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  • 1.
    Agervald, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Maturation and Regulation of Cyanobacterial Hydrogenases2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Accelerated global warming plus an increasing need for energy is an equation not easily solved, thus new forms of sustainable energy production are urgently requested. In this context hydrogen production based on a cyanobacterial system offers an environmentally friendly alternative for energy capture and conversion. Cyanobacteria can produce hydrogen gas from sun light and water through the combination of photosystems and hydrogenases, and are suitable to cultivate in large scale.

    In the present thesis the maturation process of [NiFe]-hydrogenases is investigated with special focus on transcription of the accessory genes encoding proteins needed for assembly of the large and possibly also for the small hydrogenase subunit. The cyanobacteria used are two N2-fixing, filamentous, heterocystous strains; Nostoc sp. strain PCC 7120 and Nostoc punctiforme PCC 73102.

    For a biotechnological exploration of hydrogen production tools for regulatory purposes are important. The transcription factor CalA (cyanobacterial AbrB like) (Alr0946 in the genome) in Nostoc sp. strain PCC 7120 was found to be involved in hydrogen metabolism by regulating the transcription of the maturation protein HypC. Further the bidirectional hydrogenase activity was down-regulated in the presence of elevated levels of CalA, a result important to take into account when optimizing cyanobacteria for hydrogen production.

    CalA regulates at least 25 proteins in Nostoc sp. strain PCC 7120 and one of the down-regulated proteins was superoxide dismutase, FeSOD. The characterization of FeSOD shows that it has a specific and important function in the oxidative stress tolerance of Nostoc sp. stain PCC 7120. Since CalA is involved in regulation of both the hydrogen metabolism as well as stress responses these findings indicate that Alr0946 is an important transcription factor in Nostoc sp. strain PCC 7120 active on a global level in the cell.

    This thesis adds more knowledge concerning maturation and regulation of cyanobacterial hydrogenases which might be useful for future large scale hydrogen.

    List of papers
    1. The CyAbrB transcription factor Alr0946 regulates the iron superoxide dismutase in Nostoc sp. strain PCC 7120
    Open this publication in new window or tab >>The CyAbrB transcription factor Alr0946 regulates the iron superoxide dismutase in Nostoc sp. strain PCC 7120
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    In the present investigation we analyse the results of induced over-production of the CyAbrB transcription factor Alr0946 in the cyanobacterium Nostoc sp. PCC 7120 with special focus on its effects on FeSOD. With gel based quantitative proteomics the induced over-expression of Alr0946 was shown to influence the abundance of at least 25 proteins. One of the proteins with a significant lower abundance was FeSOD, one of two types of superoxide dismutases in Nostoc sp. PCC 7120. The change in protein abundance was also followed by lower transcript as well as activity levels. Purified Alr0946 from Nostoc sp. PCC 7120 was shown to interact with the promoter region of alr2938, encoding FeSOD, indicating a transcriptional regulation of FeSOD by Alr0946. The Alr0946 over-expression strain showed a bleaching phenotype with lower growth rate and truncated filaments already two days after induction of over-expression. The phenotype was even more pronounced when illumination was increased from 35 to 125 μmol m-2s-1. This is in line with an increased need of FeSOD during a stronger oxidative stress. The results indicate that Alr0946 is involved in regulation of stress responses and that FeSOD has a specific and important function in the oxidative stress tolerance of the multicellular cyanobacterium Nostoc sp. PCC 7120.

    Keyword
    Nostoc sp. PCC 7120, FeSOD, Alr0946, transcription factor, CyAbrB
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-110862 (URN)
    Available from: 2009-11-27 Created: 2009-11-27 Last updated: 2016-04-21
    2. Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120
    Open this publication in new window or tab >>Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120
    2008 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, 69- p.Article in journal (Refereed) Published
    Abstract [en]

