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Redox Biology Aug 2023The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of...
The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo "steady state" redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was almost fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the steady state periplasmic thiol-redox potential from -228 mV to a more reducing -243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.
Topics: Escherichia coli; Protein Disulfide-Isomerases; Glutathione Disulfide; Periplasm; Protein Folding; Oxidation-Reduction; Glutathione; Proteins; Homeostasis; Disulfides; Sulfhydryl Compounds; Oxidative Stress; Escherichia coli Proteins
PubMed: 37413765
DOI: 10.1016/j.redox.2023.102800 -
BioMed Research International 2015The outer membrane (OM) of Gram-negative bacteria is asymmetric due to the presence of lipopolysaccharide (LPS) facing the outer leaflet of the OM and phospholipids... (Review)
Review
The outer membrane (OM) of Gram-negative bacteria is asymmetric due to the presence of lipopolysaccharide (LPS) facing the outer leaflet of the OM and phospholipids facing the periplasmic side. LPS is essential for bacterial viability, since it provides a permeability barrier and is a major virulence determinant in pathogenic bacteria. In Escherichia coli, several steps of LPS biosynthesis and assembly are regulated by the RpoE sigma factor and stress responsive two-component systems as well as dedicated small RNAs. LPS composition is highly heterogeneous and dynamically altered upon stress and other challenges in the environment because of the transcriptional activation of RpoE regulon members and posttranslational control by RpoE-regulated Hfq-dependent RybB and MicA sRNAs. The PhoP/Q two-component system further regulates Kdo2-lipid A modification via MgrR sRNA. Some of these structural alterations are critical for antibiotic resistance, OM integrity, virulence, survival in host, and adaptation to specific environmental niches. The heterogeneity arises following the incorporation of nonstoichiometric modifications in the lipid A part and alterations in the composition of inner and outer core of LPS. The biosynthesis of LPS and phospholipids is tightly coupled. This requires the availability of metabolic precursors, whose accumulation is controlled by sRNAs like SlrA, GlmZ, and GlmY.
Topics: Escherichia coli; Escherichia coli Proteins; Lipopolysaccharides; Periplasm; Phospholipids; RNA, Small Untranslated; Sigma Factor
PubMed: 26618164
DOI: 10.1155/2015/153561 -
Molecular Microbiology Mar 2021The bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighboring bacterial or eukaryotic cells.... (Review)
Review
The bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighboring bacterial or eukaryotic cells. Antibacterial effectors are mostly toxins that inhibit the growth of other species and help to dominate the niche. A broad variety of these toxins cause cell lysis of the prey cell by disrupting the cell envelope. Other effectors are delivered into the cytoplasm where they affect DNA integrity, cell division or exhaust energy resources. The modular nature of T6SS machinery allows different means of recruitment of toxic effectors to secreted inner tube and spike components that act as carriers. Toxic effectors can be translationally fused to the secreted components or interact with them through specialized structural domains. These interactions can also be assisted by dedicated chaperone proteins. Moreover, conserved sequence motifs in effector-associated domains are subject to genetic rearrangements and therefore engage in the diversification of the arsenal of toxic effectors. This review discusses the diversity of T6SS secreted toxins and presents current knowledge about their loading on the T6SS machinery.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Bacterial Toxins; Conserved Sequence; Cytoplasm; Microbial Interactions; Molecular Chaperones; Periplasm; Protein Domains; Type VI Secretion Systems
PubMed: 33217073
DOI: 10.1111/mmi.14648 -
FEBS Letters Dec 2020To remove xenobiotics from the periplasmic space, Gram-negative bacteria utilise unique tripartite efflux systems in which a molecular engine in the plasma membrane... (Review)
Review
To remove xenobiotics from the periplasmic space, Gram-negative bacteria utilise unique tripartite efflux systems in which a molecular engine in the plasma membrane connects to periplasmic and outer membrane subunits. Substrates bind to periplasmic sections of the engine or sometimes to the periplasmic subunits. Then, the tripartite machines undergo conformational changes that allow the movement of the substrates down the substrate translocation pathway to the outside of the cell. The transmembrane (TM) domains of the tripartite resistance-nodulation-drug-resistance (RND) transporters drive these conformational changes by converting proton motive force into mechanical motion. Similarly, the TM domains of tripartite ATP-binding cassette (ABC) transporters transmit mechanical movement associated with nucleotide binding and hydrolysis at the nucleotide-binding domains to the relevant subunits in the periplasm. In this way, metabolic energy is coupled to periplasmic alternating-access mechanisms to achieve substrate transport across the outer membrane.
Topics: ATP-Binding Cassette Transporters; Bacterial Proteins; Membrane Transport Proteins; Models, Molecular; Multidrug Resistance-Associated Proteins; Periplasm
PubMed: 32936941
DOI: 10.1002/1873-3468.13929 -
Protein Science : a Publication of the... Aug 2017Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically...
Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram-negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram-negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG , that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin-labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA-LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N-terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.
