Acrylate reductase of an anaerobic electron transport chain of the marine bacterium shewanella woodyi

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Abstract

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

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About the authors

Y. V. Bertsova

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University

Author for correspondence.
Email: bogachev@belozersky.msu.ru
Russian Federation, Moscow

M. V. Serebryakova

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University

Email: bogachev@belozersky.msu.ru
Russian Federation, Moscow

V. A. Bogachev

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University

Email: bogachev@belozersky.msu.ru
Russian Federation, Moscow

A. A. Baykov

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University

Email: bogachev@belozersky.msu.ru
Russian Federation, Moscow

A. V. Bogachev

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University

Email: bogachev@belozersky.msu.ru
Russian Federation, Moscow

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2. Fig. 1. The reaction of dimethylsulfoniopropionate decomposition catalyzed by DMSP-lyase DddY

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3. Fig. 2. dddY-associated genes of the marine bacterium Shewanella woodyi. a – Location of dddY-associated genes on the chromosome of S. woodyi: swoo_0272 – NADPH:acryloyl-CoA oxidoreductase gene; swoo_0273 – hypothetical protein with unknown functions; swoo_0274 – transcription regulator; swoo_0275 – flavin-containing subunit of flavocytochrome c; swoo_0276 – tetraheme cytochrome c; swoo_0277 – DMSP-lyase DddY (https://www.kegg.jp/kegg-bin/show_organism?org=T00676). b – Alignment of amino acid sequences of cytochrome c:fumarate oxidoreductase from S. oneidensis MR-1 (So_FccA, GenBank: AAN54044), NADH:fumarate oxidoreductase from Klebsiella pneumoniae (Kp_Frd, B5XRB0), SdhA subunit of succinate dehydrogenase from Escherichia coli (Ec_SdhA, HDZ3930178), NADH:acrylate oxidoreductase from V. harveyi (Vh_Ard, P0DW92), Swoo_0275 protein from S. woodyi (Sw_0275, ACA84576) and flavocytochrome c from H. aestuarii (Ha_flc, SHJ73509). The two alignment fragments shown contain amino acid residues (highlighted in blue and green) involved in binding, respectively, the C4- and C1-carboxylates of fumarate in fumarate reductases, as well as residues (highlighted in yellow) involved in proton transfer to fumarate.

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4. Fig. 3. Isolation of Ard from S. woodyi cells. a – Separation of the periplasmic fraction of S. woodyi cells grown anaerobically in the presence of acrylate by ion-exchange chromatography on DEAE-Sepharose. The absorbance at 405 nm is shown by the blue curve, the NaCl concentration is shown by the dotted line. The red squares indicate the acrylate reductase activity in the obtained fractions. b – SDS-PAGE of the obtained Ard preparation. 2 μg of protein were loaded into each lane. The gel was stained either for protein with Coomassie (left panel) or for heme C with tetramethylbenzidine/H2O2 (right panel). The numbered bands on the left side indicate the positions of the protein molecular weight markers. The protein bands identified by MALDI-MS are indicated on the right side.Fig. 3. Isolation of Ard from S. woodyi cells. a – Separation of the periplasmic fraction of S. woodyi cells grown anaerobically in the presence of acrylate by ion-exchange chromatography on DEAE-Sepharose. The absorbance at 405 nm is shown by the blue curve, the NaCl concentration is shown by the dotted line. The red squares indicate the acrylate reductase activity in the obtained fractions. b – SDS-PAGE of the obtained Ard preparation. 2 μg of protein were loaded into each lane. The gel was stained either for protein with Coomassie (left panel) or for heme C with tetramethylbenzidine/H2O2 (right panel). The numbered bands on the left side indicate the positions of the protein molecular weight markers. The protein bands identified by MALDI-MS are indicated on the right side.

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5. Fig. 4. Identification of Ard prosthetic groups. a – Separation of non-covalently bound flavins with Ard by HPLC. Retention volumes of FAD, FMN and riboflavin standards (Rf) are shown by arrows. b – Absorption spectra of air-oxidized (blue curve) and dithionite-reduced (red curve) Ard preparations. The spectra were measured in 100 mM Tris-HCl (pH 8.0) buffer containing 0.1 mg/ml Ard. Cytochrome c-specific absorption maxima of the γ- and α-bands are shown by arrows.

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6. Fig. 5. Catalytic properties of Ard from S. woodyi. a – Dependence of the enzymatic activity of Ard on the concentration of acrylate (blue squares) or methacrylate (red squares). The values ​​of acrylate and methacrylate reductase activities of Ard obtained by analyzing the integrated kinetics of acceptor reduction in the 0.5–50 μM (meth)acrylate concentration range are shown. The lines represent the results of fitting the obtained data using the Michaelis–Menten equation (the data for methacrylate are shown on an enlarged scale in Fig. S2 in the Appendix). b – Typical curves of MV oxidation in the presence of Ard. The additions of 1 or 0.1 mM acrylate (Acr) and 1 mM methacrylate (Mac) are indicated by arrows. The numbers above the curves indicate the residual enzymatic activity of Ard after the addition of methacrylate, where the activity before this addition was taken as 100%.

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7. Fig. 6. Anaerobic growth of S. woodyi in the presence of different electron acceptors. The initial optical density at seeding was 0.005. Cells were grown anaerobically at 25 °C for 18 h. Where indicated, 10 mM methacrylate (Mac), DMSP, or DMSO was added to the growth medium (the “none” column indicates no acceptors were added). The final optical densities of the cultures at 600 nm are shown, the error bars indicate the standard deviations of three biological replicates.

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