    Background: The maturation of hydrogenases into active enzymes is a complex process and e. g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N2fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hypgenes in Nostoc sp. strain PCC 7120. Results: RTPCRs demonstrated that the six hypgenes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between hupSL, encoding the small and large subunits of the uptake hydrogenase, and the hypoperon, and two downstream ORFs from the hypgenes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifUlike protein. Orthologues of the four ORFs asr0689alr0692, with a highly conserved genomic arrangement positioned between hupSL, and the hyp genes are found in several other N2fixing cyanobacteria, but are absent in non N2fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended hypoperon, appearing between 11 and 79 times in the genome. Conclusion: This study demonstrated that five ORFs upstream of the hypgene cluster are cotranscribed with the hypgenes, and identified three TSPs in the extended hypgene cluster in Nostoc sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-110173 (URN)10.1186/1471-2180-8-69 (DOI)000256297500001 ()
    Available from: 2009-11-05 Created: 2009-11-05 Last updated: 2017-12-12
    3. CalA, a cyanobacterial AbrB protein, interacts with the upstream region of hypC and acts as a repressor of its transcription in the cyanobacterium Nostoc sp. strain PCC 7120
    Open this publication in new window or tab >>CalA, a cyanobacterial AbrB protein, interacts with the upstream region of hypC and acts as a repressor of its transcription in the cyanobacterium Nostoc sp. strain PCC 7120
    Show others...
    2010 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 3, 880-890 p.Article in journal (Refereed) Published
    Abstract [en]

    The filamentous, heterocystous, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain, depending on growth condition, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, Alr0946, belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of hypC. Transcriptional investigations showed that alr0946 is co-transcribed with the downstream gene alr0947, which encodes a putative protease from the abortive infection superfamily, Abi. Alr0946 was shown to interact specifically not only with the upstream region of hypC but also with its own upstream region, acting as a repressor on both. The bidirectional hydrogenase activity was significant down-regulated when Alr0946 was over-expressed demonstrating a correlation to the transcription factor, either direct or indirect. In silico studies showed that homologues to both Alr0946 and Alr0947 are highly conserved proteins within cyanobacteria with a very similar physical organisation of the corresponding structural genes. Possible functions of the co-transcribed downstream protein Alr0947 are presented. In addition, we present a 3D model of the CyAbrB domain of Alr0946 and putative DNA-binding mechanisms are discussed.

    Keyword
    Nostoc sp. strain PCC 7120, hypC, Alr0946, CyAbrB, hydrogen metabolism
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-110863 (URN)10.1128/AEM.02521-09 (DOI)000274017400030 ()
    Available from: 2009-11-27 Created: 2009-11-27 Last updated: 2017-12-12
    4. Isolation and characterization of thylakoid membranes from the filamentous cyanobacterium Nostoc punctiforme
    Open this publication in new window or tab >>Isolation and characterization of thylakoid membranes from the filamentous cyanobacterium Nostoc punctiforme
    Show others...
    2007 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 131, no 4, 622-634 p.Article in journal (Refereed) Published
    Abstract [en]

    Nostoc punctiforme strain Pasteur Culture Collection (PCC) 73102, a sequenced filamentous cyanobacterium capable of nitrogen fixation, is used as a model organism for characterization of bioenergetic processes during nitrogen fixation in Nostoc. A protocol for isolating thylakoid membranes was developed to examine the biochem. and biophys. aspects of photosynthetic electron transfer. Thylakoids were isolated from filaments of N. punctiforme by pneumatic pressure-drop lysis. The activity of photosynthetic enzymes in the isolated thylakoids was analyzed by measuring oxygen evolution activity, fluorescence spectroscopy and ESR spectroscopy. Electron transfer was found functional in both PSII and PSI. Electron transfer measurements in PSII, using diphenylcarbazide as electron donor and 2,6-dichlorophenolindophenol as electron acceptor, showed that 80% of the PSII centers were active in water oxidn. in the final membrane prepn. Anal. of the membrane protein complexes was made by 2D gel electrophoresis, and identification of representative proteins was made by mass spectrometry. The ATP synthase, several oligomers of PSI, PSII and the NAD(P)H dehydrogenase (NDH)-1L and NDH-1M complexes, were all found in the gels. Some differences were noted compared with previous results from Synechocystis sp. PCC 6803. Two oligomers of PSII were found, monomeric and dimeric forms, but no CP43-less complexes. Both dimeric and monomeric forms of Cyt b6/f could be obsd. In all, 28 different proteins were identified, of which 25 are transmembrane proteins or membrane associated ones.

