-
Applied and Environmental Microbiology Jun 2021Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism....
Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in the bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species, with a preference for heavier lanthanides. Its methanol oxidation machinery is supposedly adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on transcriptome sequencing (RNA-seq) analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the cluster but potentially also a type VI secretion system. A high level of constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work with commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. The bacterium RH AL1 might be an attractive target for developing biorecovery strategies to obtain these economically highly demanded metals in environmentally friendly ways.
Topics: Bacterial Outer Membrane; Bacterial Proteins; Beijerinckiaceae; Gene Expression Regulation, Bacterial; Lanthanum; Methanol; Microscopy, Electron, Transmission; Periplasm
PubMed: 33893117
DOI: 10.1128/AEM.03144-20 -
STAR Protocols Dec 2021Bacteriophages of the family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these...
Bacteriophages of the family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these proteins (gp14, gp15, and gp16) are ejected into the host envelope forming a DNA-ejectosome for genome delivery. Here, we describe the purification and characterization of recombinant gp14, gp15, and gp16. This protocol was used for high-resolution cryo-EM structure analysis of the T7 periplasmic tunnel and can be adapted to study ejection proteins from other phages. For complete details on the use and execution of this protocol, please refer to Swanson et al. (2021).
Topics: Bacteriophage T7; Cryoelectron Microscopy; Escherichia coli; Periplasm; Recombinant Proteins; Viral Proteins
PubMed: 34825220
DOI: 10.1016/j.xpro.2021.100960 -
Nature May 2021Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year. Specialized protein...
Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year. Specialized protein transport systems-known as type VII secretion systems (T7SSs)-are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP protease. A trimer of MycP caps a central periplasmic dome-like chamber that is formed by three EccB dimers, with the proteolytic sites of MycP facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP show disruption of the EccB periplasmic assembly and increased flexibility, which highlights the importance of MycP for complex integrity. Beneath the EccB-MycP chamber, dimers of the EccC ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.
Topics: Cryoelectron Microscopy; Cytosol; Models, Molecular; Mycobacterium tuberculosis; Periplasm; Protein Domains; Protein Multimerization; Protein Stability; Tuberculosis; Type VII Secretion Systems
PubMed: 33981042
DOI: 10.1038/s41586-021-03517-z -
Nature Communications Nov 2019Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of...
Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC.
Topics: Bacterial Proteins; Biological Transport; DNA; DNA, Bacterial; Gene Transfer, Horizontal; Helicobacter pylori; Periplasm; Receptors, Cell Surface; Transformation, Bacterial
PubMed: 31767852
DOI: 10.1038/s41467-019-13352-6 -
Journal of Bacteriology Dec 2018is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors...
is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache (lcium channels and motaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpV is structurally similar to a sensor domain of a chemoreceptor with unknown function from , which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpV Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpV were in the micromolar range. An McpV variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction. Legumes share a unique association with certain soil-dwelling bacteria known broadly as rhizobia. Through concerted interorganismal communication, a legume allows intracellular infection by its cognate rhizobial species. The plant then forms an organ, the root nodule, dedicated to housing and supplying fixed carbon and nutrients to the bacteria. In return, the engulfed rhizobia, differentiated into bacteroids, fix atmospheric N into ammonium for the plant host. This interplay is of great benefit to the cultivation of legumes, such as alfalfa and soybeans, and is initiated by chemotaxis to the host plant. This study on carboxylate chemotaxis contributes to the understanding of rhizobial survival and competition in the rhizosphere and aids the development of commercial inoculants.
Topics: Amino Acids; Bacterial Proteins; Calcium Channels; Calorimetry; Carboxylic Acids; Chemotactic Factors; Chemotaxis; Fluorometry; Ligands; Medicago sativa; Models, Molecular; Periplasm; Plant Exudates; Protein Domains; Sinorhizobium meliloti; Symbiosis
PubMed: 30201781
DOI: 10.1128/JB.00519-18 -
MBio Oct 2014A defining characteristic of Chlamydia spp. is their developmental cycle characterized by outer membrane transformations of cysteine bonds among cysteine-rich outer...
UNLABELLED
A defining characteristic of Chlamydia spp. is their developmental cycle characterized by outer membrane transformations of cysteine bonds among cysteine-rich outer membrane proteins. The reduction-oxidation states of host cell compartments were monitored during the developmental cycle using live fluorescence microscopy. Organelle redox states were studied using redox-sensitive green fluorescent protein (roGFP1) expressed in CF15 epithelial cells and targeted to the cytosol, mitochondria, and endoplasmic reticulum (ER). The redox properties of chlamydiae and the inclusion were monitored using roGFP expressed by Chlamydia trachomatis following transformation. Despite the large morphological changes associated with chlamydial infection, redox potentials of the cytosol (Ψ(cyto) [average, -320 mV]), mitochondria (Ψ(mito) [average, -345 mV]), and the ER (ΨER [average, -258 mV]) and their characteristic redox regulatory abilities remained unchanged until the cells died, at which point Ψ(cyto) and Ψ(mito) became more oxidized and Ψ(ER) became more reduced. The redox status of the chlamydial cytoplasm was measured following transformation and expression of the roGFP biosensor in C. trachomatis throughout the developmental cycle. The periplasmic and outer membrane redox states were assessed by the level of cysteine cross-linking of cysteine-rich envelope proteins. In both cases, the chlamydiae were highly reduced early in the developmental cycle and became oxidized late in the developmental cycle. The production of a late-developmental-stage oxidoreductase/isomerase, DsbJ, may play a key role in the regulation of the oxidoreductive developmental-stage-specific process.
