Subdomains of the Helicobacter pylori Cag T4SS outer membrane core complex exhibit structural independence.

The Helicobacter pylori Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from ΔcagT or ΔcagM mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagY∆AP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC.

Rotation of the Fla2 flagella of Cereibacter sphaeroides requires the periplasmic proteins MotK and MotE that interact with the flagellar stator protein MotB2.

The bacterial flagellum is a complex structure formed by more than 25 different proteins, this appendage comprises three conserved structures: the basal body, the hook and filament. The basal body, embedded in the cell envelope, is the most complex structure and houses the export apparatus and the motor. In situ images of the flagellar motor in different species have revealed a huge diversity of structures that surround the well-conserved periplasmic components of the basal body. The identity of the proteins that form these novel structures in many cases has been elucidated genetically and biochemically, but in others they remain to be identified or characterized. In this work, we report that in the alpha proteobacteria Cereibacter sphaeroides the novel protein MotK along with MotE are essential for flagellar rotation. We show evidence that these periplasmic proteins interact with each other and with MotB2. Moreover, these proteins localize to the flagellated pole and MotK localization is dependent on MotB2 and MotA2. These results together suggest that the role of MotK and MotE is to activate or recruit the flagellar stators to the flagellar structure.

Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE.

Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106 s-1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<105 s-1) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance.

Monitoring Drug-Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy.

The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of ß-lactams by ß-lactamases, (ii) the interaction of drugs belonging to the ß-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

Molecular Crowding Alters the Interactions of Polymyxin Lipopeptides within the Periplasm of E. coli: Insights from Molecular Dynamics.

The cell envelope of Gram-negative bacteria is a crowded tripartite architecture that separates the cell interior from the external environment. Two membranes encapsulate the aqueous periplasm, which contains the cell wall. Little is known about the mechanisms via which antimicrobial peptides move through the periplasm from the outer membrane to their site of action, the inner membrane. We utilize all-atom molecular dynamics to study two antimicrobial peptides, polymyxins B1 and E, within models of the E. coli periplasm crowded to different extents. In a simple chemical environment, both PMB1 and PME bind irreversibly to the cell wall. The presence of specific macromolecules leads to competition with the polymyxins for cell wall interaction sites, resulting in polymyxin dissociation from the cell wall. Chemical complexity also impacts interactions between polymyxins and Braun's lipoprotein; thus, the interaction modes of lipoprotein antibiotics within the periplasm are dependent upon the nature of the other species present.

Characterization of ß-Barrel Outer Membrane Proteins and Their Interactions with Chaperones by Chemical-Crosslinking Mass Spectrometry.

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins).

Recent Advances in Modeling Membrane ß-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages.

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1ß, IL-6, IL-10, and TGF-ß1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

The Bradyrhizobium japonicum exporter ExsFGH is involved in efflux of ferric xenosiderophores from the periplasm.

The gram-negative bacterium Bradyrhizobium japonicum can take up structurally dissimilar ferric siderophores from the environment (xenosiderophores) to meet its nutritional iron requirements. Siderophore-bound iron transported into the periplasm is reduced to the ferrous form by FsrB, dissociated from the siderophore and the free ion is then transported into the cytoplasm by the ferrous iron transporter FeoAB. Here, we identified the RND family exporter genes exsFG and exsH in a selection for secondary site suppressor mutants that restore growth of an fsrB mutant on the siderophores ferrichrome or ferrioxamine. The low level of radiolabel accumulation from 55Fe-labeled ferrichrome or ferrioxamine observed in the fsrB mutant was restored to wild type levels in the fsrB exsG mutant. Moreover, the exsG mutant accumulated more radiolabel from the 55Fe-labeled siderophores than the wild type, but radiolabel accumulation from inorganic 55Fe was similar in the two strains. Thus, ExsFGH exports siderophore-bound iron, but not inorganic iron. The rescued fsrB exsG mutant required feoB for growth, indicating that ExsFGH acts on those siderophores in the periplasm. The exsG mutant was more sensitive to the siderophore antibiotic albomycin than the wild type, whereas the fsrB mutant was more resistant. This suggests ExsFGH normally exports ferrated albomycin. B. japonicum is naturally resistant to many antibiotics. The exsG strain was very sensitive to tetracycline, but not to six other antibiotics tested. We conclude that ExsFGH is a broad substrate exporter that is needed to maintain siderophore homeostasis in the periplasm.

