The Biodiversity of Hydrogenases in 141056/ آ  The Biodiversity of Hydrogenases in Frankia

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  • The Biodiversity of Hydrogenases in Frankia

    Characterization, regulation and phylogeny

    Melakeselam Leul Zerihun

    DOCTORAL DISSERTATION

    To be defended on Friday 7th December 2007, 10:00 AM at

    the Lecture Hall KB3A9, KBC, Umeå University

    Faculty opponent

    Kornel Kovacs, Professor, University of Szeged, Hungary

    Department of Plant Physiology

    Umeå Plant Science Center

    Umeå University, Sweden

  • ©Melakeselam Leul Zerihun, 2007

    Department of Plant Physiology

    Umeå Plant Science Center

    Umeå University

    SE-901 87 Umeå

    Sweden

    Doctoral Dissertation, Umeå 2007

    ISBN 978-91-7264-444-1

    Printed by VMC, KBC, Umeå University, Umeå.

  • “Life is available to anyone no matter what age. All you have to do is grab it”- Art Carney

    Dedicated to

    my beloved wife Genet F. Shawl

    my beloved daughter Abigail

    my beloved parents

  • The Biodiversity of Hydrogenases in Frankia: Characterization, regulation and

    phylogeny

    Melakeselam Leul Zerihun (2007) ISBN 978-91-7264-444-1

    Department of Plant Physiology, Umeå Plant Science Center, Umeå University, Sweden

    Dissertation abstract

    All the eighteen Frankia strains isolated from ten different actinorhizal host plants

    showed uptake hydrogenase activity. The activity of this enzyme is further increased by

    addition of nickel. Nickel also enhanced the degree of hydrogenase transfer into the

    membranes of Frankia, indicating the role of this metal in the processing of this enzyme.

    The uptake hydrogenase of Frankia is most probably a Ni-Fe hydrogenase.

    Genome characterization revealed the presence of two hydrogenase genes

    (syntons) in Frankia, which are distinctively separated in all the three available Frankia

    genomes. Both hydrogenase syntons are also commonly found in other Frankia strains.

    The structural, regulatory and accessory genes of both hydrogenase synton #1 and #2 are

    arranged closely together, but in a clearly contrasting organization. Hydrogenase synton

    #1 and #2 of Frankia are phylogenetically divergent and that hydrogenase synton #1 is

    probably ancestral among the actinobacteria. Hydrogenase synton #1 (or synton #2) of

    Frankia sp. CcI3 and F. alni ACN14a are similar in gene arrangement, content and

    orientation, while the syntons are both reduced and rearranged in Frankia sp. EANpec.

    The hydrogenases of Frankia sp. CcI3 and F. alni ACN14a are phylogenetically grouped

    together but never with the Frankia sp. EAN1pec, which is more closely related to the

    non-Frankia bacteria than Frankia itself. The tree topology is indicative of a probable

    gene transfer to or from Frankia that occurred before the emergence of Frankia. All of

    the available evidence points to hydrogenase gene duplication having occurred long

    before development of the three Frankia lineages. The uptake hydrogenase synton #1 of

    Frankia is more expressed under free-living conditions whereas hydrogenases synton #2

    is mainly involved in symbiotic interactions. The uptake hydrogenase of Frankia can also

    be manipulated to play a larger role in increasing the efficiency of nitrogen fixation in the

  • root nodules of the host plants, there by minimizing the need for environmentally

    unfriendly and costly fertilizers.

    The hydrogen-evolving hydrogenase activity was recorded in only four Frankia

    strains: F. alni UGL011101, UGL140102, Frankia sp. CcI3 and R43. After addition of

    15mM Nicl2, activity was also detected in F. alni UGL011103, Frankia sp. UGL020602,

    UGL020603 and 013105. Nickel also increased the activity of hydrogen-evolving

    hydrogenases in Frankia, indicating that Frankia may have different types of hydrogen-

    evolving hydrogenases, or that the hydrogen-evolving hydrogenases may at least be

    regulated differently in different Frankia strains. The fact that Frankia can produce

    hydrogen is reported only recently. The knowledge of the molecular biology of Frankia

    hydrogenase is, therefore, of a paramount importance to optimize the system in favor of

    hydrogen production. Frankia is an attractive candidate in search for an organism

    efficient in biological hydrogen production since it can produce a considerable amount of

    hydrogen.

    Key words: Biodiversity, Frankia, immunoblotting, gene expression, uptake

    hydrogenase, hydrogen-evolving hydrogenase, nickel, phylogeny

  • CONTENTS

    PAGE

    LIST OF PAPERS 9

    ABBREVIATIONS 10

    PREFACE 11

    INTRODUCTION 12

    Hydrogen