Comparing Inner and Outer Membrane Permeabilities for Lignin Related Aromatic Compounds

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [14C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strain’s ability to utilize aromatics and organic acids over glucose.

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Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921073117 ◽

2020 ◽

Vol 117 (17) ◽

pp. 9302-9310 ◽

Cited By ~ 7

Author(s):

Davinia Salvachúa ◽

Allison Z. Werner ◽

Isabel Pardo ◽

Martyna Michalska ◽

Brenna A. Black ◽

...

Keyword(s):

Pseudomonas Putida ◽

Outer Membrane ◽

Aromatic Compounds ◽

Value Added ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Pseudomonas Putida Kt2440 ◽

Rich Media

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

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Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds

10.1101/2020.03.26.005173 ◽

2020 ◽

Author(s):

Masaya Fujita ◽

Taichi Sakumoto ◽

Kenta Tanatani ◽

Hong Yang Yu ◽

Kosuke Mori ◽

...

Keyword(s):

Outer Membrane ◽

Aromatic Compounds ◽

Iron Uptake ◽

Iron Acquisition ◽

Essential Element ◽

Acquisition System ◽

Bacterial Degradation ◽

Iron Acquisition System ◽

Iron Transporter ◽

Outer Membrane Transporters

AbstractIron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34550), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.

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Faculty Opinions recommendation of Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737680885.793573231 ◽

2020 ◽

Author(s):

Gail Preston

Keyword(s):

Pseudomonas Putida ◽

Outer Membrane ◽

Aromatic Compounds ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Pseudomonas Putida Kt2440

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Outer membrane permeabilities in the presence of β-lactam antibiotics

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/23.5.793-a ◽

1989 ◽

Vol 23 (5) ◽

pp. 793-794

Author(s):

WRIGHT W. NICHOLS ◽

R. GLYN HEWINSON

Keyword(s):

Outer Membrane ◽

Membrane Permeabilities

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Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds

Scientific Reports ◽

10.1038/s41598-020-68984-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Masaya Fujita ◽

Taichi Sakumoto ◽

Kenta Tanatani ◽

HongYang Yu ◽

Kosuke Mori ◽

...

Keyword(s):

Outer Membrane ◽

Aromatic Compounds ◽

Iron Uptake ◽

Iron Acquisition ◽

Essential Element ◽

Acquisition System ◽

Bacterial Degradation ◽

Iron Acquisition System ◽

Iron Transporter ◽

Outer Membrane Transporters

Abstract Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34540), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.

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A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound

Communications Biology ◽

10.1038/s42003-019-0676-z ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 8

Author(s):

Masaya Fujita ◽

Kosuke Mori ◽

Hirofumi Hara ◽

Shojiro Hishiyama ◽

Naofumi Kamimura ◽

...

Keyword(s):

Vitamin B12 ◽

Outer Membrane ◽

Membrane Transport ◽

Aromatic Compounds ◽

Proton Motive Force ◽

Negative Bacterium ◽

Chemical Production ◽

Gram Negative ◽

Tonb System ◽

Important Basis

AbstractTonB-dependent receptors (TBDRs) mediate substrate-specific transport across the outer membrane, utilizing energy derived from the proton motive force transmitted from the TonB−ExbB−ExbD complex located in the inner membrane (TonB system). Although a number of TonB systems involved in the uptake of siderophores, vitamin B12 and saccharides have been identified, their involvement in the uptake and catabolism of aromatic compounds was previously unknown. Here, we show that the outer membrane transport of a biphenyl compound derived from lignin is mediated by the TonB system in a Gram-negative bacterium capable of degrading lignin-derived aromatic compounds, Sphingobium sp. strain SYK-6. Furthermore, we found that overexpression of the corresponding TBDR gene enhanced the uptake of this biphenyl compound, contributing to the improved rate of platform chemical production. Our results will provide an important basis for establishing engineered strains optimized for use in lignin valorisation.

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Molecular Structure of the Outermost Cell Wall Component of Spirillum Serpens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100079218 ◽

1977 ◽

Vol 35 ◽

pp. 342-343

Author(s):

Wah Chiu ◽

David Grano

Keyword(s):

Molecular Structure ◽

Cell Wall ◽

Outer Membrane ◽

Gel Electrophoresis ◽

Electron Microscopic Examination ◽

Electron Microscopic ◽

Cell Wall Component ◽

Protein Components ◽

Exposure Technique ◽

Structural Aspects

The periodic structure external to the outer membrane of Spirillum serpens VHA has been isolated by similar procedures to those used by Buckmire and Murray (1). From SDS gel electrophoresis, we have found that the isolated fragments contain several protein components, and that the crystalline structure is composed of a glycoprotein component with a molecular weight of ∽ 140,000 daltons (2). Under an electron microscopic examination, we have visualized the hexagonally-packed glycoprotein subunits, as well as the bilayer profile of the outer membrane. In this paper, we will discuss some structural aspects of the crystalline glycoproteins, based on computer-reconstructed images of the external cell wall fragments.The specimens were prepared for electron microscopy in two ways: negatively stained with 1% PTA, and maintained in a frozen-hydrated state (3). The micrographs were taken with a JEM-100B electron microscope with a field emission gun. The minimum exposure technique was essential for imaging the frozen- hydrated specimens.

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Cryo-Electron Microscopy and Correlation Averaging of Frozen-Hydrated, Polymorphic 2D Crystals of the Mitochondrial Outer Membrane Channel

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179257 ◽

1990 ◽

Vol 48 (1) ◽

pp. 100-101

Author(s):

Xiao-Wei Guo

Keyword(s):

Outer Membrane ◽

Hexagonal Lattice ◽

Mitochondrial Outer Membrane ◽

Rectangular Lattice ◽

Electron Microscopic ◽

Cryo Electron Microscopy ◽

Voltage Dependent ◽

Outer Membrane Channel ◽

2D Crystals ◽

Vdac Channel

Voltage-dependent, anion-selective channels (VDAC) are formed in the mitochondrial outer membrane (mitOM) by a 30-kDa polypeptide. These channels form ordered 2D arrays when mitOMs from Neurospora crassa are treated with soluble phospholipase A2. We obtain low-dose electron microscopic images of unstained specimens of VDAC crystals preserved in vitreous ice, using a Philips EM420 equipped with a Gatan cryo-transfer stage. We then use correlation analysis to compute average projections of the channel crystals. The procedure involves Fourier-filtration of a region within a crystal field to obtain a preliminary average that is subsequently cross-correlated with the entire crystal. Subregions are windowed from the crystal image at coordinates of peaks in the cross-correlation function (CCF, see Figures 1 and 2) and summed to form averages (Figure 3).The VDAC channel forms several different types of crystalline arrays in mitOMs. The polymorph first observed during phospholipase treatment is a parallelogram array (a=13 run, b=11.5 run, θ==109°) containing 6 water-filled pores per unit cell. Figure 1 shows the CCF of a sub-field of such an “oblique” array used to compute the correlation average of Figure 3A. With increased phospholipase treatment, other polymorphs are observed, often co-existing within the same crystal. For example, two distinct (but closely related) types of lattices occur in the field corresponding to the CCF of Figure 2: a “contracted” version of the parallelogram lattice (a=13 run, b=10 run, θ=99°), and a near-rectangular lattice (a=8.5 run, b=5 nm). The pattern of maxima in this CCF suggests that a third, near-hexagonal lattice (a=4.5 nm) may also be present. The correlation averages of Figures 3B-D were computed from polycrystalline fields, using peak coordinates in regions of CCFs corresponding to each of the three lattice types.

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