Aberrant Topologies of Bacterial Membrane Proteins Revealed by High Sensitivity Fluorescence Labelling.

The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods.

Single-protein Diffusion in the Periplasm of Escherichia coli.

The width of the periplasmic space of Gram-negative bacteria is only about 25-30 nm along the long axis of the cell, which affects free diffusion of (macro)molecules. We have performed single-particle displacement measurements and diffusion simulation studies to determine the impact of confinement on the apparent mobility of proteins in the periplasm of Escherichia coli. The diffusion of a reporter protein and of OsmY, an osmotically regulated periplasmic protein, is characterized by a fast and slow component regardless of the osmotic conditions. The diffusion coefficient of the fast fraction increases upon osmotic upshift, in agreement with a decrease in macromolecular crowding of the periplasm, but the mobility of the slow (immobile) fraction is not affected by the osmotic stress. We observe that the confinement created by the inner and outer membranes results in a lower apparent diffusion coefficient, but this can only partially explain the slow component of diffusion in the particle displacement measurements, suggesting that a fraction of the proteins is hindered in its mobility by large periplasmic structures. Using particle-based simulations, we have determined the confinement effect on the apparent diffusion coefficient of the particles for geometries akin the periplasmic space of Gram-negative bacteria.

Disruption of trehalose periplasmic recycling dysregulates cAMP-CRP signaling in Escherichia coli during stationary phase.

IMPORTANCE: Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such reserves. How Escherichia coli manage carbon storage systems under starvation stress, as well as transpose changes in intracellular metabolite levels into regulatory signals, is not well understood. Endogenous trehalose metabolism may be at the center of these processes, coupling carbon storage with carbon starvation responses. The coupled transport to the periplasm and subsequent hydrolysis of trehalose back to glucose for transport to the cytoplasm may function as a crucial metabolic signaling pathway. Although trehalose has been characterized as a stress protectant in E. coli, the disaccharide also functions as both an energy storage compound and a regulator of carbohydrate metabolism in fungi, plants, and other bacteria. Our research explores the metabolic regulatory properties of trehalose in E. coli and a potential mechanism by which the intracellular carbon pool is interconnected with regulatory circuits, enabling long-term survival.

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide.

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

Microbial Primer: Multidrug efflux pumps.

Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.

The inhibitory mechanism of a small protein reveals its role in antimicrobial peptide sensing.

A large number of small membrane proteins have been uncovered in bacteria, but their mechanism of action has remained mostly elusive. Here, we investigate the mechanism of a physiologically important small protein, MgrB, which represses the activity of the sensor kinase PhoQ and is widely distributed among enterobacteria. The PhoQ/PhoP two-component system is a master regulator of the bacterial virulence program and interacts with MgrB to modulate bacterial virulence, fitness, and drug resistance. A combination of cross-linking approaches with functional assays and protein dynamic simulations revealed structural rearrangements due to interactions between MgrB and PhoQ near the membrane/periplasm interface and along the transmembrane helices. These interactions induce the movement of the PhoQ catalytic domain and the repression of its activity. Without MgrB, PhoQ appears to be much less sensitive to antimicrobial peptides, including the commonly used C18G. In the presence of MgrB, C18G promotes MgrB to dissociate from PhoQ, thus activating PhoQ via derepression. Our findings reveal the inhibitory mechanism of the small protein MgrB and uncover its importance in antimicrobial peptide sensing.

Bacterial extracellular electron transfer components are spin selective.

Metal-reducing bacteria have adapted the ability to respire extracellular solid surfaces instead of soluble oxidants. This process requires an electron transport pathway that spans from the inner membrane, across the periplasm, through the outer membrane, and to an external surface. Multiheme cytochromes are the primary machinery for moving electrons through this pathway. Recent studies show that the chiral-induced spin selectivity (CISS) effect is observable in some of these proteins extracted from the model metal-reducing bacteria, Shewanella oneidensis MR-1. It was hypothesized that the CISS effect facilitates efficient electron transport in these proteins by coupling electron velocity to spin, thus reducing the probability of backscattering. However, these studies focused exclusively on the cell surface electron conduits, and thus, CISS has not been investigated in upstream electron transfer components such as the membrane-associated MtrA, or periplasmic proteins such as small tetraheme cytochrome (STC). By using conductive probe atomic force microscopy measurements of protein monolayers adsorbed onto ferromagnetic substrates, we show that electron transport is spin selective in both MtrA and STC. Moreover, we have determined the spin polarization of MtrA to be ∼77% and STC to be ∼35%. This disparity in spin polarizations could indicate that spin selectivity is length dependent in heme proteins, given that MtrA is approximately two times longer than STC. Most significantly, our study indicates that spin-dependent interactions affect the entire extracellular electron transport pathway.

