Biology subjects

Erhardt, M.

Publications and source records attributed to Erhardt, M..

33 records · Page 2Linked to original sources

bioRxiv2023

Structure and mechanism of Zorya anti-phage defense system

Zorya is a recently identified and widely distributed bacterial immune system, which protects against phage invasion. It consists of a predicted membrane-embedded complex (ZorAB) and soluble components that differ among Zorya subtypes, notably ZorC and ZorD, in type I Zorya systems. Here, we reveal the molecular basis of the Zorya defense system using cryo-electron microscopy, mutagenesis, fluorescence microscopy, proteomics, and functional studies. We demonstrate that ZorAB shares the stoichiometry of other 5:2 inner membrane ion-driven rotary motors. Additionally, ZorA5B2 features a dimeric ZorB peptidoglycan binding domain and a pentameric -helical coiled-coil tail made of ZorA that projects approximately 700 [A] into the cytoplasm. We further characterize the structure and function of the soluble Zorya components, ZorC and ZorD, and find that they harbour DNA binding and nuclease activity, respectively. Comprehensive functional and mutational analyses demonstrates that all Zorya components work in concert to protect bacterial cells against invading phages. We present evidence that ZorAB operates as an ion-driven motor that becomes activated and anchors to the cell wall upon sensing of cell envelope perturbations during phage invasion. Subsequently, ZorAB transfers the phage invasion signal through the ZorA cytoplasmic tail to the soluble effectors, which function to prevent phage propagation. In summary, our study elucidates the foundational mechanisms of Zorya function and reveals a novel triggering signal for the rapid activation of an anti-phage defense system.

molecular biology↗

bioRxiv2023

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a new model for conjugation that revises our understanding of the DNA transport mechanism and the dissemination of drug resistance in complex bacterial communities. One-Sentence SummaryPlasmid DNA passes through the F pilus during conjugational transfer between physically distant bacteria.

microbiology↗

bioRxiv2023

SPI-1 virulence gene expression modulates motility of Salmonella Typhimurium in a proton motive force- and adhesins-dependent manner

Both the bacterial flagellum and the evolutionary related injectisome encoded on the Salmonella pathogenicity island 1 (SPI-1) play crucial roles during the infection cycle of Salmonella species. The interplay of both is highlighted by the complex cross-regulation that includes transcriptional control of the flagellar master regulatory operon flhDC by HilD, the master regulator of SPI-1 gene expression. Contrary to the HilD-dependent activation of flagellar gene expression, we report here that activation of HilD resulted in a dramatic loss of motility, which was dependent on the presence of SPI-1. Single cell analyses revealed that HilD-activation results in a SPI-1-dependent induction of the stringent response and a pronounced decrease of proton motive force (PMF), while flagellation was not affected. We further found that activation of HilD enhanced the adhesion of Salmonella to epithelial cells. A transcriptome analysis revealed a concomitant upregulation of several adhesin systems, which when overproduced, phenocopied the HilD-induced motility defect. We propose that a combination of SPI-1-dependent depletion of the PMF and upregulation of adhesins upon HilD-activation allows flagellated Salmonella to rapidly modulate their motility during infection, thereby enabling efficient adhesion to host cells and delivery of effector proteins.

microbiology↗

bioRxiv2022

A regulatory toolkit of arabinose-inducible artificial transcription factors for Gram-negative bacteria

The Gram-negative bacteria Salmonella Typhimurium and Escherichia coli are important model organisms, powerful prokaryotic expression platforms for biotechnological applications, and pathogenic strains constitute major public health threats. To facilitate new approaches for research, biomedicine, and biotechnological applications, we developed a set of arabinose-inducible artificial transcription factors (ATFs) using CRISPR/dCas9 and Arabidopsis-derived DNA-binding proteins, allowing to control gene expression in E. coli and Salmonella over a wide inducer concentration range. As a proof-of-concept, we employed the developed ATFs to engineer a Salmonella biosensor strain, SALSOR 0.2 (SALmonella biosenSOR 0.2), which responds to the presence of alkaloid drugs with quantifiable fluorescent output. We demonstrated that SALSOR 0.2 was able to detect the presence of the antitussive noscapine alkaloid with ~2.3-fold increased fluorescent signal over background noise compared to a previously described biosensor. Moreover, we used plant-derived ATFs to control {beta}-carotene biosynthesis in E. coli, which resulted in ~1.6-fold higher {beta}-carotene production compared to expression of the biosynthesis pathway using a strong constitutive promoter. The arabinose-inducible ATFs reported here thus enhance the synthetic biology repertoire of transcriptional regulatory modules that allow tuning protein expression in the Gram-negative model organisms Salmonella and E. coli.

