toxin domains

Toxin-Antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids, as well as bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets, and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analysed the conservation of genomic neighbourhoods using our computational tool FlaGs (for Flanking Genes), which allows high-throughput detection of TA-like operons. Focussing on the widespread but poorly experimentally characterised antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have renamed the domain to Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralised TA pairs. While the majority of validated PanA-neutralised toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises replication and translation, as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralisation mechanisms employed by Type II TA systems. Finally, through directed evolution we have selected PanA variants that can neutralise non-cognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain. SignificanceToxin-antitoxin systems are enigmatic and diverse elements of bacterial and bacteriophage genomes. We have uncovered remarkable versatility of an antitoxin protein domain, that has evolved to neutralise dozens of different toxin domains. We find that antitoxins carrying this domain - Panacea - form complexes with their cognate toxins, indicating a direct neutralisation mechanism, and that Panacea can be evolved to neutralise a non-cognate and non-homologous toxin with just two amino acid substitutions. This raises the possibility that this domain could be an adaptable universal, or semi-universal protein neutraliser with significant biotechnological and medical potential.

microbiology↗

bioRxiv2019

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase-separation process induced by low fluidity is biologically reversible.

microbiology↗

bioRxiv2019

A new family of Type VI secretion system-delivered effector proteins displays ion-selective pore-forming activity

Type VI secretion systems (T6SSs) are nanomachines widely used by bacteria to compete with rivals. T6SSs deliver multiple toxic effector proteins directly into neighbouring cells and play key roles in shaping diverse polymicrobial communities. A number of families of T6SS-dependent anti-bacterial effectors have been characterised, however the mode of action of others remains unknown. Here we report that Ssp6, an anti-bacterial effector delivered by the Serratia marcescens T6SS, is an ion-selective pore-forming toxin. In vivo, Ssp6 inhibits growth by causing depolarisation of the inner membrane of intoxicated cells and also leads to increased outer membrane permeability, whilst reconstruction of Ssp6 activity in vitro demonstrated that it forms cation-selective pores. A survey of bacterial genomes revealed that Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 represents a new family of T6SS-delivered anti-bacterial effectors, further diversifying the portfolio of weapons available for deployment during inter-bacterial conflict.

microbiology↗

bioRxiv2018

A membrane-depolarising toxin substrate of the Staphylococcus aureus Type VII protein secretion system targets eukaryotes and bacteria

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues. Significance statementStaphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a Type VII secretion system (T7SS) that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a T7 substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo.

microbiology↗

bioRxiv2017

Membrane curvature and the Tol-Pal complex determine polar localization of the chemoreceptor Tar in E. coli

Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years different mechanisms have been put forward to explain this polar localization; from stochastic clustering, membrane curvature driven localization, interactions with the Tol-Pal complex, to nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at cell poles. This finding also implies that the curvature of cell poles does not attract chemoreceptor complexes. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterised by strong membrane curvature. Chemoreceptors, like Tar, form trimer-of-dimers that bend the cell membrane due to a rigid tripod structure with an estimated curvature of approximately 37 nm. This curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favourable as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or cell poles. These findings favour a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex.\n\nImportanceBacteria have exquisite mechanisms to sense and to adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli, and found that membrane curvature at cell division sites and interaction with the Tal-pol protein complex, localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

microbiology↗

bioRxiv2017

The Gram-positive model organism Bacillus subtilis does not form detectable cardiolipin-specific lipid domains

Rather than being a homogenous diffusion-dominated structure, biological membranes can exhibit areas with distinct composition and characteristics commonly termed as lipid domains. Arguably the most comprehensively studied examples in bacteria are domains formed by cardiolipin, which have been functionally linked to protein targeting, cell division process, and mode of action of membrane targeting antimicrobials. Cardiolipin domains were originally identified in the Gram-negative model organism Escherichia coli based on preferential staining by the fluorescent membrane dye nonyl acridine orange (NAO), and later reported to exist also in other Gram-negative and -positive bacteria. Recently, the lipid-specificity of NAO has been questioned based on studies conducted in E. coli. This prompted us to re-analyse cardiolipin domains also in the Gram-positive model organism Bacillus subtilis. Here we show that logarithmically growing B. subtilis does not form microscopically detectable cardiolipin-specific lipid domains, and that NAO is not a specific stain for cardiolipin in this organism.\n\nAbbreviations

microbiology↗