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Biochemical Society Transactions Dec 2012Bacterial anaerobic respiration using selenium oxyanions as the sole electron acceptor primarily result in the precipitation of selenium biominerals observed as either... (Review)
Review
Bacterial anaerobic respiration using selenium oxyanions as the sole electron acceptor primarily result in the precipitation of selenium biominerals observed as either intracellular or extracellular selenium deposits. Although a better understanding of the enzymology of bacterial selenate reduction is emerging, the processes by which the selenium nanospheres are constructed, and in some cases secreted, has remained poorly studied. Thauera selenatis is a Gram-negative betaproteobacterium that is capable of respiring selenate due to the presence of a periplasmic selenate reductase (SerABC). SerABC is a molybdoenzyme that catalyses the reduction of selenate to selenite by accepting electrons from the Q-pool via a dihaem c-type cytochrome (cytc4). The product selenite is presumed to be reduced in the cytoplasm, forming intracellular selenium nanospheres that are ultimately secreted into the surrounding medium. The secretion of the selenium nanospheres is accompanied by the export of a ~95 kDa protein SefA (selenium factor A). SefA has no cleavable signal peptide, suggesting that it is also exported directly for the cytoplasmic compartment. It has been suggested that SefA functions to stabilize the formation of the selenium nanospheres before secretion, possibly providing reaction sites for selenium nanosphere creation or providing a shell to prevent subsequent selenium aggregation. The present paper draws on our current knowledge of selenate respiration and selenium biomineralization in T. selenatis and other analogous systems, and extends the application of nanoparticle tracking analysis to determine the size distribution profile of the selenium nanospheres secreted.
Topics: Amino Acid Sequence; Bacterial Proteins; Chemical Precipitation; Electron Transport; Molecular Sequence Data; Nanospheres; Oxidation-Reduction; Oxidoreductases; Periplasm; Selenium; Sequence Homology, Amino Acid; Thauera
PubMed: 23176461
DOI: 10.1042/BST20120087 -
Methods in Molecular Biology (Clifton,... 2013Many proteins secreted to the bacterial cell envelope contain cysteine residues that are involved in disulfide bonds. These disulfides either play a structural role,...
Many proteins secreted to the bacterial cell envelope contain cysteine residues that are involved in disulfide bonds. These disulfides either play a structural role, increasing protein stability, or reversibly form in the catalytic site of periplasmic oxidoreductases. Monitoring the in vivo redox state of cysteine residues, i.e., determining whether those cysteines are oxidized to a disulfide bond or not, is therefore required to fully characterize the function and structural properties of numerous periplasmic proteins. Here, we describe a reliable and rapid method based on trapping reduced cysteine residues with 4'-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), a maleimide compound. We use the Escherichia coli DsbA protein to illustrate the method, which can be applied to all envelope proteins.
Topics: Chromatography, Gel; Cysteine; Disulfides; Electrophoresis, Polyacrylamide Gel; Oxidation-Reduction; Periplasm; Proteins; Spectrophotometry, Ultraviolet
PubMed: 23299744
DOI: 10.1007/978-1-62703-245-2_20 -
Nature Sep 2017Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet...
Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.
Topics: ATP-Binding Cassette Transporters; Adenosine Diphosphate; Bacterial Proteins; Biological Transport; Cell Membrane; Cryoelectron Microscopy; Escherichia coli; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Lipopolysaccharides; Models, Molecular; Nanostructures; Periplasm; Protein Binding; Protein Domains
PubMed: 28869968
DOI: 10.1038/nature23649 -
Extremophiles : Life Under Extreme... Jul 2017Thermotogales are rod-shaped, Gram-negative, anaerobic, (hyper) thermophiles distinguished by an outer sheath-like toga, which comprises an outer membrane (OM) and an...
Thermotogales are rod-shaped, Gram-negative, anaerobic, (hyper) thermophiles distinguished by an outer sheath-like toga, which comprises an outer membrane (OM) and an amorphous layer (AL). Thermosipho globiformans bacteria can transform into spheroids with multiple cells concurrently with AL disintegration during early growth; the cell is defined as the cytoplasmic membrane (CM) plus the entity surrounded by the CM. Spheroids eventually produce rapidly moving periplasmic 'progenies' through an unknown mechanism. Here, we used high-temperature microscopy (HTM) to directly observe spheroid generation and growth. Rod OMs abruptly inflated to form ~2 μm-diameter balloons. Concurrently, multiple globular cells emerged in the balloons, suggesting their translocation and transformation from the rod state. During spheroid growth, the cells elongated and acquired a large dish shape by possible fusion. Spheroids with dish-shaped cells further enlarged to ~12 μm in diameter. HTM and epifluorescence-microscopy results collectively indicated that the nucleoids of dish-shaped cells transformed to form a ring shape, which then distorted to form a lip shape as the spheroid enlarged. HTM showed that 'progenies' were produced in the spheroid periplasm. Transmission electron microscopy results suggested that the 'progenies' represented immature progenies lacking togas, which were acquired subsequently.
