The gram-negative bacterial periplasm: Size matters.

Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allows more efficient and diverse mechanisms of protein oxidation, folding, and quality control. The periplasm also contains structural elements and important environmental sensing modules, and it allows complex nanomachines to span the cell envelope. Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer. This periplasm intermembrane distance is critical for sensing outer membrane damage and dictates length of the flagellar periplasmic rotor, which controls motility. These exciting results resolve longstanding debates about whether the periplasmic distance has a biological function and raise the possibility that the mechanisms for maintenance of periplasmic size could be exploited for antibiotic development.

Communication across the bacterial cell envelope depends on the size of the periplasm.

The cell envelope of gram-negative bacteria, a structure comprising an outer (OM) and an inner (IM) membrane, is essential for life. The OM and the IM are separated by the periplasm, a compartment that contains the peptidoglycan. The OM is tethered to the peptidoglycan via the lipoprotein, Lpp. However, the importance of the envelope's multilayered architecture remains unknown. Here, when we removed physical coupling between the OM and the peptidoglycan, cells lost the ability to sense defects in envelope integrity. Further experiments revealed that the critical parameter for the transmission of stress signals from the envelope to the cytoplasm, where cellular behaviour is controlled, is the IM-to-OM distance. Augmenting this distance by increasing the length of the lipoprotein Lpp destroyed signalling, whereas simultaneously increasing the length of the stress-sensing lipoprotein RcsF restored signalling. Our results demonstrate the physiological importance of the size of the periplasm. They also reveal that strict control over the IM-to-OM distance is required for effective envelope surveillance and protection, suggesting that cellular architecture and the structure of transenvelope protein complexes have been evolutionarily co-optimised for correct function. Similar strategies are likely at play in cellular compartments surrounded by 2 concentric membranes, such as chloroplasts and mitochondria.

Extracellular reduction of solid electron acceptors by Shewanella oneidensis.

Shewanella oneidensis is the best understood model organism for the study of dissimilatory iron reduction. This review focuses on the current state of our knowledge regarding this extracellular respiratory process and highlights its physiologic, regulatory and biochemical requirements. It seems that we have widely understood how respiratory electrons can reach the cell surface and what the minimal set of electron transport proteins to the cell surface is. Nevertheless, even after decades of work in different research groups around the globe there are still several important questions that were not answered yet. In particular, the physiology of this organism, the possible evolutionary benefit of some responses to anoxic conditions, as well as the exact mechanism of electron transfer onto solid electron acceptors are yet to be addressed. The elucidation of these questions will be a great challenge for future work and important for the application of extracellular respiration in biotechnological processes.

Thiol Starvation Induces Redox-Mediated Dysregulation of Escherichia coli Biofilm Components.

A hallmark of bacterial biofilms is the production of an extracellular matrix (ECM) that encases and protects the community from environmental stressors. Biofilm formation is an integral portion of the uropathogenic Escherichia coli (UPEC) life cycle. Approximately 2% of UPEC isolates are cysteine auxotrophs. Here, we investigated how cysteine homeostasis impacted UPEC UTI89 strain biofilm formation and, specifically, the production of the ECM components curli and cellulose. Cysteine auxotrophs produced less cellulose and slightly more curli compared to wild-type (WT) strains, and cysteine auxotrophs formed smooth, nonrugose colonies. Cellulose production was restored in cysteine auxotrophs when YfiR was inactivated. YfiR is a redox-sensitive regulator of the diguanylate cyclase, YfiN. The production of curli, a temperature-regulated appendage, was independent of temperature in UTI89 cysteine auxotrophs. In a screen of UPEC isolates, we found that ∼60% of UPEC cysteine auxotrophs produced curli at 37°C, but only ∼2% of cysteine prototrophic UPEC isolates produced curli at 37°C. Interestingly, sublethal concentrations of amdinocillin and trimethoprim-sulfamethoxazole inhibited curli production, whereas strains auxotrophic for cysteine continued to produce curli even in the presence of amdinocillin and trimethoprim-sulfamethoxazole. The dysregulation of ECM components and resistance to amdinocillin in cysteine auxotrophs may be linked to hyperoxidation, since the addition of exogenous cysteine or glutathione restored WT biofilm phenotypes to mutants unable to produce cysteine and glutathione.IMPORTANCE Uropathogenic Escherichia coli (UPEC) bacteria are the predominant causative agent of urinary tract infections (UTIs). UTIs account for billions of dollars of financial burden annually to the health care industry in the United States. Biofilms are an important aspect of the UPEC pathogenesis cascade and for the establishment of chronic infections. Approximately 2% of UPEC isolates from UTIs are cysteine auxotrophs, yet there is relatively little known about the biofilm formation of UPEC cysteine auxotrophs. Here we show that cysteine auxotrophs have dysregulated biofilm components due to a change in the redox state of the periplasm. Additionally, we show the relationship between cysteine auxotrophs, biofilms, and antibiotics frequently used to treat UTIs.