    Keyword
    Nostoc punctiforme, isolation, membranes, thylakoids, proteomics, photosystem II, photosystem I
    National Category
    Chemical Sciences Biological Sciences
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-12478 (URN)10.1111/j.1399-3054.2007.00982.x (DOI)000250763500010 ()18251853 (PubMedID)
    Available from: 2012-05-08 Created: 2007-12-27 Last updated: 2017-12-11Bibliographically approved
    5. Transcript analysis of the extended hyp-operons in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133
    Open this publication in new window or tab >>Transcript analysis of the extended hyp-operons in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133
    Show others...
    2011 (English)In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 4, no 186Article in journal (Other academic) Published
    Abstract [en]

    The ability of cyanobacteria to capture solar energy, via oxygenic photosynthesis, and convert that energy to molecular hydrogen (H2) has made them an interesting group of organisms with potential as future energy producers. There are three types of enzymes directly involved in the cyanobacterial hydrogen metabolism; nitrogenases that produce H2 as a by-product when fixating atmospheric nitrogen, uptake hydrogenases that catalyze the oxidation of H2,thereby preventing energy losses from the cells, and bidirectional hydrogenases that has the capacity to both oxidize and reduce H2. Hydrogenases are complex metalloenzymes, and the insertion of ligands and correct folding of the proteins require assistance of accessory proteins, the Hyp proteins. Cyanobacterial hydrogenases are NiFe-type hydrogenases and consist of a large and a small subunit. Today, the maturation process of the large subunit has been uncovered to a large extent in cyanobacteria, mostly by analogy assumptions from studies done in other bacteria such as Escherichia coli but also from mutational analyses in cyanobacteria, while the maturation process of the small subunit is still unknown. Recently a set of genes, putatively involved in the maturation process of the small subunit, was discovered in Nostoc sp. PCC 7120 and Nostoc punctiforme ATCC 29133. These five ORFs, encoding unknown proteins, are located in between the uptake hydrogenase structural genes and the hyp-genes were shown to be transcribed together with the hyp-genes in Nostoc PCC 7120. The ORFs upstream the hyp-genes can be found in the same genomic arrangement in other filamentous, nitrogen fixing cyanobacterial strains but are interestingly missing in strains incapable of nitrogen fixation. In this study we have further investigated the function of the ORFs upstream the hyp-genes by studying their transcription pattern after nitrogen depletion in the filamentous, nitrogen fixing strains Nostoc PCC 7120 and N. punctiforme. The transcription pattern were compared to the transcription pattern of hupS and hoxY, encoding the uptake and bidirectional hydrogenase small subunits, nifD, encoding a nitrogenase subunit and hypC and hypF, encoding the maturation process accessory proteins HypC and HypF. All the five ORFs upstream the hyp-genes, in both organisms, were upregulated after nitrogen step down in accordance with the transcription pattern for hupS, nifD, hypC and hypF which support the theory that these genes might be involved in the maturation of the small subunit.

    Place, publisher, year, edition, pages
    London: BioMed Central, 2011
    Keyword
    Nostoc sp.strain PCC 7120, Nostoc punctiforme ATCC29133, hyp, hydrogenase maturation
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-110865 (URN)10.1186/1756-0500-4-186 (DOI)
    Available from: 2009-11-27 Created: 2009-11-27 Last updated: 2017-12-12
  • 2.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Baebprasert, Wipawee
    Program of Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    The CyAbrB transcription factor Alr0946 regulates the iron superoxide dismutase in Nostoc sp. strain PCC 7120Manuscript (preprint) (Other academic)
    Abstract [en]

    In the present investigation we analyse the results of induced over-production of the CyAbrB transcription factor Alr0946 in the cyanobacterium Nostoc sp. PCC 7120 with special focus on its effects on FeSOD. With gel based quantitative proteomics the induced over-expression of Alr0946 was shown to influence the abundance of at least 25 proteins. One of the proteins with a significant lower abundance was FeSOD, one of two types of superoxide dismutases in Nostoc sp. PCC 7120. The change in protein abundance was also followed by lower transcript as well as activity levels. Purified Alr0946 from Nostoc sp. PCC 7120 was shown to interact with the promoter region of alr2938, encoding FeSOD, indicating a transcriptional regulation of FeSOD by Alr0946. The Alr0946 over-expression strain showed a bleaching phenotype with lower growth rate and truncated filaments already two days after induction of over-expression. The phenotype was even more pronounced when illumination was increased from 35 to 125 μmol m-2s-1. This is in line with an increased need of FeSOD during a stronger oxidative stress. The results indicate that Alr0946 is involved in regulation of stress responses and that FeSOD has a specific and important function in the oxidative stress tolerance of the multicellular cyanobacterium Nostoc sp. PCC 7120.