Topics: Binding Sites; Carrier Proteins; Cloning, Molecular; Electron Spin Resonance Spectroscopy; Escherichia coli; Escherichia coli Proteins; Gene Expression; Kinetics; Lipopolysaccharides; Models, Molecular; Mutation; Periplasm; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Tertiary; Recombinant Proteins; Spin Labels
PubMed: 28419595
DOI: 10.1002/pro.3177 -
ACS Infectious Diseases Sep 2020Antibiotic resistance has emerged as a serious threat to global public health in recent years. Lack of novel antimicrobials, especially new classes of compounds, further... (Review)
Review
Antibiotic resistance has emerged as a serious threat to global public health in recent years. Lack of novel antimicrobials, especially new classes of compounds, further aggravates the situation. For Gram-negative bacteria, their double layered cell envelope and an array of efflux pumps act as formidable barriers for antimicrobials to penetrate. While cytoplasmic targets are hard to reach, proteins in the periplasm are clearly more accessible, as the drug only needs to breach the outer membrane. In this review, we summarized recent efforts on the validation and testing of periplasmic proteins as potential antimicrobial targets and the development of related inhibitors that either inhibit the growth of a bacterial pathogen or reduce its virulence during interaction with host cells. We conclude that the periplasm contains a promising pool of novel antimicrobial targets that should be scrutinized more closely for the development of effective treatment against multidrug-resistant Gram-negative bacteria.
Topics: Anti-Bacterial Agents; Bacteria; Drug Resistance, Multiple, Bacterial; Gram-Negative Bacteria; Periplasm
PubMed: 32786281
DOI: 10.1021/acsinfecdis.0c00384 -
Molecular Oral Microbiology Apr 2021Porphyromonas gingivalis, a pathogen of chronic periodontitis, is an asaccharolytic microorganism that solely utilizes nutritional amino acids as its energy source and... (Review)
Review
Porphyromonas gingivalis, a pathogen of chronic periodontitis, is an asaccharolytic microorganism that solely utilizes nutritional amino acids as its energy source and cellular constituents. The bacterium is considered to incorporate proteinaceous nutrients mainly as dipeptides, thus exopeptidases that produce dipeptides from polypeptides are critical for survival and proliferation. We present here an overview of dipeptide production by P. gingivalis mediated by dipeptidyl-peptidases (DPPs), e.g., DPP4, DPP5, DPP7, and DPP11, serine exopeptidases localized in periplasm, which release dipeptides from the N-terminus of polypeptides. Additionally, two other exopeptidases, acylpeptidyl-oligopeptidase (AOP) and prolyl tripeptidyl-peptidase A (PTP-A), which liberate N-terminal acylated di-/tri-peptides and tripeptides with Pro at the third position, respectively, provide polypeptides in an acceptable form for DPPs. Hence, a large fraction of dipeptides is produced from nutritional polypeptides by DPPs with differential specificities in combination with AOP and PTP-A. The resultant dipeptides are then incorporated across the inner membrane mainly via a proton-dependent oligopeptide transporter (POT), a member of the major facilitator superfamily. Recent studies also indicate that DPP4 and DPP7 directly link between periodontal and systemic diseases, such as type 2 diabetes mellitus and coagulation abnormality, respectively. Therefore, these dipeptide-producing and incorporation molecules are considered to be potent targets for prevention and treatment of periodontal and related systemic diseases.
Topics: Diabetes Mellitus, Type 2; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Humans; Periplasm; Porphyromonas gingivalis; Proteins
PubMed: 33006264
DOI: 10.1111/omi.12317 -
Protein Science : a Publication of the... Apr 2023Outer membrane protein (OMP) biogenesis in gram-negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones...
Outer membrane protein (OMP) biogenesis in gram-negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones bind unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress aggregation, facilitate diffusion across the periplasm, and enhance folding. FkpA primarily responds to heat-shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function in the context of OMP folding, we monitored the folding of three OMPs and found that FkpA, unlike other periplasmic chaperones, increases the folded yield but decreases the folding rate of OMPs. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to influence the OMP folding trajectory. Consistent with the folding assay results, FkpA binds all three uOMPs as determined by sedimentation velocity (SV) and photo-crosslinking experiments. We determine the binding affinity between FkpA and uOmpA by globally fitting SV titrations and find it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggesting an extensive binding interface. Initial characterizations of the complex using photo-crosslinking indicate that the binding interface spans the entire FkpA molecule. In contrast to prior findings, folding and binding experiments performed using subdomain constructs of FkpA demonstrate that the full-length chaperone is required for full activity. Together these results support that FkpA has a distinct and direct effect on OMP folding that it achieves by utilizing an extensive chaperone-client interface to tightly bind clients.
Topics: Humans; Carrier Proteins; Peptidylprolyl Isomerase; Escherichia coli Proteins; Escherichia coli; Bacterial Outer Membrane Proteins; Protein Folding; Molecular Chaperones; Periplasm
PubMed: 36775935
DOI: 10.1002/pro.4592 -
Proceedings of the National Academy of... Sep 2021Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel...
Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal-tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near-atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.
Topics: Bacteriophage T7; Capsid; Capsid Proteins; Cell Membrane; Cryoelectron Microscopy; DNA, Viral; Lipid Bilayers; Models, Molecular; Periplasm; Structure-Activity Relationship; Transduction, Genetic; Viral Proteins
PubMed: 34504014
DOI: 10.1073/pnas.2102003118 -
Philosophical Transactions of the Royal... Oct 2015Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering... (Review)
Review
Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Biological Transport, Active; Cell Membrane; Gram-Negative Bacteria; Lipopolysaccharides; Membrane Lipids; Models, Biological; Models, Molecular; Periplasm
PubMed: 26370939
DOI: 10.1098/rstb.2015.0027