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP-transport system.

In this study, we investigated an SBP (DctPAm ) of a tripartite ATP-independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7T . Deletion of dctPAm as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7T , if cultivated on mineral salt medium supplemented with d-glucose, d-galactose, l-arabinose, d-fucose, d-xylose or d-gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctPAm using the broad host vector pBBR1MCS-5::dctPAm . Furthermore, an uptake assay with radiolabeled [14 C(U)]-d-glucose clearly showed that the deletion of dctPAm , dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of KD performing thermal shift assays showed a shift in the melting temperature of DctPAm in the presence of d-gluconic acid (KD 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above-mentioned carbohydrates d-galactonate (KD 10.72 ± 1.4 µM), d-fuconic acid (KD 13.50 ± 1.6 µM) and d-xylonic acid (KD 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.

Compartmentalization of the Carbaryl Degradation Pathway: Molecular Characterization of Inducible Periplasmic Carbaryl Hydrolase from Pseudomonas spp.

Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA, encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of Escherichia coli The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent Km and Vmax in the range of 98 to 111 µM and 69 to 73 µmol · min-1 · mg-1, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol.IMPORTANCE Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of Pseudomonas spp. Predicted transmembrane domain and signal peptide sequences of Pseudomonas were found to be functional in Escherichia coli and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.

Harnessing the Periplasm of Bacterial Cells To Develop Biocatalysts for the Biosynthesis of Highly Pure Chemicals.

Although biocatalytic transformation has shown great promise in chemical synthesis, there remain significant challenges in controlling high selectivity without the formation of undesirable by-products. For instance, few attempts to construct biocatalysts for de novo synthesis of pure flavin mononucleotide (FMN) have been successful, due to riboflavin (RF) accumulating in the cytoplasm and being secreted with FMN. To address this problem, we show here a novel biosynthesis strategy, compartmentalizing the final FMN biosynthesis step in the periplasm of an engineered Escherichia coli strain. This construct is able to overproduce FMN with high specificity (92.4% of total excreted flavins). Such a biosynthesis approach allows isolation of the final biosynthesis step from the cytoplasm to eliminate undesirable by-products, providing a new route to develop biocatalysts for the synthesis of high-purity chemicals.IMPORTANCE The periplasm of Gram-negative bacterial hosts is engineered to compartmentalize the final biosynthesis step from the cytoplasm. This strategy is promising for the overproduction of high-value products with high specificity. We demonstrate the successful implementation of this strategy in microbial production of highly pure FMN.

Genetic and Mechanistic Analyses of the Periplasmic Domain of the Enterohemorrhagic Escherichia coli QseC Histidine Sensor Kinase.

The histidine sensor kinase (HK) QseC senses autoinducer 3 (AI-3) and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators (RRs) to regulate the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC). The QseB, QseF, and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain and aligned the sequences from 12 different species to identify the most conserved amino acids. We selected eight amino acids conserved in all of these QseC homologues. The corresponding QseC site-directed mutants were expressed and still able to autophosphorylate; however, four mutants demonstrated an increased basal level of phosphorylation. These mutants have differential flagellar, motility, LEE, and Shiga toxin expression phenotypes. We selected four mutants for more in-depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE, and QseF. This suggests that these mutations in the periplasmic sensing domain affected the region downstream of the QseC signaling cascade and therefore can influence which pathway QseC regulates.IMPORTANCE In the foodborne pathogen EHEC, QseC senses AI-3, epinephrine, and norepinephrine, increases its autophosphorylation, and then transfers its phosphate to three RRs: QseB, QseF, and KdpE. QseB controls expression of flagella and motility, KdpE controls expression of the LEE region, and QseF controls the expression of Shiga toxin. This tripartite signaling pathway must be tightly controlled, given that flagella and the type three secretion system (T3SS) are energetically expensive appendages and Shiga toxin expression leads to bacterial cell lysis. Our data suggest that mutations in the periplasmic sensing loop of QseC differentially affect the expression of the three arms of this signaling cascade. This suggests that these point mutations may change QseC's phosphotransfer preferences for its RRs.

Flagellar motility of the pathogenic spirochetes.

