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Journal of Bacteriology Sep 2018Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to... (Review)
Review
Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to secrete an array of biologically diverse proteins-from small, <10-kDa bacteriocins to gigantic adhesins with a mass >1 MDa. For the last several decades, T1SSs have been characterized as a one-step translocation strategy whereby the secreted substrate is transported directly into the extracellular environment from the cytoplasm with no periplasmic intermediate. Recent phylogenetic, biochemical, and genetic evidences point to a distinct subgroup of T1SS machinery linked with a bacterial transglutaminase-like cysteine proteinase (BTLCP), which uses a two-step secretion mechanism. BTLCP-linked T1SSs transport a class of repeats-in-toxin (RTX) adhesins that are critical for biofilm formation. The prototype of this RTX adhesin group, LapA of Pf0-1, uses a novel N-terminal retention module to anchor the adhesin at the cell surface as a secretion intermediate threaded through the outer membrane-localized TolC-like protein LapE. This secretion intermediate is posttranslationally cleaved by the BTLCP family LapG protein to release LapA from its cognate T1SS pore. Thus, the secretion of LapA and related RTX adhesins into the extracellular environment appears to be a T1SS-mediated two-step process that involves a periplasmic intermediate. In this review, we contrast the T1SS machinery and substrates of the BLTCP-linked two-step secretion process with those of the classical one-step T1SS to better understand the newly recognized and expanded role of this secretion machinery.
Topics: Adhesins, Bacterial; Bacterial Proteins; Biofilms; Cell Membrane; Computational Biology; Cysteine Proteases; Periplasm; Phylogeny; Pseudomonas fluorescens; Transglutaminases; Type I Secretion Systems
PubMed: 29866808
DOI: 10.1128/JB.00168-18 -
BioMed Research International 2019All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable... (Review)
Review
All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an "off" to an "on" state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.
Topics: Biosensing Techniques; Periplasm; Periplasmic Binding Proteins; Protein Domains; Protein Engineering
PubMed: 30719443
DOI: 10.1155/2019/4798793 -
MBio Jun 2021We demonstrate here that the acquisition of DNase resistance by transforming DNA, often assumed to indicate transport to the cytoplasm, reflects uptake to the periplasm,...
We demonstrate here that the acquisition of DNase resistance by transforming DNA, often assumed to indicate transport to the cytoplasm, reflects uptake to the periplasm, requiring a reevaluation of conclusions about the roles of several proteins in transformation. The new evidence suggests that the transformation pilus is needed for DNA binding to the cell surface near the cell poles and for the initiation of uptake. The cellular distribution of the membrane-anchored ComEA of Bacillus subtilis does not dramatically change during DNA uptake as does the unanchored ComEA of and . Instead, our evidence suggests that ComEA stabilizes the attachment of transforming DNA at localized regions in the periplasm and then mediates uptake, probably by a Brownian ratchet mechanism. Following that, the DNA is transferred to periplasmic portions of the channel protein ComEC, which plays a previously unsuspected role in uptake to the periplasm. We show that the transformation endonuclease NucA also facilitates uptake to the periplasm and that the previously demonstrated role of ComFA in the acquisition of DNase resistance derives from the instability of ComGA when ComFA is deleted. These results prompt a new understanding of the early stages of DNA uptake for transformation. Transformation is a widely distributed mechanism of bacterial horizontal gene transfer that plays a role in the spread of antibiotic resistance and virulence genes and more generally in evolution. Although transformation was discovered nearly a century ago and most, if not all the proteins required have been identified in several bacterial species, much remains poorly understood about the molecular mechanism of DNA uptake. This study uses epifluorescence microscopy to investigate the passage of labeled DNA into the compartment between the cell wall and the cell membrane of Bacillus subtilis, a necessary early step in transformation. The roles of individual proteins in this process are identified, and their modes of action are clarified.