  • 3.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Baebprasert, Wipawee
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Zhang, Xiaohui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Incharoensakdi, Aran
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    The CyAbrB transcription factor CalA regulates the iron superoxide dismutase in Nostoc sp. strain PCC 71202010In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 12, no 10, 2826-2837 p.Article in journal (Refereed)
    Abstract [en]

    P>In the present investigation the results of induced over-production of the CyAbrB transcription factor CalA (Cyanobacterial AbrB-like, annotated as Alr0946) in the cyanobacterium Nostoc sp. PCC 7120 were analysed. The CalA overexpression strain showed a bleaching phenotype with lower growth rate and truncated filaments 2 days after induction of overexpression. The phenotype was even more pronounced when illumination was increased from 35 to 125 mu mol m-2 s-1. Using gel-based quantitative proteomics, the induced overexpression of CalA was shown to downregulate the abundance of FeSOD, one of two types of superoxide dismutases in Nostoc sp. PCC 7120. The change in protein abundance was also accompanied by lower transcript as well as activity levels. Purified recombinant CalA from Nostoc sp. PCC 7120 was shown to interact with the promoter region of alr2938, encoding FeSOD, indicating a transcriptional regulation of FeSOD by CalA. The bleaching phenotype is in line with a decreased tolerance against oxidative stress and indicates that CalA is involved in regulation of cellular responses in which FeSOD has an important and specific function in the filamentous cyanobacterium Nostoc sp. PCC 7120.

  • 4.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Camsund, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    CRISPR in the extended hyp-operon of the cyanobacterium Nostoc sp. strain PCC 7120, characteristics and putative function(s)2012In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 37, no 10, 8828-8833 p.Article in journal (Refereed)
    Abstract [en]

    The presence of small RNAs (sRNA) and their functions in transcriptional regulation has lately turned into a hot topic. Since cyanobacteria often face changes in the surrounding environment, they need to have a well working system for stress response. Quick adaption is necessary, and an RNA-based regulatory system is thus useful. One example of these sRNAs is CRISPRs. In this work we report the existence of a CRISPR within the hyp-operon (hyp genes encode proteins responsible for the maturation of hydrogenases) of the filamentous cyanobacterium Nostoc sp. strain PCC 7120. We present data concerning its characteristics and putative function(s) and raise the question concerning the importance of this CRISPR array and other CRISPR systems in general. In addition, we discuss the use of the CRISPR system as a potential bacterial genetic defence mechanism to achieve robust, cyanobacterial cultures in large scale, commercial production units.

  • 5.
    Agervald, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Zhang, Xiaohui
    Department of Biological Sciences, Purdue University.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Devine, Ellenor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    CalA, a cyanobacterial AbrB protein, interacts with the upstream region of hypC and acts as a repressor of its transcription in the cyanobacterium Nostoc sp. strain PCC 71202010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 3, 880-890 p.Article in journal (Refereed)
    Abstract [en]

    The filamentous, heterocystous, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain, depending on growth condition, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, Alr0946, belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of hypC. Transcriptional investigations showed that alr0946 is co-transcribed with the downstream gene alr0947, which encodes a putative protease from the abortive infection superfamily, Abi. Alr0946 was shown to interact specifically not only with the upstream region of hypC but also with its own upstream region, acting as a repressor on both. The bidirectional hydrogenase activity was significant down-regulated when Alr0946 was over-expressed demonstrating a correlation to the transcription factor, either direct or indirect. In silico studies showed that homologues to both Alr0946 and Alr0947 are highly conserved proteins within cyanobacteria with a very similar physical organisation of the corresponding structural genes. Possible functions of the co-transcribed downstream protein Alr0947 are presented. In addition, we present a 3D model of the CyAbrB domain of Alr0946 and putative DNA-binding mechanisms are discussed.

  • 6. Baebprasert, Wipawee
    et al.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Incharoensakdi, Aran
    Response of H-2 production and Hox-hydrogenase activity to external factors in the unicellular cyanobacterium Synechocystis sp strain PCC 68032010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 13, 6611-6616 p.Article in journal (Refereed)
    Abstract [en]