synthetic biology↗

bioRxiv2022

Mechanisms of ion selectivity and rotor coupling in the bacterial flagellar sodium-driven stator unit

Bacteria swim using a flagellar motor that is powered by stator units. These stator units are energized by an ionic gradient across the membrane, typically proton or sodium. The presumed monodirectional rotation of the stator units allows the bidirectional rotation of the flagellar motor. However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the motor remain largely unclear. Here we have determined by cryo-electron microscopy the structure of the Na+-driven type stator unit PomAB from the gram-negative bacterium Vibrio alginolyticus in both lipidic and detergent environments, at a resolution up to 2.5 [A]. The structure is in a plugged, auto-inhibited state consisting of five PomA subunits surrounding two PomB subunits. The electrostatic potential map uncovers sodium ion binding sites within the transmembrane domain, which together with functional experiments and explicit solvent molecular dynamics simulations, suggest a mechanism for ion translocation and selectivity. Resolved conformational isomers of bulky hydrophobic residues from PomA, in the vicinity of key determinant residues for sodium ion coupling of PomB, prime PomA for clockwise rotation. The rotation is tightly blocked by the trans-mode organization of the PomB plug motifs. The structure also reveals a conformationally dynamic helical motif at the C-terminus of PomA, which we propose regulates the distance between PomA subunit cytoplasmic domains and is involved in stator unit-rotor interaction, concomitant stator unit activation, and torque transmission. Together, our studies provide mechanistic insight for understanding flagellar stator unit ion selectivity and incorporation of the stator units into the motor.

microbiology↗

bioRxiv2022

Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest

Initiation of bacterial DNA replication takes place at the origin of replication, a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response. Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be a valuable tool for biotechnological applications. ImportanceEven though bacteria are constantly replicating under laboratory conditions, natural environments expose them to various stresses like lack of nutrients, high salinity, and pH changes, which can keep them in non-replicating states. Non-replicating states can allow bacteria to become less sensitive or tolerant to antibiotics (persisters), remain inactive in specific niches for an extended period (dormancy), and adapt to some hostile ecosystems. Non-replicating states have been studied due to the possibility of repurposing energy to produce additional metabolites or proteins. Using CRISPRi targeting bacterial replication initiation sequences, we successfully arrested the replication of B. subtilis. We observed that non-replicating cells continued growing but not dividing, and the initial arrest did not induce global stress conditions such as SOS or stringent response. Notably, these cells continued their metabolic activity and translation. This study provides comprehensive insights into the physiological response of replication initiation blockage in B. subtilis.

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

BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

The widespread bacterial second messenger bis-(3-5)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups. ImportanceThe second messenger c-di-GMP controls various bacterial functions including development of resistant biofilm communities and transition into dormant spores. In vivo detection of c-di-GMP levels is therefore crucial for a better understanding of how intracellular c-di-GMP levels induce changes of bacterial physiology. Here, we describe the design of a novel c-di-GMP biosensor and demonstrate its effective application in investigating fluctuations in intracellular c-di-GMP levels in Escherichia coli and Salmonella enterica serovar Typhimurium on a population-based and single-cell level.

microbiology↗

bioRxiv2020

Controlling membrane barrier during bacterial type-III protein secretion

Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigated how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.

microbiology↗

bioRxiv2020

The impact of plasma membrane lipid composition on flagella-mediated adhesion of enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC on host tissues is the first step enabling bacterial colonization. Adhesins like fimbriae and flagella mediate this mechanism. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using Giant Unilamellar Vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components including proteins and glycoproteins. In contrast, with GUVs we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild type EHEC but not of a non-flagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagella-mediated adhesion to plasma membranes has broad implications to host-pathogen interactions.Importance Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues and form biofilm. Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagella-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagella adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts are important in bacterial adhesion. These findings significantly strengthen plasma membrane adhesion via bacterial flagella in an important human pathogen. This mechanism represents a promising target for the development of novel anti-adhesion therapies.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2020

