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The Biodiversity of Hydrogenases in Frankia: Characterization, regulation and phylogeny
Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
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 [en]
Biodiversity, Frankia, immunoblotting, gene expression, uptake hydrogenase, hydrogen-evolving hydrogenase, nickel, phylogeny
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1435ISBN: 978-91-7264-444-1 (print)OAI: oai:DiVA.org:umu-1435DiVA: diva2:141056
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
List of papers
1. Biodiversity of Hydrogenases in Frankia
Open this publication in new window or tab >>Biodiversity of Hydrogenases in Frankia
2005 (English)In: Current Microbiology, ISSN 0343-8651, E-ISSN 1432-0991, Vol. 50, no 1, 17-23 p.Article in journal (Refereed) Published
Abstract [en]

Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.

Place, publisher, year, edition, pages
New York: Springer, 2005
Keyword
Biodiversity, Cryoultramicrotomy, Frankia/*enzymology, Hydrogen/metabolism, Hydrogenase/*metabolism, Nickel/pharmacology
Identifiers
urn:nbn:se:umu:diva-2781 (URN)doi:10.1007/s00284-004-4323-6 (DOI)15696261 (PubMedID)
Available from: 2007-11-16 Created: 2007-11-16 Last updated: 2015-04-29Bibliographically approved
2. Molecular characterization of uptake hydrogenase in Frankia
Open this publication in new window or tab >>Molecular characterization of uptake hydrogenase in Frankia
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
Keyword
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:nbn:se:umu:diva-2782 (URN)15667266 (PubMedID)
Available from: 2007-11-16 Created: 2007-11-16 Last updated: 2015-03-18Bibliographically approved
3. The organization, regulation and phylogeny of uptake hydrogenase genes in Frankia
Open this publication in new window or tab >>The organization, regulation and phylogeny of uptake hydrogenase genes in Frankia
2007 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 130, no 3, 464-470 p.Article in journal (Refereed) Published
Abstract [en]

Frankia alni ACN14a, Frankia sp. CcI3 and Frankia sp. EAN1pec, which have different host specificity and geographical distribution, have two uptake hydrogenase syntons in their genome: hydrogenase synton#1 and hydrogenase synton#2. The organization of hydrogenase genes on these syntons also varies. Phylogenetic analysis of the structural genes of these syntons showed that they were significantly divergent and that hydrogenase synton#1 subunits of these Frankia strains were probably ancestral among the actinobacteria. Hydrogenase gene duplication might have occurred long before emergence of the three Frankia lineages. The structural subunits of hydrogenase HupS2 and HupL2 (synton#2) of F. alni ACN14a and Frankia sp. CcI3, which belong to phylogenetic Frankia cluster 1, were grouped closely together but away from Frankia sp. EAN1pec, which belongs to Frankia cluster 3. Phylogenetic analysis showed the occurrence of lateral transfer of hupL2 in Frankia sp. EAN1pec to or from Geobacter sulfurreducens. The transcript levels of hupS1 and hupL1 relative to hupS2 and hupL2 were higher in F. alni ACN14a grown under free-living conditions. Under symbiotic conditions, transcript levels of hupS2 and hupL2 were higher than those of hupS1 and hupL1. Hydrogenase subunits of synton#1 are more expressed under free-living conditions, whereas those of synton#2 are mainly involved in symbiotic interactions.

Place, publisher, year, edition, pages
Copenhagen: Munksgaard, 2007
Identifiers
urn:nbn:se:umu:diva-16001 (URN)10.1111/j.1399-3054.2007.00861.x (DOI)
Available from: 2007-08-09 Created: 2007-08-09 Last updated: 2015-04-29Bibliographically approved
4. A hydrogen-evolving enzyme is present in Frankia sp. R43
Open this publication in new window or tab >>A hydrogen-evolving enzyme is present in Frankia sp. R43
2004 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 236, no 2, 235-240 p.Article in journal (Refereed) Published
Abstract [en]

The ability to evolve hydrogen using methyl viologen as an electron donor was assayed in the nitrogen-fixing actinomycetes Frankia sp. R43 and Frankia sp. KB5. To further examine the nature of hydrogen-evolving enzymes that may be present in these organisms immunological studies were performed. Under anaerobic conditions (both nitrogen-limiting and nitrogen-containing) Frankia sp. R43 but not Frankia sp. KB5 evolved hydrogen,which was not linked to NAD-reducing activity. Immunological analysis of total protein from Frankia sp. R43 and Frankia sp. KB5 using an antiserum raised against Ralstonia eutropha HoxF, recognized an antigen in Frankia sp. R43 but not in Frankia sp. KB5. Immunogold labeling using antibodies raised against the R. eutropha HoxH recognized sites in both hyphae and vesicles of Frankia sp. R43, but not in Frankia sp. KB5. Based on these physiological and immunological findings, we conclude that Frankia sp. R43 has a hydrogen-evolving hydrogenase.

