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Functional characterization of xanthorhodopsin in Salinivibrio socompensis, a novel halophile isolated from modern stromatolites

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Abstract

A putative xanthorhodopsin-encoding gene, XR34, was found in the genome of the moderately halophilic gammaproteobacterium Salinivibrio socompensis S34, isolated from modern stromatolites found on the shore of Laguna Socompa (3570 m), Argentina Puna. XR-encoding genes were clustered together with genes encoding X-carotene, retinal (vitamin-A aldehyde), and carotenoid biosynthesis enzymes while the carotene ketolase gene critical for the salinixanthin antenna compound was absent. To identify its functional behavior, we herein overexpressed and characterized this intriguing microbial rhodopsin. Recombinant XR34 showed all the salient features of canonical microbial rhodopsin and covalently bound retinal as a functional chromophore with λmax = 561 nm (εmax ca. 60,000 M−1 cm−1). Two canonical counterions with pK values of around 6 and 3 were identified by pH titration of the recombinant protein. With a recovery time of approximately half an hour in the dark, XR34 shows light–dark adaptation shifting the absorption maximum from 551 to 561 nm. Laser-flash induced photochemistry at pH 9 (deprotonated primary counterion) identified a photocycle starting with a K-like intermediate, followed by an M-state (λmax ca. 400 nm, deprotonated Schiff base), and a final long wavelength-absorbing N- or O-like intermediate before returning to the parental 561 nm-state. Initiating the photocycle at pH 5 (protonated counterion) yields only bathochromic intermediates, due to the lacking capacity of the counterion to accept the Schiff base proton. Illumination of the membrane-embedded protein yielded a capacitive transport current. The presence of the M-intermediate under these conditions was demonstrated by a blue light-induced shunt process.

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Data availability

The sequence of the xantorhodopsin protein-coding gene from Salinivibrio socompensis sp. S34 is available at GenBank Acc. AGR66236.1 together with all sequences of rhodopsin-related proteins retrieved from public databases via Uniprot (https://www.uniprot.org/blast/) and MicRhoDE (http://application.sb-roscoff.fr/micrhode/). All other raw data from results presented in the manuscript are available upon request.

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Acknowledgements

The authors acknowledge the generous financial support by PICT RAICES 2019–3216 projects. CB thanks Anja Becker for technical assistance and is grateful for the continuous support from the Max-Planck-Society. WG thanks the University of Leipzig for hosting him as a visitor. VHA is a staff researcher from the National Research Council (CONICET) in Argentina. DA and MG were the recipients of doctoral/postdoctoral fellowships from CONICET.

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Authors and Affiliations

  1. Laboratorio de Investigaciones Microbiológicas de Lagunas Andinas (LIMLA), Planta Piloto de Procesos Industriales y Microbiológicos (PROIMI), CCT, CONICET, Av. Belgrano y Pje. Caseros, San Miguel de Tucumán, 4000, Tucumán, Argentina

    Marta F. Gorriti & María Eugenia Farías

  2. Max-Planck-Institute for Biophysics, Max-von-Laue-Straße 3, Frankfurt am Main, 60438, Germany

    Christian Bamann, Phillip Wood & Ernst Bamberg

  3. Laboratorio de Microbiología Ultraestructural y Molecular, Centro Integral de Microscopía Electrónica (CIME, CONICET, UNT) CCT, CONICET, Facultad de Agronomía, Zootecnia y Veterinaria, Finca El Manantial, UNT, Camino de Sirga s/n (4107), Yerba Buena, Tucumán, Argentina

    Daniel Gonzalo Alonso-Reyes & Virginia Helena Albarracín

  4. Institute for Analytical Chemistry, University of Leipzig, Johannisallee 29, Leipzig, 04103, Germany

    Daniel Gonzalo Alonso-Reyes & Wolfgang Gärtner

  5. Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán, Miguel Lillo 205, San Miguel de Tucumán, 4000, Tucumán, Argentina

    Virginia Helena Albarracín

  6. Facultad de Agronomía, Zootecnia y Veterinaria, Universidad Nacional de Tucumán, Centro Universitario Ing. R. Herrera (Ex Quinta Agronómica), Avda. Pte. N. Kirchner 1900., San Miguel de Tucumán, 4000, Tucumán, Argentina

    Virginia Helena Albarracín

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  1. Marta F. Gorriti
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Contributions

MG, DA, PW, VHA, and CB performed the experiments. VHA, MEF, WG, and EB designed the project. All authors contributed to the generation of the manuscript.

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Correspondence to Virginia Helena Albarracín.

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Additional information

This paper is dedicated to Silvia Braslavsky, a pioneer in photobiology and photobiophysics, on the occasion of her 80th birthday. The authors want to congratulate Silvia on her anniversary and like to acknowledge her great contribution to the establishment of a German-Argentinean research group in the photo-microbiology of Andean extremophiles. Likewise, we are thankful for her warm hospitality in her house in Mulheim an der Ruhr, where delicious food, perfect wine, and science tertulia were always a lovely blend.

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Gorriti, M.F., Bamann, C., Alonso-Reyes, D.G. et al. Functional characterization of xanthorhodopsin in Salinivibrio socompensis, a novel halophile isolated from modern stromatolites. Photochem Photobiol Sci 22, 1809–1823 (2023). https://doi.org/10.1007/s43630-023-00412-6

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