    The effects of external factors on both H-2 production and bidirectional Hox-hydrogenase activity were examined in the non-N-2-fixing cyanobacterium Synechocystis PCC 6803. Exogenous glucose and increased osmolality both enhanced H-2 production with optimal production observed at 0.4% and 20 mosmol kg(-1), respectively. Anaerobic condition for 24 h induced significant higher H(2)ase activity with cells in BC11(0) showing highest activities. Increasing the pH resulted in an increased Hox-hydrogenase activity with an optimum at pH 7.5. The Hox-hydrogenase activity gradually increased with increasing temperature from 30 degrees C to 60 degrees C with the highest activity observed at 70 degrees C. A low concentration at 100 mu M of either DTT or beta-mercaptoethanol resulted in a minor stimulation of H-2 production. beta-Mercaptoethanol added to nitrogen- and sulfur-deprived cells stimulated H-2 production significantly. The highest Hox-hydrogenase activity was observed in cells in BG11(0)-S-deprived condition and 750 mu M beta-mercaptoethanol measured at a temperature of 70 degrees C; 14.32 mu mol H-2 mg chl alpha(-1) min(-1).

  • 7.
    Camsund, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Devine, Ellenor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Peter, Yohanoun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    A HupS-GFP fusion protein demonstrates a heterocyst specific localisation of the uptake hydrogenase in the cyanobacterium Nostoc punctiformeIn: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968Article in journal (Refereed)
  • 8.
    Camsund, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Devine, Ellenor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Yohanoun, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    A HupS-GFP fusion protein demonstrates a heterocyst-specific localization of the uptake hydrogenase in Nostoc punctiforme2011In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 316, no 2, 152-159 p.Article in journal (Refereed)
    Abstract [en]

    All diazotrophic filamentous cyanobacteria contain an uptake hydrogenase that is involved in the reoxidation of H-2 produced during N-2-fixation. In Nostoc punctiforme ATCC 29133, N-2-fixation takes place in the microaerobic heterocysts, catalysed by a nitrogenase. Although the function of the uptake hydrogenase may be closely connected to that of nitrogenase, the localization in cyanobacteria has been under debate. Moreover, the subcellular localization is not understood. To investigate the cellular and subcellular localization of the uptake hydrogenase in N. punctiforme, a reporter construct consisting of the green fluorescent protein (GFP) translationally fused to HupS, within the complete hupSL operon, was constructed and transferred into N. punctiforme on a self-replicative vector by electroporation. Expression of the complete HupS-GFP fusion protein was confirmed by Western blotting using GFP antibodies. The N. punctiforme culture expressing HupS-GFP was examined using laser scanning confocal microscopy, and fluorescence was exclusively detected in the heterocysts. Furthermore, the fluorescence in mature heterocysts was localized to several small or fewer large clusters, which indicates a specificity of the subcellular localization of the uptake hydrogenase.

  • 9.
    Cardona, Tanai
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Battchikova, Natalia
    Department of Biology, University of Turku.
    Zhang, Pengpeng
    Department of Biology, University of Turku.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Aro, Eva-Mari
    Department of Biology, University of Turku.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Magnuson, Ann
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme2009In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1787, no 4, 252-263 p.Article in journal (Refereed)
    Abstract [en]

    Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes.We could identify asignificant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled PhotosystemII complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.

  • 10. Dasgupta, Chitralekha Nag
    et al.
    Gilbert, J. Jose
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Heidorn, Thorsten
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Borgvang, Stig A.
    Skjanes, Kari
    Das, Debabrata
    Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production2010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 19, 10218-10238 p.Article in journal (Refereed)
    Abstract [en]

    Hydrogen production through biological routes is promising because they are environmentally friendly. Hydrogen production through biophotolysis or photofermentation is usually a two stage process. In the first stage CO2 is utilized for biomass production which is followed by hydrogen production in the second stage in anaerobic/sulfur-deprived conditions. In addition, one-stage photobiological hydrogen production process can be achieved using selected cyanobacterial strains. The major challenges confronting the large scale production of biomass/hydrogen are limited not only on the performance of the photobioreactors in which light penetration in dense cultures is a major bottleneck but also on the characteristics of the organisms. Other dependable factors include area/volume (AN) ratio, mode of agitation, temperature and gas exchange. Photobioreactors of different geometries are reported for biohydrogen production: Tubular, Flat plate, Fermentor type etc. Every reactor has its own advantages and disadvantages. Airlift, helical tubular and flat plate reactors are found most suitable with respect to biomass production. These bioreactors may be employed for hydrogen production with necessary modifications to overcome the existing bottlenecks like gas hold up, oxygen toxicity and poor agitation. This review article attempts to focus on existing photobioreactors with respect to biomass generation and hydrogen production and the steps taken to improve its performance through engineering innovation that definitely help in the future design and construction of photobioreactors.