Topics: Bacillus subtilis; Biological Transport; Cell Membrane; DNA, Bacterial; Membrane Proteins; Periplasm; Transformation, Bacterial
PubMed: 34126763
DOI: 10.1128/mBio.01061-21 -
Microbial Cell Factories Mar 2016Protein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies. In this approach, a signal...
BACKGROUND
Protein secretion to the periplasm of Escherichia coli offers an attractive route for producing heterologous proteins including antibodies. In this approach, a signal peptide is fused to the N-terminus of the heterologous protein. The signal peptide mediates translocation of the heterologous protein from the cytoplasm to the periplasm and is cleaved during the translocation process. It was previously shown that optimization of the translation initiation region (TIR) which overlaps with the nucleotide sequence of the signal sequence improves the production of heterologous proteins. Despite the progress, there is still room to improve yields using secretion as a means to produce protein complexes such as full-length monoclonal antibodies (mAbs).
RESULTS
In this study we identified the inefficient secretion of heavy chain as the limitation for full-length mAb accumulation in the periplasm. To improve heavy chain secretion we investigated the effects of various signal peptides at controlled TIR strengths. The signal peptide of disulfide oxidoreductase (DsbA) mediated more efficient secretion of heavy chain than the other signal peptides tested. Mutagenesis studies demonstrated that at controlled translational levels, hydrophobicity of the hydrophobic core (H-region) of the signal peptide is a critical factor for heavy chain secretion and full-length mAb accumulation in the periplasm. Increasing the hydrophobicity of a signal peptide enhanced heavy chain secretion and periplasmic levels of assembled full-length mAbs, while decreasing the hydrophobicity had the opposite effect.
CONCLUSIONS
This study demonstrates that under similar translational strengths, the hydrophobicity of the signal peptide plays an important role in heavy chain secretion. Increasing the hydrophobicity of the H-region and controlling TIR strengths can serve as an approach to improve heavy chain secretion and full-length mAb production in E. coli.
Topics: Amino Acid Sequence; Antibodies; Antibodies, Monoclonal; Escherichia coli; Hydrophobic and Hydrophilic Interactions; Immunoglobulin Heavy Chains; Inclusion Bodies; Molecular Sequence Data; Peptide Chain Initiation, Translational; Periplasm; Protein Engineering; Protein Sorting Signals
PubMed: 26935575
DOI: 10.1186/s12934-016-0445-3 -
Molecular Microbiology Sep 2018Shewanella oneidensis is the best understood model organism for the study of dissimilatory iron reduction. This review focuses on the current state of our knowledge... (Review)
Review
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.
Topics: Cell Membrane; Cytochromes; Electron Transport; Electrons; Flavins; Heme; Iron; Oxygen; Periplasm; Shewanella; Succinate Dehydrogenase
PubMed: 29995975
DOI: 10.1111/mmi.14067 -
Scientific Reports Oct 2020OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including...
OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.
Topics: Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Periplasm; Porins; Protein Transport; SEC Translocation Channels
PubMed: 33004891
DOI: 10.1038/s41598-020-73185-y -
Journal of Structural Biology Sep 2022The CusS histidine kinase is a member of Escherichia coli two-component signal transduction system, engaged in a response to copper ions excess in the cell periplasm....
The CusS histidine kinase is a member of Escherichia coli two-component signal transduction system, engaged in a response to copper ions excess in the cell periplasm. The periplasmic sensor domain of CusS binds the free copper ions and the CusS kinase core phosphorylates the cognate CusR which regulates transcription of the efflux pomp CusCBA. A small amount of copper ions is indispensable for the aerobic cell metabolism. Nonetheless, its excess in the cytoplasm generates damaging and reactive hydroxyl radicals. For that reason, understanding the bacterial copper sensing mechanisms can contribute to reducing bacterial copper-resistance and developing bactericidal copper-based materials. The crystal structure of the CusS kinase core was solved at the resolution of 1.4 Å. The cytoplasmic catalytic core domains formed a homodimer. Based on the obtained structure, the intramolecular and intermolecular interactions crucial for the mechanism of CusS autophosphorylation were described.