Bacterial pathogens are often classified by their toxicity and invasiveness. The invasiveness of a given bacterium is determined by how capable the bacterium is at invading a broad range of tissues in its host. Of mammalian pathogens, some of the most invasive come from a group of bacteria known as the spirochetes, which cause diseases, such as syphilis, Lyme disease, relapsing fever and leptospirosis. Most of the spirochetes are characterized by their distinct shapes and unique motility. They are long, thin bacteria that can be shaped like flat-waves, helices, or have more irregular morphologies. Like many other bacteria, the spirochetes use long, helical appendages known as flagella to move; however, the spirochetes enclose their flagella in the periplasm, the narrow space between the inner and outer membranes. Rotation of the flagella in the periplasm causes the entire cell body to rotate and/or undulate. These deformations of the bacterium produce the force that drives the motility of these organisms, and it is this unique motility that likely allows these bacteria to be highly invasive in mammals. This review will describe the current state of knowledge on the motility and biophysics of these organisms and provide evidence on how this knowledge can inform our understanding of spirochetal diseases.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate.

UNLABELLED: We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. IMPORTANCE: Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger.

Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa.

In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.

Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily.

AmtB, a member of the Rh/Amt/MEP superfamily, is responsible for ammonia transport in Escherichia coli. The ammonia pathway in AmtB consists of a narrow hydrophobic lumen in between hydrophilic periplasmic and cytoplasmic vestibules. A series of molecular dynamics simulations (greater than 0.4 µs in total) were performed to determine the mechanism of solute recruitments and selectivity by the periplasmic vestibule. The results show that the periplasmic vestibule plays a crucial role in solute selectivity, and its solute preferences follow the order of NH4(+) > NH3 > CO2. Based on our results, NH4(+) recruitment is initiated by its interaction with either E70 or E225, highly conserved residues located at the entrance of the vestibule. Subsequently, the backbone carbonyl groups at the periplasmic vestibule direct NH4(+) to the conserved aromatic cage at the bottom of the vestibule (known as the Am1 site). The umbrella sampling simulations suggest that the conserved residue D160 is not directly involved in the ammonia conduction; rather its main function is to keep the structure of periplasmic vestibule intact. The MD simulations also revealed that two partially stacked phenyl rings of F107 and F215, separating the periplasmic vestibule from the hydrophobic lumen, flip open and closed simultaneously with a frequency of approximately 10(8) flipping events per second. These results show how the periplasmic vestibule selectively recruits NH4(+) to the Am1 site, and also that the synchronized flipping of two phenyl rings potentially facilitates the solute transition from the periplasmic vestibule to the hydrophobic lumen in the Rh/Amt/MEP superfamily.

Ultrastructure and histochemistry of the periplasm in oocytes of sturgeons during egg envelope formation.

In the cytoplasm of oocytes (ooplasm) located in ovarian follicles with diameters 2000 microm and 2150 microm in Acipenser gueldenstaedtii, and 2000 microm and 2350 microm in A. baerii, periplasm containing a basophilic compartment and endoplasm containing reserve materials was formed. Vesicles involved in polyspermy blocking and in the formation of the embryo were located in the periplasm. These included compact (cCGs), low-electron-dense cortical granules (lCGs), and lamellar bodies. The cCGs were bounded by a membrane, comprised fibrillar material, fibrils and rod-shaped components. The lCGs were membrane-bounded and contained fibrillar material and granular inclusions. Endoplasmic reticulum (ER) and Golgi complexes were involved in the formation of cCG and lCG. The basophilic compartment, ER and Golgi vesicles participated in the formation of lamellar bodies. They comprised numerous membranes and fibrillar material. It is assumed that they transfer membranes and their precursors to the growing furrow during cleavage and release their content to organize the extracellular matrix. The location of compounds in the developing egg envelope of A. gueldenstaedtii was presented and discussed. Ovaries of both investigated species represented the first pubertal stages of development. Such fish should not be used for reproduction.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

The lipopolysaccharide transport system of Gram-negative bacteria.

The cell envelope of Gram-negative bacteria consists of two distinct membranes, the inner (IM) and the outer membrane (OM) separated by the periplasm. The OM contains in the outer leaflet the lipopolysaccharide (LPS), a complex lipid with important biological activities. In the host it elicits the innate immune response whereas in the bacterium it is responsible for the peculiar permeability barrier properties exhibited by the OM. The chemical structure of LPS and its biosynthetic pathways have been fully elucidated. By contrast only recently details of the transport and assembly of LPS into the OM have emerged. LPS is synthesized in the cytoplasm and at the inner leaflet of the IM and needs to cross two different compartments, the IM and the periplasm, to reach its final destination at the OM. This review focuses on recent studies that led to our present understanding of the protein machine implicated in LPS transport and in assembly at the cell surface.

