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Heterocystous cyanobacteria, Dps proteins and H2 production
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

To mitigate climate change, CO2-emitting technologies have to be exchanged by renewable alternatives such as H2. H2 can be produced by cyanobacteria, but major efforts to enhance H2 production yields by genetic modifications or optimised cultivation conditions resulted in energetic photo-to-H2 conversion efficiencies of 4.0 %, far from its theoretical maximum. New concepts to enhance photobiological H2 production are described in two separate thesis chapters.

In photobioreactors, photosynthesis can lead to high O2 concentrations promoting oxidative stress that decreases the photosynthetic efficiency. Genetic modifications could potentially increase the cellular robustness facing oxidative stress e.g. by the introduction of Dps proteins. This protein class is known to mitigate oxidative stress, but cyanobacterial Dps proteins are fairly unexplored. In the 1st thesis chapter, I searched to identify the function of five Dps proteins from the filamentous and heterocystous cyanobacterium Nostoc punctiforme. Since the physiologically active Dps proteins are twelve subunit complexes, various methods were utilised to verify their multimeric state and stability. All five NpDps formed high multimeric complexes that allowed for further enzymatic characterisations. In spectroscopic analyses NpDps1-3 were found to utilise H2O2 for Fe2+ oxidation, whereas NpDps4 only used O2. NpDps4 crystal structures revealed an uncommon ferroxidase center (FOC) with a His character. This His-type FOC was found across the cyanobacterial phylum. Based on their O2 and H2O2 consumption, all four NpDps display interesting candidates to enhance the cellular robustness in photobioreactors.

To enhance H2 production yields, a reallocation of photosynthetic energy from cell growth to H2 production is required. In the 2nd thesis chapter, Nostoc PCC 7120 ΔhupW was set under iron starvation to evaluate this cultivation strategy for the purpose of H2 production. The Fe-limited culture comprised a ~ 5.3 fold lower chlorophyll a and a ~ 4.5 fold higher specific carbohydrate concentration as compared to the control. The Fe-limited cells retained long filaments with high heterocyst frequency of ~ 6 %. The microoxic environment inside heterocysts enables efficient H2-production from O2-senstive photo fermentative pathways. Therefore, iron-starvation could display the basis of enhanced H2 production on the cost of growth. For this purpose a biofilm-containing photobioreactor was designed.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 89
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1837
Keywords [en]
H2 production, Cyanobacteria, Dps, ferritin-like proteins, iron, nutrient limitation, oxidative stress, biofilm, photobioreactor, iron homeostasis, H2O2
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-390471ISBN: 978-91-513-0716-9 (print)OAI: oai:DiVA.org:uu-390471DiVA, id: diva2:1342579
Public defence
2019-10-02, Lecture hall 80101, Ångström Laboratory, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-09-10 Created: 2019-08-13 Last updated: 2019-09-10
List of papers
1. Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions
Open this publication in new window or tab >>Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions
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2018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 43, p. 16635-16646Article in journal (Refereed) Published
Abstract [en]

DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and H2O2 detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium Nostoc punctiforme displays an unusually high number of five Dps proteins (NpDps1-5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them in vitro Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of H2O2 However, no iron oxidation with O2 as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from N. punctiforme and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

Keywords
DNA binding protein, DNA-binding proteins from starved cells, bacterial metabolism, cyanobacteria, ferritin, iron, metal homeostasis, multifunctional protein, oxidative stress, reactive oxygen species (ROS)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-365084 (URN)10.1074/jbc.RA118.002425 (DOI)000448515100008 ()30171072 (PubMedID)
Funder
NordForsk, 82845Swedish Energy Agency, 11674-5Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2019-08-13Bibliographically approved
2. The Dps4 from Nostoc punctiforme ATCC 29133 is a member of His-type FOC containing Dps protein class that can be broadly found among cyanobacteria
Open this publication in new window or tab >>The Dps4 from Nostoc punctiforme ATCC 29133 is a member of His-type FOC containing Dps protein class that can be broadly found among cyanobacteria
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2019 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 8, article id e0218300Article in journal (Refereed) Published
Abstract [en]

Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe2+ to reduce H2O2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe2+ utilizing O2, but no evidence for its use of the oxidant H2O2 could be found. We identified Zn2+ to be an effective inhibitor of the O2-mediated Fe2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390470 (URN)10.1371/journal.pone.0218300 (DOI)
Funder
NordForsk, 82845Swedish Energy Agency, 11674-5Lars Hierta Memorial Foundation
Available from: 2019-08-10 Created: 2019-08-10 Last updated: 2019-08-16Bibliographically approved
3. Structural diffusion properties of two atypical Dps from the cyanobacterium Nostoc punctiforme disclose interactions with ferredoxins and DNA
Open this publication in new window or tab >>Structural diffusion properties of two atypical Dps from the cyanobacterium Nostoc punctiforme disclose interactions with ferredoxins and DNA
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(English)Manuscript (preprint) (Other academic)
Keywords
Cyanobacteria, Ferredoxin, Ferritin-like proteins, Fluorescence correlation spectroscopy, FCS, Iron, Protein-protein interaction
National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-390709 (URN)
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-15Bibliographically approved
4. Iron limitation as a growth-restricted cultivation strategy in the diazotrophic cyanobacterium Nostoc PCC 7120 ΔhupW for a new perspective on H2 production systems
Open this publication in new window or tab >>Iron limitation as a growth-restricted cultivation strategy in the diazotrophic cyanobacterium Nostoc PCC 7120 ΔhupW for a new perspective on H2 production systems
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(English)Manuscript (preprint) (Other academic)
Keywords
Cyanobacteria, H2 production, Growth limitation, biofuels, iron, magnesium, nutrient depletion, heterocyst, cultivation strategies, iron deprivation
National Category
Natural Sciences Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-390473 (URN)
Available from: 2019-08-11 Created: 2019-08-11 Last updated: 2019-08-14

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