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Research Article | Molecular Biology and Physiology

First Betalain-Producing Bacteria Break the Exclusive Presence of the Pigments in the Plant Kingdom

Luis Eduardo Contreras-Llano, M. Alejandra Guerrero-Rubio, José Daniel Lozada-Ramírez, Francisco García-Carmona, Fernando Gandía-Herrero
Lotte Søgaard-Andersen, Editor
Luis Eduardo Contreras-Llano
Department of Biochemistry and Molecular Biology A, Faculty of Biology, Regional Campus of International Excellence, Campus Mare Nostrum, University of Murcia, Murcia, SpainDepartment of Health Sciences, Universidad de las Americas Puebla, Puebla, Mexico
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  • ORCID record for Luis Eduardo Contreras-Llano
M. Alejandra Guerrero-Rubio
Department of Biochemistry and Molecular Biology A, Faculty of Biology, Regional Campus of International Excellence, Campus Mare Nostrum, University of Murcia, Murcia, Spain
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José Daniel Lozada-Ramírez
Department of Health Sciences, Universidad de las Americas Puebla, Puebla, Mexico
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Francisco García-Carmona
Department of Biochemistry and Molecular Biology A, Faculty of Biology, Regional Campus of International Excellence, Campus Mare Nostrum, University of Murcia, Murcia, Spain
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Fernando Gandía-Herrero
Department of Biochemistry and Molecular Biology A, Faculty of Biology, Regional Campus of International Excellence, Campus Mare Nostrum, University of Murcia, Murcia, Spain
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  • ORCID record for Fernando Gandía-Herrero
Lotte Søgaard-Andersen
Max Planck Institute for Terrestrial Microbiology
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DOI: 10.1128/mBio.00345-19
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  • FIG 1
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    FIG 1

    Betalain production and characterization of the novel dioxygenase from Gluconacetobacter diazotrophicus. (A to D) Detection of dopaxanthin in Gluconacetobacter diazotrophicus cultures supplemented with l-DOPA. Standard dopaxanthin was monitored by HPLC-DAD at a λ of 480 nm (A) and by HPLC-ESI-TOF MS at an extracted-ion chromatogram (EIC) of 391.1136 m/z (B). The same peak was detected in an EIC of G. diazotrophicus transformations in water (C) and culture medium (D), both supplemented with 7.6 mM l-DOPA. (E) Electrophoretic analysis for the expression and purification of recombinant GdDODA from E. coli culture. Lane 1, molecular weight markers; lane 2, soluble protein content of cells harvested prior to IPTG induction; lane 3, soluble protein content of cells harvested 20 h after IPTG induction (0.5 mM); lane 4, eluted protein after affinity chromatography purification. (F) Analysis of GdDODA by gel filtration chromatography. The profile shows a single peak corresponding to a homodimeric form. (Inset) Calibration curve and molecular mass determination. Kav, distribution coefficient; MW, molecular weight. (G) Sequence comparison of GdDODA with the structurally characterized PDB protein 2NYH from Burkholderia xenovorans. Sequence alignment using structural information was performed with Expresso (18). Conserved blocks of amino acids are squared and strictly conserved residues are shown in red. Information was displayed with ESPript program (19). (H) Structural model for the DODA from G. diazotrophicus (orange and dark gray) (GdDOD) superimposed on the YgiD protein from E. coli (blue and pale gray). Orange and blue portions of the proteins indicate structural similarity according to the combinatorial extension (CE) algorithm. (I) Phylogenetic analysis of all characterized 4,5-DOPA-dioxygenases known to produce betalamic acid. Multiple-sequence alignment was performed using ClustalW2. The unrooted tree was obtained with the Phylogeny inference package from EMBL (neighbor-joining algorithm) (17, 34) using the conserved block among residues His91 and Asp122. This block contains one of the three strictly conserved histidines in the plant enzymes, which also appears in the novel dioxygenase from G. diazotrophicus as His101. AmDOD, GdDOD, MjDOD, PgDOD, and BvDOD, the DODAs from Amanita muscaria, G. diazotrophicus, Mirabilis jalapa, Portulaca grandiflora, and Beta vulgaris, respectively.

  • FIG 2
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    FIG 2

    G. diazotrophicus dioxygenase activity characterization. (A) Spectral evolution of the transformation of DOPA (2.5 mM) by the addition of pure 4,5-DODA enzyme to the reaction medium. Spectra were recorded at 10-min intervals for 180 min, using a scanning speed of 2,000 nm · min–1. (B) Effect of pH on dioxygenase activity. Reactions were performed with 2.5 mM l-DOPA in 50 mM sodium acetate buffer for pH values ranging from 3.5 to 5.5 and in 50 mM sodium phosphate for pH values ranging from 5.5 to 8.5. (C) Enzyme activity dependence on l-DOPA concentration measured in 50 mM sodium phosphate buffer, pH 6.5. AU, arbitrary units.