IMPORTANCE
Infectious Chlamydia organisms have highly oxidized and cysteine cross-linked membrane proteins that confer environmental stability when outside their host cells. Once these organisms infect a new host cell, the proteins become reduced and remain reduced during the active growth stage. These proteins become oxidized at the end of their growth cycle, wherein infectious organisms are produced and released to the environment. How chlamydiae mediate and regulate this key step in their pathogenesis is unknown. Using biosensors specifically targeted to different compartments within the infected host cell and for the chlamydial organisms themselves, the oxidoreductive states of these compartments were measured during the course of infection. We found that the host cell redox states are not changed by infection with C. trachomatis, whereas the state of the chlamydial organisms remains reduced during infection until the late developmental stages, wherein the organisms' cytosol and periplasm become oxidized and they acquire environmental resistance and infectivity.
Topics: Cell Line; Cell Membrane; Chlamydia trachomatis; Cytoplasm; Epithelial Cells; Genes, Reporter; Humans; Organelles; Oxidation-Reduction; Periplasm
PubMed: 25352618
DOI: 10.1128/mBio.01924-14 -
PloS One 2015Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to...
Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.
Topics: Bacterial Adhesion; Bacterial Proteins; Cellulose; Fibrobacter; Fimbriae Proteins; Models, Biological; Periplasm; Proteomics; Transcriptome
PubMed: 26629814
DOI: 10.1371/journal.pone.0143809 -
MBio Nov 2019Photoferrotrophy is a form of anoxygenic photosynthesis whereby bacteria utilize soluble or insoluble forms of ferrous iron as an electron donor to fix carbon dioxide...
Photoferrotrophy is a form of anoxygenic photosynthesis whereby bacteria utilize soluble or insoluble forms of ferrous iron as an electron donor to fix carbon dioxide using light energy. They can also use poised electrodes as their electron donor via phototrophic extracellular electron uptake (phototrophic EEU). The electron uptake mechanisms underlying these processes are not well understood. Using TIE-1 as a model, we show that a single periplasmic decaheme cytochrome , PioA, and an outer membrane porin, PioB, form a complex allowing extracellular electron uptake across the outer membrane from both soluble iron and poised electrodes. We observe that PioA undergoes postsecretory proteolysis of its N terminus to produce a shorter heme-attached PioA (holo-PioA, where PioA represents the C terminus of PioA), which can exist both freely in the periplasm and in a complex with PioB. The extended N-terminal peptide controls heme attachment, and its processing is required to produce wild-type levels of holo-PioA and holo-PioAB complex. It is also conserved in PioA homologs from other phototrophs. The presence of PioAB in these organisms correlate with their ability to perform photoferrotrophy and phototrophic EEU. Some anoxygenic phototrophs use soluble iron, insoluble iron minerals (such as rust), or their proxies (poised electrodes) as electron donors for photosynthesis. However, the underlying electron uptake mechanisms are not well established. Here, we show that these phototrophs use a protein complex made of an outer membrane porin and a periplasmic decaheme cytochrome (electron transfer protein) to harvest electrons from both soluble iron and poised electrodes. This complex has two unique characteristics: (i) it lacks an extracellular cytochrome , and (ii) the periplasmic decaheme cytochrome undergoes proteolytic cleavage to produce a functional electron transfer protein. These characteristics are conserved in phototrophs harboring homologous proteins.
Topics: Bacterial Proteins; Biological Transport; Carbon Dioxide; Cytochromes c; Electrodes; Electron Transport; Electrons; Iron; Periplasm; Photosynthesis; Porins; Rhodopseudomonas
PubMed: 31690680
DOI: 10.1128/mBio.02668-19 -
Scientific Reports Apr 2017A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to...
A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining "Structure Intracellular Accumulation Activity Relationship" for the development of new therapeutic candidates.
Topics: Anti-Bacterial Agents; Ceftazidime; Cell Membrane; Gram-Negative Bacteria; Microbial Sensitivity Tests; Microspectrophotometry; Molecular Structure; Periplasm; Permeability
PubMed: 28428543
DOI: 10.1038/s41598-017-00945-8 -
The Journal of Biological Chemistry Nov 2023Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas...
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.
Topics: Acetylation; Alginates; Bacterial Proteins; Biofilms; Periplasm; Protein Processing, Post-Translational; Pseudomonas aeruginosa
PubMed: 37797696
DOI: 10.1016/j.jbc.2023.105314