Topics: Bacteria; Microscopy; Periplasm; Temperature
PubMed: 28577249
DOI: 10.1007/s00792-017-0944-0 -
Journal of Bacteriology Nov 2008Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive...
Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.e., the periplasm), it is still unclear what structures (or mechanism) sustain a gram-positive periplasmic space. Here we have used cryo-EM of frozen-hydrated sections in combination with various labels to probe the model gram-positive organism Bacillus subtilis for major periplasmic components. Incubation of cells with positively charged gold nanoparticles showed almost similar levels of gold binding to the periplasm and the cell wall. On cells whose cell walls were enzymatically hydrolyzed (i.e., on protoplasts), a surface diffuse layer extending approximately 30 nm from the membrane was revealed. The thickness and density of this layer were not significantly altered after treatment with a nonspecific protease, whereas it was labeled with anti-lipoteichoic acid (LTA) antibodies conjugated to nanogold. Further, the LTA layer spans most of the thickness of the periplasmic space, which strongly suggests that LTA is a major component of the B. subtilis periplasm.
Topics: Bacillus subtilis; Cell Wall; Cryoelectron Microscopy; Lipopolysaccharides; Periplasm; Teichoic Acids
PubMed: 18790869
DOI: 10.1128/JB.00581-08 -
Methods in Molecular Biology (Clifton,... 2011While Escherichia coli is in wide use as a host organism for preparative protein production, problems with the folding of the recombinant gene product as well as protein... (Review)
Review
While Escherichia coli is in wide use as a host organism for preparative protein production, problems with the folding of the recombinant gene product as well as protein aggregation, i.e., formation of inclusion bodies, are frequently encountered. This is particularly true for proteins that carry structural disulfide bonds, including antibody fragments, cytokines, growth factors, and extracellular fragments of eukaryotic cell surface receptors. In these cases, secretion into the oxidizing milieu of the bacterial periplasm in principle enables disulfide bond formation, resulting in a correctly folded and soluble protein. However, this process often occurs at low efficiency, depending on the nature of the recombinant gene product. Therefore, we have developed the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and/or folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC, which catalyze the formation and isomerization of disulfide bridges, and two peptidyl-prolyl cis/trans isomerases with chaperone activity, FkpA and SurA. Here, we present a detailed protocol how to use this system for the bacterial secretion of recombinant proteins, including human EGF as a new example, and we give hints on optimization of the expression procedure.
Topics: Disulfides; Epidermal Growth Factor; Escherichia coli; Escherichia coli Proteins; Humans; Molecular Chaperones; Oxidation-Reduction; Periplasm; Periplasmic Proteins; Plasmids; Protein Disulfide-Isomerases; Protein Folding; Recombinant Proteins
PubMed: 21125388
DOI: 10.1007/978-1-61737-967-3_12 -
Critical Reviews in Microbiology 1998When subject to an osmotic 'up-shock', water flows outward from bacterial cytoplasm of the bacterium. Lipid bilayers can shrink very little in area and therefore must... (Review)
Review
When subject to an osmotic 'up-shock', water flows outward from bacterial cytoplasm of the bacterium. Lipid bilayers can shrink very little in area and therefore must wrinkle to accommodate the smaller volume. The usual consequence is that all the layers of the cell envelope must become wrinkled together because they adhere to each other and must now cover a smaller surface. Plasmolysis spaces are formed if the cytoplasmic membrane (CM) separates from the other components of the wall. However, because the CM bilayer is essentially an incompressible two-dimensional liquid, this constraint restricts the location and shape of plasmolysis spaces. With mild up-shocks they form at the pole and around constricting regions in the cell. Elsewhere their creation requires the formation of endocytotic or exocytotic vesicles. The formation of endocytotic vesicles occurs in animal and plant cells as well as in bacterial cells. With stronger up-shocks tubular structures (Bayer adhesion sites), or other special geometric shapes (e.g., Scheie structures) allow the bilayer to surround an irregular shaped cytoplast. Periosmotic agents, that is, those that extract water from the periplasm as well as the cytoplasm, are molecules such as poly-vinyl-pyrrolidone and alpha-cyclodextrin that are too large to pass through the porins in the outer membrane. They were found to significantly inhibit the formation of plasmolysis spaces. Presumably, they inhibit the plasmolysis process, which requires that extracellular fluid enter between the CM and the outer membrane (OM). In the extreme case, with the dehydrating action of both osmotic agents and periosmotic agents, periplasmic space formation tends to be prevented and a new kind of space develops within the cytoplasm. We have designated these as 'cytoplasmic voids'. These novel structures are not bounded by lipid bilayers, in contrast to the endocytotic vesicles. These new spaces appear to result from the negative turgor pressure generated by the application of the combination of osmotic and periosmotic agents causing bubble formation. Several ideas in the literature about the wall biology (periseptal annuli, leading edge, osmotic pressure in the periplasm) are presented and critiqued. The basic criticism of these is that much of the phenomena can be explained because of the physics of the phospholipid bilayers and osmotic forces and thus does not imply the existence of a special control mechanism to regulate growth and division.