  • 11.
    Devine, Ellenor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Cyanobacterial Hydrogen Metabolism: Regulation and Maturation of Hydrogenases2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In times with elevated CO2 levels and global warming there is a need of finding alternatives to carbon based energy carriers. One such environmental friendly solution could be H2 produced by living organisms. Cyanobacteria are good candidates since they can produce H2 from sunlight and water through the combination of photosynthesis and H2 producing enzymes i.e. nitrogenases and/or [NiFe]-hydrogenases. This thesis investigates the maturation and transcriptional regulation of [NiFe]-hydrogenases in cyanobacteria, with a special focus on hydrogenase specific proteases.

    The core of all hydrogenases consists of the small and large subunit. The large subunit in which the catalytic site is located goes through an extenstive maturation process which ends with a proteolytic cleavage performed by a hydrogenase specific protease (HupW/HoxW). This thesis shows that within the maturation process of hydrogenases, the proteolytic cleavage is probably the only step that is specific with respect to different types of hydrogenases i.e. one type of protease cleaves only one type of hydrogenase. Further in-silico analysis revealed that these proteases and the hydrogenases might have co-evolved since ancient time and that the specificity observed could be the result of a conserved amino acid sequence which differs between the two types of proteases (HupW/HoxW).

    A number of different transcription factors were revealed and shown to interact with the promoter regions of several of the genes encoding maturation proteins. The results indicate that the hydrogenase specific proteases are regulated on a transcriptional level in a similar manner as the hydrogenases they cleave. This thesis contributes with knowledge concerning transcriptional regulation and protein regulation of hydrogenases which will be useful for designing genetically engineered cyanobacteria with an improved and adjustable H2 production.

    List of papers
    1. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120
    Open this publication in new window or tab >>Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120
    2009 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 9, 53- p.Article in journal (Refereed) Published
    Abstract [en]

    Background: The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results: In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for[NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion: Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the[NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-129076 (URN)10.1186/1471-2180-9-53 (DOI)000265847000001 ()
    Available from: 2010-08-06 Created: 2010-08-05 Last updated: 2017-12-12
    2. The protease HupW is specific for the uptake hydrogenase in Nostoc sp. strain PCC 7120
    Open this publication in new window or tab >>The protease HupW is specific for the uptake hydrogenase in Nostoc sp. strain PCC 7120
    (English)Manuscript (preprint) (Other academic)
    Keyword
    HupW, cyanobacteria, hydrogenase, hydrogenase specific protease
    Identifiers
    urn:nbn:se:uu:diva-134247 (URN)
    Available from: 2010-11-23 Created: 2010-11-23 Last updated: 2012-09-12
    3. Transcriptional analysis of the hydrogenase specific proteases in Nostoc punctiforme and Nostoc sp. strain PCC 7120
    Open this publication in new window or tab >>Transcriptional analysis of the hydrogenase specific proteases in Nostoc punctiforme and Nostoc sp. strain PCC 7120
    (English)Manuscript (preprint) (Other academic)
    Keyword
    transcription, regulation, cyanobacteria, hydrogenase
    National Category
    Biological Sciences
    Research subject
    Chemistry with specialization in Microbial Chemistry
    Identifiers
    urn:nbn:se:uu:diva-134245 (URN)
    Available from: 2010-11-23 Created: 2010-11-23 Last updated: 2012-09-12
    4. A HupS-GFP fusion protein demonstrates a heterocyst specific localisation of the uptake hydrogenase in the cyanobacterium Nostoc punctiforme
    Open this publication in new window or tab >>A HupS-GFP fusion protein demonstrates a heterocyst specific localisation of the uptake hydrogenase in the cyanobacterium Nostoc punctiforme
    Show others...
    (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968Article in journal (Refereed) Submitted
    Keyword
    GFP, hydrogenase, HupL, cyanobacteria
    National Category
    Biological Sciences
    Research subject
    Chemistry with specialization in Microbial Chemistry
    Identifiers
    urn:nbn:se:uu:diva-133876 (URN)
    Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2017-12-12Bibliographically approved
    5. An in-silico study of the NtcA transcription factor family in filamentous cyanobacteria
    Open this publication in new window or tab >>An in-silico study of the NtcA transcription factor family in filamentous cyanobacteria
    (English)Manuscript (preprint) (Other academic)
    Keyword
    NtcA, cyanobacteria, ntirogen control
    National Category
    Biochemistry and Molecular Biology Biological Sciences
    Research subject
    Chemistry with specialization in Microbial Chemistry
    Identifiers
    urn:nbn:se:uu:diva-134241 (URN)
    Available from: 2010-11-23 Created: 2010-11-23 Last updated: 2012-09-12
    6.
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  • 12.
    Devine, Ellenor
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Transcriptional analysis of the hydrogenase specific proteases in Nostoc punctiforme and Nostoc sp. strain PCC 7120Manuscript (preprint) (Other academic)
  • 13.
    Devine, Ellenor
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    The protease HupW is specific for the uptake hydrogenase in Nostoc sp. strain PCC 7120Manuscript (preprint) (Other academic)
  • 14.
    Devine, Ellenor
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    An in-silico study of the NtcA transcription factor family in filamentous cyanobacteriaManuscript (preprint) (Other academic)
  • 15.
    Ekman, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Ow, Saw Yen
    ChELSI Institute, Department of Chemical and Process Engineering, The University of Sheffield.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Zhang, Xiaohui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    van Wagenen, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Wright, Phillip C.
    ChELSI Institute, Department of Chemical and Process Engineering, The University of Sheffield.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Metabolic Adaptations in a H-2 Producing Heterocyst-Forming Cyanobacterium: Potentials and Implications for Biological Engineering2011In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 4, 1772-1784 p.Article in journal (Refereed)
    Abstract [en]

    Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the ability to fix atmospheric nitrogen and photoproduce hydrogen through the enzyme nitrogenase. The H-2 produced is reoxidized by an uptake hydrogenase. Inactivation of the uptake hydrogenase in N. punctiforme leads to increased H-2 release but unchanged rates of N-2 fixation, indicating redirected metabolism. System-wide understanding of the mechanisms of this metabolic redirection was obtained using complementary quantitative proteomic approaches, at both the filament and the heterocyst level. Of the total 1070 identified and quantified proteins, 239 were differentially expressed in the uptake hydrogenase mutant (NHM5) as compared to wild type. Our results indicate that the inactivation of uptake hydrogenase in N. punctiforme changes the overall metabolic equilibrium, affecting both oxygen reduction mechanisms in hetcrocysts as well as processes providing reducing equivalents for metabolic functions such as N-2 fixation. We identify specific metabolic processes used by NHM5 to maintain a high rate of N-2 fixation, and thereby potential targets for further improvement of nitrogenase based H-2 photogeneration. These targets include, but are not limited to, components of the oxygen scavenging capacity and cell envelope of heterocysts and proteins directly or indirectly involved in reduced carbon transport from vegetative cells to heterocysts.

  • 16.
    Ekman, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Sandh, Gustaf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Nenninger, Anja
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Oliveira, Paulo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.2014In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 16, no 3, 829-844 p.Article in journal (Refereed)
    Abstract [en]

    Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium.

  • 17.
    Heidorn, Thorsten
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Camsund, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Huang, Hsin-Ho
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Oliveira, Paulo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions2011In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 497, 539-579 p.Article, review/survey (Refereed)
    Abstract [en]

    Cyanobacteria are the only prokaryotes capable of using sunlight as their energy, water as an electron donor, and air as a source of carbon and, for some nitrogen-fixing strains, nitrogen. Compared to algae and plants, cyanobacteria are much easier to genetically engineer, and many of the standard biological parts available for Synthetic Biology applications in Escherichia coli can also be used in cyanobacteria. However, characterization of such parts in cyanobacteria reveals differences in performance when compared to E. coli, emphasizing the importance of detailed characterization in the cellular context of a biological chassis. Furthermore, cyanobacteria possess special characteristics (e.g., multiple copies of their chromosomes, high content of photosynthetically active proteins in the thylakoids, the presence of exopolysaccharides and extracellular glycolipids, and the existence of a circadian rhythm) that have to be taken into account when genetically engineering them. With this chapter, the synthetic biologist is given an overview of existing biological parts, tools and protocols for the genetic engineering, and molecular analysis of cyanobacteria for Synthetic Biology applications.