  • FIG 3
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    FIG 3

    HPLC analysis of reaction products formed by G. diazotrophicus dioxygenase and derived compounds. (A, B, C) Chromatograms obtained at a λ of 360 nm (A), a λ of 405 nm (B), and a λ of 480 nm (C) for a reaction medium containing 2.5 mM l-DOPA and 10 mM solvent AA in phosphate buffer (50 mM, pH 6.5) at 25°C 3.3 h after the addition of the purified enzyme. (D, E, F) The same reaction monitored at a λ of 360 nm (D), a λ of 405 nm (E), and a λ of 480 nm (F) 31.5 h after the reaction was triggered. (G to K) Normalized spectra shown correspond to peak 1 (4,5-seco-DOPA) (G), peak 2 (2,3-seco-DOPA) (H), peak 3 (betalamic acid) (I), peak 4 (muscaflavin) (J), and peak 5 (dopaxanthin) (K). r.u., relative units.

  • FIG 4
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    FIG 4

    Enzymatic-chemical mechanism in the formation of betalains. (A) Time evolution of the reaction`s products formed by G. diazotrophicus dioxygenase activity and their derived compounds. Normalized data are based on pigment content determination by HPLC at the wavelengths 360 nm (seco-DOPAs), 405 nm (betalamic acid and muscaflavin), and 480 nm (dopaxanthin). (B) Proposed chemical reactions underlying the spontaneous cyclization of the enzyme-generated 4,5-seco-DOPA intermediate into betalamic acid. (C) Transformation of betalamic acid. (Left) Time course for the formation of dopaxanthin from betalamic acid (100 µM) and l-DOPA (3.5 mM) at pH 5.0. (Center) pH effect on the rate of dopaxanthin synthesis. (Right) Effect of betalamic acid concentration on the formation of dopaxanthin at a fixed concentration of l-DOPA (3.5 mM). Rates were calculated based on dopaxanthin concentration in the reaction medium after 12 h.

  • FIG 5
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    FIG 5

    Biosynthetic scheme of betalains. The enzyme-catalyzed reactions and the chemical spontaneous cyclization and condensation ultimately yielding the flower pigment dopaxanthin are shown. The flower shown corresponds to Glottiphyllum oligocarpum, which produces dopaxanthin as the only pigment responsible for its coloration. Numbers in brackets correspond to the intermediates and final products described in Fig. 3.

Tables

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  • Supplemental Material
  • TABLE 1

    Expression and purification of G. diazotrophicus dioxygenase

    StepVol (ml)Protein
    (mg/ml)
    Total protein
    (mg)
    Activitya
    (µM · min−1)
    Sp act (µmol ·
    min−1 · mg−1)
    Purification
    fold
    Yield (%)
    Crude extractb6.015.995.41.5780.5951.0100
    Ni2+ chromatography7.04.531.80.8231.0871.861
    • ↵a Activity was determined using a 50-µl protein solution under the assay conditions.

    • ↵b Crude extract was obtained from a cellular paste harvested from a 0.5-liter culture.

  • TABLE 2

    Main peptides identified to fully characterize the proteina

    Peptide identifiedm/z
    (–)MTPVPEPIRQIGTIGSYHAHVYFDGPDGR(A)3,210.58
    (R)QIGTIGSYHAHVYFDGPDGR(A)2,190.04
    (R)DGIWLGQPRALLGSR(L)1,638.91
    (R)DGIWLGQPR(A)1,041.55
    (–)MTPVPEPIR(Q)1,039.56
    (R)AAIADR(F)616.34
    (R)DHLR(D)540.29
    • ↵a Its peptide mass fingerprint (PMF) was determined by MALDI-TOF analysis after trypsin digestion. Amino acids in parentheses correspond to the theoretical residue after trypsin digestion. (–), the beginning of the sequence.

