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Molecular characterization of uptake hydrogenase in Frankia
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
2005 (English)In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 33, no 1, 64-66 p.Article in journal (Refereed) Published
Abstract [en]

A molecular characterization of uptake hydrogenase in Fronkia was performed by using two-dimensional gel electrophoresis, matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, PCR amplification and Southern blotting. A polypeptide of approx. 60 kDa was recognized in Frankia UGL011102, AVCI1 and KBS on the two-dimensional gel by blotting with Ralstonia eutropha (Hox G) antibody. Further analysis by MS resulted in a peptide 'fingerprint', which was similar to the membrane-bound hydrogenase 2 large subunit (HYD2) in Escherichia coli. in addition, a 127 bp PCR fragment could also be amplified from Frankia AVCI1, which gave a 76% similarity with the large subunit of hydrogenase in, e.g., Azotobacter chrococcum, Bradyrhizobium japonicum and Rhizobium leguminosorum. Although immunological similarity between the small subunit of Frankia hydrogenase and that of other organisms has not yet been found, a PCR product of 500 bp could be amplified from the local source of Fronkia, the analysis of which gave 69 and 67% identity with the small subunit of hydrogenases in B. japonicum and R. leguminosorum respectively. A Southern-blot analysis further indicated evidence for the presence of the small hydrogenase subunit in other Fronkia strains, i.e. KBS, Avcl1 and Ccl3.

Place, publisher, year, edition, pages
London: Biochemical Society , 2005. Vol. 33, no 1, 64-66 p.
Keyword [en]
Amino Acid Sequence, Base Sequence, Blotting; Southern, DNA Primers, Electrophoresis; Gel; Two-Dimensional, Frankia/*enzymology, Hydrogenase/chemistry/*metabolism, Molecular Sequence Data, Sequence Homology; Amino Acid
Identifiers
URN: urn:nbn:se:umu:diva-2782PubMedID: 15667266OAI: oai:DiVA.org:umu-2782DiVA: diva2:141051
Available from: 2007-11-16 Created: 2007-11-16 Last updated: 2017-12-14Bibliographically approved
In thesis
1. The Biodiversity of Hydrogenases in Frankia: Characterization, regulation and phylogeny
Open this publication in new window or tab >>The Biodiversity of Hydrogenases in Frankia: Characterization, regulation and phylogeny
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Fysiologisk botanik, 2007. 63 p.
Keyword
Biodiversity, Frankia, immunoblotting, gene expression, uptake hydrogenase, hydrogen-evolving hydrogenase, nickel, phylogeny
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1435 (URN)978-91-7264-444-1 (ISBN)
Public defence
2007-12-07, KB3A9, KBC, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2007-11-16 Created: 2007-11-16 Last updated: 2011-03-14Bibliographically approved

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