Topics: Copper; Escherichia coli; Escherichia coli Proteins; Histidine Kinase; Periplasm
PubMed: 35907487
DOI: 10.1016/j.jsb.2022.107883 -
Cell Jul 2016It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the...
It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client's affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins.
Topics: Carrier Proteins; Entropy; Escherichia coli; Escherichia coli Proteins; Hydrophobic and Hydrophilic Interactions; Molecular Chaperones; Periplasm; Periplasmic Proteins; Protein Folding; Static Electricity
PubMed: 27293188
DOI: 10.1016/j.cell.2016.05.054 -
Scientific Reports Sep 2018The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal...
The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal structures have trapped the protein in sugar-bound states facing the periplasm, but with narrow openings unable to accommodate sugar. Therefore, the molecular details of sugar uptake remain elusive. In this work, we have used extended simulations and metadynamics sampling to explore a putative sugar-uptake pathway and associated free energy landscape. We found an entrance at helix-pair 2 and 11, which involved lipid head groups and residues Gln 241 and Gln 359. Furthermore, the protein displayed high flexibility on the periplasmic side of Phe 27, which is located at the narrowest section of the pathway. Interactions to Phe 27 enabled passage into the binding site, which was associated with a 24 ± 4 kJ/mol binding free energy in excellent agreement with an independent binding free energy calculation and experimental data. Two free energy minima corresponding to the two possible binding poses of the lactose analog β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) were aligned with the crystal structure-binding pocket. This work outlines the chemical environment of a putative periplasmic sugar pathway and paves way for understanding substrate affinity and specificity in LacY.
Topics: Cell Membrane; Lipid Bilayers; Membrane Transport Proteins; Molecular Dynamics Simulation; Periplasm; Protein Conformation; Protein Transport; Thermodynamics
PubMed: 30254312
DOI: 10.1038/s41598-018-32624-7 -
MBio Oct 2022In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM)...
In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein Wza of Escherichia coli is an octamer in which the eight C-terminal domains form an α-helical OM pore and the eight copies of the three N-terminal domains (D1 to D3) form a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18-stranded β-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS (exopolysaccharide) is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments, phylogenomics, and computational structural biology, we identify and characterize EpsX as an OM 18-stranded β-barrel protein important for EPS synthesis and identify AlgE, a β-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, consists only of the periplasmic D1 and D2 domains and completely lacks the domain for spanning the OM (herein termed a OPX protein). , EpsX and EpsY mutually stabilize each other and interact in pulldown experiments supporting their direct interaction. Based on these observations, we propose that EpsY and EpsX make up and represent a third type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning β-barrel translocon together with the periplasmic OPX protein EpsY. Based on computational genomics, similar composite systems are widespread in Gram-negative bacteria. Bacteria secrete a wide variety of polysaccharides that have critical functions in, e.g., fitness, surface colonization, and biofilm formation and in beneficial and pathogenic human-, animal-, and plant-microbe interactions. In Gram-negative bacteria, export of these chemically diverse polysaccharides across the outer membrane depends on two known translocons, i.e., an outer membrane OPX protein in Wzx/Wzy- and ABC transporter-dependent pathways and an outer membrane 16- to 18-stranded β-barrel protein in synthase-dependent pathways. Here, using a combination of experiments in Myxococcus xanthus, phylogenomics, and computational structural biology, we provide evidence supporting that a third type of translocon can export polysaccharides across the outer membrane. Specifically, in this translocon, an outer membrane-spanning β-barrel protein functions together with an entirely periplasmic OPX protein that completely lacks the domain for spanning the OM. Computational genomics support that similar composite systems are widespread in Gram-negative bacteria.
Topics: ATP-Binding Cassette Transporters; Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Periplasm; Polysaccharides, Bacterial
PubMed: 35972145
DOI: 10.1128/mbio.02032-22