scholarly journals Uptake of monoaromatic hydrocarbons during biodegradation by FadL channel-mediated lateral diffusion

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kamolrat Somboon ◽  
Anne Doble ◽  
David Bulmer ◽  
Arnaud Baslé ◽  
Syma Khalid ◽  
...  
Keyword(s):  
Channel Wall ◽  
Lateral Diffusion ◽  
Computational Approach ◽  
Facilitated Diffusion ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Modus Operandi ◽  
Gram Negative ◽  
Hydrophobic Pollutants ◽  
Monoaromatic Hydrocarbons

AbstractIn modern societies, biodegradation of hydrophobic pollutants generated by industry is important for environmental and human health. In Gram-negative bacteria, biodegradation depends on facilitated diffusion of the pollutant substrates into the cell, mediated by specialised outer membrane (OM) channels. Here we show, via a combined experimental and computational approach, that the uptake of monoaromatic hydrocarbons such as toluene in Pseudomonas putida F1 (PpF1) occurs via lateral diffusion through FadL channels. Contrary to classical diffusion channels via which polar substrates move directly into the periplasmic space, PpF1 TodX and CymD direct their hydrophobic substrates into the OM via a lateral opening in the channel wall, bypassing the polar barrier formed by the lipopolysaccharide leaflet on the cell surface. Our study suggests that lateral diffusion of hydrophobic molecules is the modus operandi of all FadL channels, with potential implications for diverse areas such as biodegradation, quorum sensing and gut biology.

scholarly journals Uptake of monoaromatic hydrocarbons during biodegradation by FadL channel-mediated lateral diffusion

2020 ◽  
Author(s):  
Kamolrat Somboon ◽  
Anne Doble ◽  
David Bulmer ◽  
Arnaud Baslé ◽  
Syma Khalid ◽  
...  
Keyword(s):  
Channel Wall ◽  
Lateral Diffusion ◽  
Computational Approach ◽  
Facilitated Diffusion ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Modus Operandi ◽  
Gram Negative ◽  
Hydrophobic Pollutants ◽  
Monoaromatic Hydrocarbons

AbstractIn modern societies, biodegradation of hydrophobic pollutants generated by industry is important for environmental and human health. In Gram-negative bacteria, biodegradation depends on facilitated diffusion of the pollutant substrates into the cell, mediated by specialised outer membrane (OM) channels. Here we show, via a combined experimental and computational approach, that the uptake of monoaromatic hydrocarbons such as toluene in Pseudomonas putida F1 (PpF1) occurs via lateral diffusion through FadL channels. Contrary to classical diffusion channels via which polar substrates move directly into the periplasmic space, PpF1 TodX and CymD direct their hydrophobic substrates into the OM via a lateral opening in the channel wall, bypassing the polar barrier formed by the lipopolysaccharide leaflet on the cell surface. Our study suggests that lateral diffusion of hydrophobic molecules is the modus operandi of all FadL channels, with potential implications for diverse areas such as biodegradation, quorum sensing and gut biology.

Ultrastructure of periplasmic bodies in gram-negative bacteria

1990 ◽  
Vol 48 (3) ◽  
pp. 640-641
Author(s):  
Xie Nianming ◽  
Ding Shaoqing ◽  
Wang Luping ◽  
Yuan Zenglin ◽  
Zhan Guolai ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Campylobacter Jejuni ◽  
Proteus Mirabilis ◽  
Shigella Flexneri ◽  
Morphological Description ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Clostridium Tetani ◽  
Gram Negative ◽  
Brucella Canis

Perhaps the data about periplasmic enzymes are obtained through biochemical methods but lack of morphological description. We have proved the existence of periplasmic bodies by electron microscope and described their ultrastructures. We hope this report may draw the attention of biochemists and mrophologists to collaborate on researches in periplasmic enzymes or periplasmic bodies with each other.One or more independent bodies may be seen in the periplasmic space between outer and inner membranes of Gram-negative bacteria, which we called periplasmic bodies. The periplasmic bodies have been found in seven species of bacteria at least, including the Pseudomonas aeroginosa. Shigella flexneri, Echerichia coli. Yersinia pestis, Campylobacter jejuni, Proteus mirabilis, Clostridium tetani. Vibrio cholerae and Brucella canis.

scholarly journals A novel chemical biology and computational approach to expedite the discovery of new-generation polymyxins against life-threatening Acinetobacter baumannii

2021 ◽  
Author(s):  
Xukai Jiang ◽  
Nitin A. Patil ◽  
Mohammad A. K. Azad ◽  
Hasini Wickremasinghe ◽  
Heidi Yu ◽  
...  
Keyword(s):  
Public Health ◽  
Chemical Biology ◽  
Multidrug Resistant ◽  
Computational Approach ◽  
Gram Negative Bacteria ◽  
Global Public Health ◽  
Gram Negative ◽  
Life Threatening ◽  
New Generation ◽  
Novel Antibiotics

Multidrug-resistant Gram-negative bacteria have been an urgent threat to global public health. Novel antibiotics are desperately needed to combat these 'superbugs'.