Place, publisher, year, edition, pages
Amsterdam: Elsevier/North-Holland, 2004
Keyword
Anaerobiosis, Bacterial Proteins/immunology/isolation & purification/metabolism, Blotting; Western, Cupriavidus necator/enzymology/immunology, Frankia/chemistry/*enzymology/immunology, Hydrogen/*metabolism, Hydrogenase/immunology/*metabolism, Hyphae/enzymology, Immunohistochemistry, NAD/metabolism, Paraquat/metabolism
Identifiers
urn:nbn:se:umu:diva-2784 (URN)doi:10.1016/j.femsle.2004.05.049 (DOI)15251202 (PubMedID)
Available from: 2007-11-16 Created: 2007-11-16 Last updated: 2015-04-29Bibliographically approved
5. Occurrence and characterisation of the hydrogen-evolving enzyme in Frankia sp.
Open this publication in new window or tab >>Occurrence and characterisation of the hydrogen-evolving enzyme in Frankia sp.
2006 (English)In: International journal of hydrogen energy, ISSN 0360-3199, Vol. 31, no 11, 1445-1451 p.Article in journal (Refereed) Published
Abstract [en]

An increase in hydrogen evolution from the hydrogen-evolving enzyme in the actinomycete Frankia was recorded in the presence of nickel. Immunogold localisation analysis of the intracellular distribution of hydrogenase proteins indicated that they were evenly distributed in the membranes and cytosol of both hyphae and vesicles. In addition, molecular characterisation of the hydrogen-evolving enzyme at the proteomic level, using two-dimensional gel electrophoresis combined with mass spectrometry, confirmed that the Frankia hydrogen-evolving enzyme is similar to the cyanobacterial bidirectional hydrogenase of Anabena siamensis.

Place, publisher, year, edition, pages
Oxford: Pergamon, 2006
Keyword
Frankia; Hydrogen; Hydrogen-evolving hydrogenase; Nickel
Identifiers
urn:nbn:se:umu:diva-15951 (URN)10.1016/j.ijhydene.2006.06.009 (DOI)
Available from: 2007-08-06 Created: 2007-08-06 Last updated: 2015-04-29Bibliographically approved
6. The phylogeny of uptake hydrogenases in Frankia
Open this publication in new window or tab >>The phylogeny of uptake hydrogenases in Frankia
2009 (English)In: International Microbiology, ISSN 1139-6709, E-ISSN 1618-1905, Vol. 12, no 1, 23-28 p.Article in journal (Refereed) Published
Abstract [en]

Uptake hydrogenase is an enzyme that is beneficial for nitrogen fixation in bacteria. Recent studies have shown that Frankia sp. has two sets of uptake hydrogenase genes, organized in synton 1 and synton 2. In the present study, phylogenetic analysis of the structural subunits of hydrogenase syntons 1 and 2 showed a distinct clustering pattern between the proteins of Frankia strains that were isolated from different host plants and non-Frankia organisms. The structural subunits of hydrogenase synton 1 of Frankia sp. CpI1, Frankia alni ACN14a, and F. alni AvCI1 were grouped together while those of Frankia spp. CcI3, KB5, UGL140104, and UGL011102 formed another group. The structural subunits of hydrogenase synton 2 of F. alni ACN14a and Frankia spp. CcI3 and BCU110501 grouped together, but those of Frankia spp. KB5 and CpI1, F. alni ArI3, and F. alniAvCI1 comprised a separate group. The structural subunits of hydrogenase syntons 1 and 2 of Frankia sp. EAN1pec were more closely related to those of non-Frankia bacteria, i.e., Streptomyces avermitilis and Anaeromyxobacter sp., respectively, than to those of other Frankia strains, suggesting the occurrence of lateral gene transfer between these organisms. In addition, the accessory Hyp proteins of hydrogenase syntons 1 and 2 of F. alni ACN14a and Frankia sp. CcI3 were shown to be phylogenetically more related to each other than to those of Frankia EAN1pec.

Keyword
Frankia cluster, lateral gene transfer, hydrogenase syntons
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-23628 (URN)10.2436/20.1501.01.78 (DOI)19440980 (PubMedID)
Available from: 2009-06-29 Created: 2009-06-29 Last updated: 2017-12-13Bibliographically approved

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