  • TABLE 3

    HPLC-ESI-TOF MS analysis of the reaction products formed by G. diazotrophicus dioxygenase activity in water supplemented with l-DOPA at 7.6 mM

    CompoundChemical
    formula
    [M + H]+
    (m/z)
    Main-
    daughter
    ion (m/z)
    Secondary-
    daughter
    ion(s) (m/z)
    TOF exact
    mass (m/z)
    (exptl)
    Calculated
    mass (m/z)
    (theoretical)
    Δppm
    4,5-Seco-DOPAC9H11NO6230.2140.0187.1, 94.1230.0065230.06592.52
    2,3-Seco-DOPAC9H11NO6230.2140.094.1230.0065230.06592.52
    Betalamic acidC9H9NO5212.0166.1138.0212.0562212.05532.07
    MuscaflavinC9H9NO5212.0166.0149.0212.0562212.05532.07
    DopaxanthinC18H18N2O8391.3347.1301.1, 255.1391.1141391.11361.25

Supplemental Material

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  • FIG S1

    Dopaxanthin accumulation in G. diazotrophicus cultures at different concentrations of added l-DOPA. Samples were analyzed at 24 and 48 h and showed accumulation of dopaxanthin even in the absence of exogenous l-DOPA. Download FIG S1, PDF file, 0.07 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TEXT S1

    Details about structure modelling. Download Text S1, PDF file, 0.1 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Sequence coverage of the detected fragments identified in the peptide mass fingerprint of GdDODA. Download FIG S2, PDF file, 0.06 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    Effect of aeration and inert atmosphere in the formation of dopaxanthin by GdDODA. Enzyme assays without stirring were considered standard conditions (not saturated air). Inert atmosphere was obtained with nitrogen gas. Download FIG S3, PDF file, 0.07 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S4

    Kinetic analysis of GdDODA. (A) Activity measured for the enzyme under growing concentrations of the substrates l-DOPA, dihydrocaffeic acid, 4-methyl-catechol, and catechol. l-DOPA behaves as a Michaelis-Menten substrate, while dihydrocaffeic acid, 4-methyl-catechol, and catechol present substrate inhibition kinetics. (B) Kinetic mechanism and rate equation for inhibition by excess of substrate. Download FIG S4, PDF file, 0.1 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S1

    Kinetic analysis of GdDODA with different substrates. Strong inhibition by an excess of substrate was shown for dihydrocaffeic acid, 4-methyl-catechol, and catechol. Download Table S1, PDF file, 0.1 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S5

    ESI-MS fragment spectra of betalains and intermediate compounds identified in this work. MS2 spectra of all compounds are provided with structures and annotations. Download FIG S5, PDF file, 0.4 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S6

    Evolution of betalamic acid, muscaflavin, and dopaxanthin in an enzymatic assay with GdDODA. Absolute concentrations are expressed in micromolar units, and conditions are as those described in the legend of Fig. 4. Download FIG S6, PDF file, 0.1 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S7

    Extended phylogenetic analysis of the novel betalain-forming dioxygenase from G. diazotrophicus. (A) The novel enzyme was searched against similar enzymes, and the 100 most similar sequences were used to construct a neighbor-joining phylogenetic tree (S. F. Altschul, J. C. Wootton, E. M. Gertz, R. Agarwala, et al., FEBS J 272:5101–5109, 2005, https://doi.org/10.1111/j.1742-4658.2005.04945.x; W. Li, A. Cowley, M. Uludag, T. Gur, et al., Nucleic Acids Res 43:W580–W584, 2015, https://doi.org/10.1093/nar/gkv279). The tree shows the presence of three main clades comprising species of Bradyrhizobium (red), Komagataeibacter (orange), and Mesorhizobium (yellow). Sequences corresponding to Inquilinus are shown in blue, and those corresponding to Acetobacter are shown in pink. (B) The block among residues His91 and Asp122 in GdDODA (see the main text for details) was used to construct a larger tree with all the characterized betalain-forming enzymes and including bacterial members of the different classes identified in panel A. In this tree, experimentally validated enzymes are labeled. The presence of betalamic acid derivatives (betalains) is also indicated. Additional sequences corresponding to bacterial members are WP_027577986 (Bradyrhizobium), WP_003618779 (Komagataeibacter), RWL99607 (Mesorhizobium), and WP_034835604 (Inquilinus). Download FIG S7, PDF file, 0.5 MB.

    Copyright © 2019 Contreras-Llano et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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First Betalain-Producing Bacteria Break the Exclusive Presence of the Pigments in the Plant Kingdom
Luis Eduardo Contreras-Llano, M. Alejandra Guerrero-Rubio, José Daniel Lozada-Ramírez, Francisco García-Carmona, Fernando Gandía-Herrero
mBio Mar 2019, 10 (2) e00345-19; DOI: 10.1128/mBio.00345-19

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First Betalain-Producing Bacteria Break the Exclusive Presence of the Pigments in the Plant Kingdom
Luis Eduardo Contreras-Llano, M. Alejandra Guerrero-Rubio, José Daniel Lozada-Ramírez, Francisco García-Carmona, Fernando Gandía-Herrero
mBio Mar 2019, 10 (2) e00345-19; DOI: 10.1128/mBio.00345-19
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KEYWORDS

betalains
betalamic acid
dioxygenase
enzyme mining
pigments

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