Bacterial cleanup: lateral diffusion of hydrophobic molecules through protein channel walls

2010 ◽  
Vol 1 (3-4) ◽  
pp. 263-270 ◽  
Author(s):  
Bert van den Berg
Keyword(s):  
Diffusion Mechanism ◽  
Lateral Diffusion ◽  
Biofuel Production ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Hydrophobic Compounds ◽  
Protein Channel ◽  
Biochemical Research ◽  
Established Role ◽  
Hydrophilic And Hydrophobic Compounds

AbstractThe outer membrane (OM) of Gram-negative bacteria forms a very efficient barrier against the permeation of both hydrophilic and hydrophobic compounds, owing to the presence of lipopolysaccharides on the outside of the cell. Although much is known about the OM passage of hydrophilic molecules, it is much less clear how hydrophobic molecules cross this barrier. Members of the FadL channel family, which are widespread in Gram-negative bacteria, are so far the only proteins with an established role in the uptake of hydrophobic molecules across the OM. Recent structural and biochemical research has shown that these channels operate according to a unique lateral diffusion mechanism, in which the substrate moves from the lumen of the barrel into the OM via an unusual opening in the wall of the barrel. Understanding how hydrophobic molecules cross the OM is not only of fundamental importance but could also have applications in the design of novel, hydrophobic drugs, biofuel production and the generation of more efficient bacterial biodegrader strains.

scholarly journals The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

2021 ◽  
Vol 119 (1) ◽  
pp. e2114997119
Author(s):  
Ashton N. Combs ◽  
Thomas J. Silhavy
Keyword(s):  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Adaptor Protein ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Timely Manner ◽  
Gram Negative ◽  
Bam Complex ◽  
Periplasmic Chaperone ◽  
Chaperone Network

The biogenesis of integral β-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the β-barrel assembly machine (Bam) complex. We find that β-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam–OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

scholarly journals Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
J. Wendler ◽  
D. Ehmann ◽  
L. Courth ◽  
B. O. Schroeder ◽  
N. P. Malek ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Outer Membrane ◽  
Specific Activity ◽  
Redox System ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Redox Systems ◽  
Native Form ◽  
Gram Negative ◽  
Content Type

ABSTRACTThe antimicrobial peptide human β-defensin 1 (hBD1) is continuously produced by epithelial cells in many tissues. Compared to other defensins, hBD1 has only minor antibiotic activity in its native state. After reduction of its disulfide bridges, however, it becomes a potent antimicrobial agent against bacteria, while the oxidized native form (hBD1ox) shows specific activity against Gram-negative bacteria. We show that the killing mechanism of hBD1ox depends on aerobic growth conditions and bacterial enzymes. We analyzed the different activities of hBD1 using mutants ofEscherichia colilacking one or more specific proteins of their outer membrane, cytosol, or redox systems. We discovered that DsbA and DsbB are essential for the antimicrobial activity of hBD1ox but not for that of reduced hBD1 (hBD1red). Furthermore, our results strongly suggest that hBD1ox uses outer membrane protein FepA to penetrate the bacterial periplasm space. In contrast, other bacterial proteins in the outer membrane and cytosol did not modify the antimicrobial activity. Using immunogold labeling, we identified the localization of hBD1ox in the periplasmic space and partly in the outer membrane ofE. coli. However, in resistant mutants lacking DsbA and DsbB, hBD1ox was detected mainly in the bacterial cytosol. In summary, we discovered that hBD1ox could use FepA to enter the periplasmic space, where its activity depends on presence of DsbA and DsbB. HBD1ox concentrates in the periplasm in Gram-negative bacteria, which finally leads to bleb formation and death of the bacteria. Thus, the bacterial redox system plays an essential role in mechanisms of resistance against host-derived peptides such as hBD1.

scholarly journals Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Förster Resonance Energy Transfer Assay in Escherichia coli

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Nils Y. Meiresonne ◽  
René van der Ploeg ◽  
Mark A. Hink ◽  
Tanneke den Blaauwen
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gram Negative Bacteria ◽  
Periplasmic Protein ◽  
Periplasmic Space ◽  
Gram Negative ◽  
Conformation Changes ◽  
Antibiotic Screening

ABSTRACT One of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies. IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies.

scholarly journals Mechanistic aspects of maltotriose-conjugate translocation to the Gram-negative bacteria cytoplasm

2018 ◽  
Vol 2 (1) ◽  
pp. e201800242 ◽  
Author(s):  
Estelle Dumont ◽  
Julia Vergalli ◽  
Jelena Pajovic ◽  
Satya P Bhamidimarri ◽  
Koldo Morante ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Small Molecule ◽  
Single Channel ◽  
Efflux Pumps ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Gram Negative ◽  
Entry Pathway ◽  
Pass Through ◽  
Novel Antibiotics

Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin–fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of theEscherichia colimaltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathwayviathe activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.

scholarly journals Structure and Stoichiometry of the Ton Molecular Motor

2020 ◽  
Vol 21 (2) ◽  
pp. 375 ◽  
Author(s):  
Herve Celia ◽  
Nicholas Noinaj ◽  
Susan K Buchanan
Keyword(s):  
Outer Membrane ◽  
Mode Of Action ◽  
Molecular Motor ◽  
Proton Gradient ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Inner Membrane ◽  
Gram Negative ◽  
X Ray ◽  
X Ray Crystallography

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite decades of investigation and the recent flurry of structures being reported by X-ray crystallography and cryoEM, the mode of action of the Ton molecular motor has remained elusive, and the precise stoichiometry of its subunits is still a matter of debate. This review summarizes the latest findings on the Ton system by presenting the recently reported structures and related reports on the stoichiometry of the fully assembled complex.

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