Approaches for high-throughput quantification of periplasmic recombinant proteins.

The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide bond formation, required by some proteins (such as antibody fragments) for correct folding and function. It also permits simpler protein release and downstream processing than cytoplasmic accumulation. As such, targeting of recombinant proteins to the E. coli periplasm is a key strategy in biologic manufacture. However, expression and translocation of each recombinant protein requires optimisation including selection of the best signal peptide and growth and production conditions. Traditional methods require separation and analysis of protein compositions of periplasmic and cytoplasmic fractions, a time- and labour-intensive method that is difficult to parallelise. Therefore, approaches for high throughput quantification of periplasmic protein accumulation offer advantages in rapid process development.

Structure of an endogenous mycobacterial MCE lipid transporter.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

Periplasmic biomineralization for semi-artificial photosynthesis.

Semiconductor-based biointerfaces are typically established either on the surface of the plasma membrane or within the cytoplasm. In Gram-negative bacteria, the periplasmic space, characterized by its confinement and the presence of numerous enzymes and peptidoglycans, offers additional opportunities for biomineralization, allowing for nongenetic modulation interfaces. We demonstrate semiconductor nanocluster precipitation containing single- and multiple-metal elements within the periplasm, as observed through various electron- and x-ray-based imaging techniques. The periplasmic semiconductors are metastable and display defect-dominant fluorescent properties. Unexpectedly, the defect-rich (i.e., the low-grade) semiconductor nanoclusters produced in situ can still increase adenosine triphosphate levels and malate production when coupled with photosensitization. We expand the sustainability levels of the biohybrid system to include reducing heavy metals at the primary level, building living bioreactors at the secondary level, and creating semi-artificial photosynthesis at the tertiary level. The biomineralization-enabled periplasmic biohybrids have the potential to serve as defect-tolerant platforms for diverse sustainable applications.

Goethite Formed in the Periplasmic Space of Pseudomonas sp. JM-7 during Fe Cycling Enhances Its Denitrification in Water.

Denitrification-driven Fe(II) oxidation is an important microbial metabolism that connects iron and nitrogen cycling in the environment. The formation of Fe(III) minerals in the periplasmic space has a significant effect on microbial metabolism and electron transfer, but direct evidence of iron ions entering the periplasm and resulting in periplasmic mineral precipitation and electron conduction properties has yet to be conclusively determined. Here, we investigated the pathways and amounts of iron, with different valence states and morphologies, entering the periplasmic space of the denitrifier Pseudomonas sp. JM-7 (P. JM-7), and the possible effects on the electron transfer and the denitrifying ability. When consistently provided with Fe(II) ions (from siderite (FeCO3)), the dissolved Fe(II) ions entered the periplasmic space and were oxidized to Fe(III), leading to the formation of a 25 nm thick crystalline goethite crust, which functioned as a semiconductor, accelerating the transfer of electrons from the intracellular to the extracellular matrix. This consequently doubled the denitrification rate and increased the electron transport capacity by 4-30 times (0.015-0.04 µA). However, as the Fe(II) concentration further increased to above 4 mM, the Fe(II) ions tended to preferentially nucleate, oxidize, and crystallize on the outer surface of P. JM-7, leading to the formation of a densely crystallized goethite layer, which significantly slowed down the metabolism of P. JM-7. In contrast to the Fe(II) conditions, regardless of the initial concentration of Fe(III), it was challenging for Fe(III) ions to form goethite in the periplasmic space. This work has shed light on the likely effects of iron on environmental microorganisms, improved our understanding of globally significant iron and nitrogen geochemical cycles in water, and expanded our ability to study and control these important processes.