Structure and function of stator units of the bacterial flagellar motor

Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 [A]-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction. One Sentence SummaryStructural basis of torque generation in the bidirectional bacterial flagellar motor

biophysics↗

bioRxiv2020

Controlling minimal and maximal hook-length of the bacterial flagellum

Hook-length control is a central checkpoint during assembly of the bacterial flagellum. During hook growth, a 405 amino acids (aa) protein, FliK, is intermittently secreted and thought to function as a molecular measuring tape that, in Salmonella, controls hook-length to 55 nm {+/-} 6 nm. The underlying mechanism involves interactions of both the -helical, N-terminal domain of FliK (FliKN) with the hook and hook cap, and of its C-terminal domain with a component of the export apparatus. However, various deletion mutants of FliKN display uncontrolled hook-length, which is not consistent with a ruler mechanism. Here, we carried out an extensive deletion analysis of FliKN to investigate its contribution in the hook-length control mechanism. We identified FliKN mutants deleted for up to 80 aa that retained wildtype motility. However, the short FliK variants did not produce shorter hook-lengths as expected from a physical ruler. Rather, the minimal length of the hook depends on the level of hook protein production and secretion. Our results thus support a model in which FliK functions as a hook growth terminator protein that limits the maximal length of the hook, and not as a molecular ruler that physically measures hook-length.

microbiology↗

bioRxiv2019

RADA is the main branch migration factor in plant mitochondrial recombination and its defect leads to mtDNA instability and cell cycle arrest

The mitochondria of flowering plants have large and complex genomes whose structure and segregation are modulated by recombination activities. The late steps of mitochondrial recombination are still poorly characterized: while the loss of mitochondrial recombination is not viable, a deficiency in RECG1-dependent branch migration has little impact on plant development, implying the existence of alternative pathways. Here we present RADA, an ortholog of bacterial RadA/Sms, which is required for the processing of organellar recombination intermediates. While bacterial RadA is dispensable, RADA-deficient plants are severely impacted in their development and fertility, correlating with increased mtDNA ectopic recombination and replication of recombination-generated subgenomes. The radA mutation is epistatic to recG1, indicating that RADA drives the main branch migration pathway of plant mitochondria. In contrast, the double mutation radA recA3 is lethal, underlining the importance of an alternative RECA3-dependent pathway. Although RADA is dually targeted to mitochondria and chloroplasts, we found little to no effects of radA on the stability of the plastidial genome. The stunted growth of radA mutants could not be correlated with obvious defects in mitochondrial gene expression. Rather, it seems that is partially caused by a retrograde signal that activates nuclear genes repressing cell cycle progression.

plant biology↗

bioRxiv2019

Flagella methylation promotes bacterial adhesion and host cell invasion

The flagellum is the motility device of many bacteria and the long external filament is made of several thousand copies of a single protein, flagellin. While posttranslational modifications of flagellin are common among bacterial pathogens, the role of lysine methylation remained unknown. Here, we show that both flagellins of Salmonella enterica, FliC and FljB, are methylated at surface-exposed lysine residues. A Salmonella mutant deficient in flagellin methylation was outcompeted for gut colonization in a gastroenteritis mouse model. In support, methylation of flagellin promoted invasion of epithelial cells in vitro. Lysine methylation increased the surface hydrophobicity of flagellin and enhanced flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization and successful infection of the host.

microbiology↗

bioRxiv2018

Hook length of the bacterial flagellum is controlled to nanometer-scale for optimal motility performance

Most bacteria swim in liquid environments by rotating one or several flagella. The long external filament of the flagellum is connected to a membrane-embedded basal-body by a flexible universal joint, the hook, which allows the transmission of motor torque to the filament. The length of the hook is controlled on a nanometer-scale by a sophisticated molecular ruler mechanism. However, why its length is stringently controlled has remained elusive. We engineered and studied a diverse set of hook-length variants of Salmonella enterica. Measurements of plate-assay motility, single-cell swimming speed and directional persistence in quasi 2D and population-averaged swimming speed and body angular velocity in 3D revealed that the motility performance is optimal around the wild type hook-length. We conclude that too short hooks may be too stiff to function as a junction and too long hooks may buckle and create instability in the flagellar bundle. Accordingly, peritrichously flagellated bacteria move most efficiently as the distance travelled per body rotation is maximal and body wobbling is minimized. Thus, our results suggest that the molecular ruler mechanism evolved to control flagellar hook growth to the optimal length consistent with efficient bundle formation. The hook-length control mechanism is therefore a prime example of how bacteria evolved elegant, but robust mechanisms to maximize their fitness under specific environmental constraints.\n\nAuthor summaryMany bacteria use flagella for directed movement in liquid environments. The flexible hook connects the membrane-embedded basal-body of the flagellum to the long, external filament. Flagellar function relies on self-assembly processes that define or self-limit the lengths of major parts. The length of the hook is precisely controlled on a nanometer-scale by a molecular ruler mechanism. However, the physiological benefit of tight hook-length control remains unclear. Here, we show that the molecular ruler mechanism evolved to control the optimal length of the flagellar hook, which is consistent with efficient motility performance. These results highlight the evolutionary forces that enable flagellated bacteria to optimize their fitness in diverse environments and might have important implications for the design of swimming micro-robots.

microbiology↗