Topics: Biophysical Phenomena; Biophysics; Cell Membrane Permeability; Gram-Negative Bacteria; Microscopy, Electron; Osmotic Pressure; Periplasm
PubMed: 9561823
DOI: 10.1080/10408419891294172 -
New Biotechnology Nov 2023The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide...
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.
Topics: Periplasmic Proteins; Escherichia coli; Periplasm; Biological Products; Recombinant Proteins
PubMed: 37708933
DOI: 10.1016/j.nbt.2023.09.003 -
Proceedings of the National Academy of... Apr 2013Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often...
Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often associated with the ordered conformation, for some of these conditionally unstructured proteins, the opposite applies: Their activation is associated with their unfolding. An example is the small periplasmic chaperone HdeA, which is critical for the ability of enteric bacterial pathogens like Escherichia coli to survive passage through extremely acidic environments, such as the human stomach. At neutral pH, HdeA is a chaperone-inactive dimer. On a shift to low pH, however, HdeA monomerizes, partially unfolds, and becomes rapidly active in preventing the aggregation of substrate proteins. By mutating two aspartic acid residues predicted to be responsible for the pH-dependent monomerization of HdeA, we have succeeded in isolating an HdeA mutant that is active at neutral pH. We find this HdeA mutant to be substantially destabilized, partially unfolded, and mainly monomeric at near-neutral pH at a concentration at which it prevents aggregation of a substrate protein. These results provide convincing evidence for direct activation of a protein by partial unfolding.
Topics: Amino Acid Sequence; Base Sequence; Circular Dichroism; Escherichia coli Proteins; Hydrogen-Ion Concentration; Models, Molecular; Molecular Chaperones; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis; Periplasm; Protein Binding; Protein Conformation; Protein Unfolding; Sequence Alignment; Sequence Analysis, DNA; Ultracentrifugation
PubMed: 23487787
DOI: 10.1073/pnas.1222458110 -
The Journal of Biological Chemistry May 2012The Escherichia coli NhaA antiporter couples the transport of H(+) and Na(+) (or Li(+)) ions to maintain the proper pH range and Na(+) concentration in cells. A crystal...
The Escherichia coli NhaA antiporter couples the transport of H(+) and Na(+) (or Li(+)) ions to maintain the proper pH range and Na(+) concentration in cells. A crystal structure of NhaA, solved at pH 4, comprises 12 transmembrane helices (TMs), arranged in two domains, with a large cytoplasm-facing funnel and a smaller periplasm-facing funnel. NhaA undergoes conformational changes, e.g. after pH elevation to alkaline ranges, and we used two computational approaches to explore them. On the basis of pseudo-symmetric features of the crystal structure, we predicted the structural architecture of an alternate, periplasm-facing state. In contrast to the crystal structure, the model presents a closed cytoplasmic funnel, and a periplasmic funnel of greater volume. To examine the transporter functional direction of motion, we conducted elastic network analysis of the crystal structure and detected two main normal modes of motion. Notably, both analyses predicted similar trends of conformational changes, consisting of an overall rotational motion of the two domains around a putative symmetry axis at the funnel centers, perpendicular to the membrane plane. This motion, along with conformational changes within specific helices, resulted in closure at the cytoplasmic end and opening at the periplasmic end. Cross-linking experiments, performed between segments on opposite sides of the cytoplasmic funnel, revealed pH-dependent interactions consistent with the proposed conformational changes. We suggest that the model-structure and predicted motion represent alkaline pH-induced conformational changes, mediated by a cluster of evolutionarily conserved, titratable residues, at the cytoplasmic ends of TMs II, V, and IX.
Topics: Escherichia coli Proteins; Hydrogen-Ion Concentration; Models, Molecular; Periplasm; Protein Conformation; Sodium-Hydrogen Exchangers
PubMed: 22431724
DOI: 10.1074/jbc.M111.336446