Measurement of the Cell-Body Rotation of Leptospira.

Leptospira spp. swim in liquid and crawl on surfaces with two periplasmic flagella. The periplasmic flagella attach to the protoplasmic cylinder via basal rotary motors (flagellar motors) and transform the ends of the cell body into spiral or hook shape. The rotations of the periplasmic flagella are thought to gyrate the cell body and rotate the protoplasmic cylinder for propelling the cell; however, the motility mechanism has not been fully elucidated. Since the motility is a critical virulence factor for pathogenic leptospires, the kinematic insight is valuable to understand the mechanism of infection. This chapter describes microscopic methodologies to measure the motility of Leptospira, focusing on rotation of the helical cell body.

The flat-ribbon configuration of the periplasmic flagella of Borrelia burgdorferi and its relationship to motility and morphology.

Electron cryotomography was used to analyze the structure of the Lyme disease spirochete, Borrelia burgdorferi. This methodology offers a new means for studying the native architecture of bacteria by eliminating the chemical fixing, dehydration, and staining steps of conventional electron microscopy. Using electron cryotomography, we noted that membrane blebs formed at the ends of the cells. These blebs may be precursors to vesicles that are released from cells grown in vivo and in vitro. We found that the periplasmic space of B. burgdorferi was quite narrow (16.0 nm) compared to those of Escherichia coli and Pseudomonas aeruginosa. However, in the vicinity of the periplasmic flagella, this space was considerably wider (42.3 nm). In contrast to previous results, the periplasmic flagella did not form a bundle but rather formed a tight-fitting ribbon that wraps around the protoplasmic cell cylinder in a right-handed sense. We show how the ribbon configuration of the assembled periplasmic flagella is more advantageous than a bundle for both swimming and forming the flat-wave morphology. Previous results indicate that B. burgdorferi motility is dependent on the rotation of the periplasmic flagella in generating backward-moving waves along the length of the cell. This swimming requires that the rotation of the flagella exerts force on the cell cylinder. Accordingly, a ribbon is more beneficial than a bundle, as this configuration allows each periplasmic flagellum to have direct contact with the cell cylinder in order to exert that force, and it minimizes interference between the rotating filaments.

The Pseudomonas aeruginosa T6SS Delivers a Periplasmic Toxin that Disrupts Bacterial Cell Morphology.

The type VI secretion system (T6SS) is crucial in interbacterial competition and is a virulence determinant of many Gram-negative bacteria. Several T6SS effectors are covalently fused to secreted T6SS structural components such as the VgrG spike for delivery into target cells. In Pseudomonas aeruginosa, the VgrG2b effector was previously proposed to mediate bacterial internalization into eukaryotic cells. In this work, we find that the VgrG2b C-terminal domain (VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein. We resolve the structure of VgrG2bC-ter and confirm it is a member of the zinc-metallopeptidase family of enzymes. We show that this effector causes membrane blebbing at midcell, which suggests a distinct type of T6SS-mediated growth inhibition through interference with cell division, mimicking the impact of ß-lactam antibiotics. Our study introduces a further effector family to the T6SS arsenal and demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells.

Stochastic binding of Ca2+ ions in the dyadic cleft; continuous versus random walk description of diffusion.

Ca(2+) signaling in the dyadic cleft in ventricular myocytes is fundamentally discrete and stochastic. We study the stochastic binding of single Ca(2+) ions to receptors in the cleft using two different models of diffusion: a stochastic and discrete Random Walk (RW) model, and a deterministic continuous model. We investigate whether the latter model, together with a stochastic receptor model, can reproduce binding events registered in fully stochastic RW simulations. By evaluating the continuous model goodness-of-fit for a large range of parameters, we present evidence that it can. Further, we show that the large fluctuations in binding rate observed at the level of single time-steps are integrated and smoothed at the larger timescale of binding events, which explains the continuous model goodness-of-fit. With these results we demonstrate that the stochasticity and discreteness of the Ca(2+) signaling in the dyadic cleft, determined by single binding events, can be described using a deterministic model of Ca(2+) diffusion together with a stochastic model of the binding events, for a specific range of physiological relevant parameters. Time-consuming RW simulations can thus be avoided. We also present a new analytical model of bimolecular binding probabilities, which we use in the RW simulations and the statistical analysis.