  • 18.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    The Cyanobacterial Uptake Hydrogenase: Regulation, Maturation and Function2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    With accellerating global warming and pollution problems a change of energy regime is necessary. Solar energy offers a clean and unlimited energy source of enormous potential. Due to it’s intermittenet nature solar energy must be stored - ideally in the chemical bond of a carrier molecule. Hydrogen gas, H2, an energy carrier with water as only emission when used in a fuel cell, is considered to be the choise for the future. In this context cyanobacteria show promising potential as future H2 factories since they can produce H2 from solar energy and water. The main enzymes directly involved in cyanobacterial hydrogen metabolism are nitrogenases and hydrogenases. Cyanobacterial hydrogenases are either uptake hydrogenases or bidirectional hydrogenases and their maturation requires assistance of six maturation proteins and two hydrogenase specific proteases. In this thesis the transcriptional regulation, maturation and function of the cyanobacterial uptake hydrogenases were investigated in the filamentous, heterocyst forming strains Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120. Five genes, encoding proteins putatively involved in the maturation of the uptake hydrogenase were identified upstream the known maturation genes. Two transcription factors, CalA and CalB, were found interacting with the stretch of DNA forming the upstream regions of the uptake hydrogenase structural genes and the novel maturation genes. The expression of the uptake hydrogenase were  heterocysts specific and the specificity mapped to a short promoter region starting -57 bp upstream the transcription start point. In addition, the function of the uptake hydrogenase was inserted in a metabolic context. Among the proteases, a conserved region was discovered possibly involved in determining the hydrogenase specificity. This thesis has given valuable information about the transcriptional regulation, maturation and function of the uptake hydrogenase in filamentous, heterocystous cyanobacteria and identified new targets for bioengineering of mutant strains with higher H2 production rates.

    List of papers
    1. Characterization of the hupSL promoter activity in Nostoc punctiforme ATCC 29133
    Open this publication in new window or tab >>Characterization of the hupSL promoter activity in Nostoc punctiforme ATCC 29133
    Show others...
    2009 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 9, 54- p.Article in journal (Refereed) Published
    Abstract [en]

    Background: In cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H-2, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H-2 and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H-2. Nostoc punctiforme ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. Generally, little is known about the transcriptional regulation of the cyanobacterial uptake hydrogenases. In this study gel shift assays showed that NtcA has a specific affinity to a region of the hupSL promoter containing a predicted NtcA binding site. The predicted NtcA binding site is centred at 258.5 bp upstream the transcription start point (tsp). To further investigate the hupSL promoter, truncated versions of the hupSL promoter were fused to either gfp or luxAB, encoding the reporter proteins Green Fluorescent Protein and Luciferase, respectively. Results: Interestingly, all hupsSL promoter deletion constructs showed heterocyst specific expression. Unexpectedly the shortest promoter fragment, a fragment covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. Deletion of the NtcA binding site neither affected the expression to any larger extent nor the heterocyst specificity. Conclusion: Obtained data suggest that the hupSL promoter in N. punctiforme is not strictly dependent on the upstream NtcA cis element and that the shortest promoter fragment (- 57 to tsp) is enough for a high and heterocyst specific expression of hupSL. This is highly interesting because it indicates that the information that determines heterocyst specific gene expression might be confined to this short sequence or in the downstream untranslated leader sequence.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-129139 (URN)10.1186/1471-2180-9-54 (DOI)000265028800001 ()
    Funder
    EU, FP7, Seventh Framework Programme, 212508
    Available from: 2010-08-09 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
    2. Transcript analysis of the extended hyp-operons in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133
    Open this publication in new window or tab >>Transcript analysis of the extended hyp-operons in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133
    Show others...
    2011 (English)In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 4, no 186Article in journal (Other academic) Published
    Abstract [en]

    The ability of cyanobacteria to capture solar energy, via oxygenic photosynthesis, and convert that energy to molecular hydrogen (H2) has made them an interesting group of organisms with potential as future energy producers. There are three types of enzymes directly involved in the cyanobacterial hydrogen metabolism; nitrogenases that produce H2 as a by-product when fixating atmospheric nitrogen, uptake hydrogenases that catalyze the oxidation of H2,thereby preventing energy losses from the cells, and bidirectional hydrogenases that has the capacity to both oxidize and reduce H2. Hydrogenases are complex metalloenzymes, and the insertion of ligands and correct folding of the proteins require assistance of accessory proteins, the Hyp proteins. Cyanobacterial hydrogenases are NiFe-type hydrogenases and consist of a large and a small subunit. Today, the maturation process of the large subunit has been uncovered to a large extent in cyanobacteria, mostly by analogy assumptions from studies done in other bacteria such as Escherichia coli but also from mutational analyses in cyanobacteria, while the maturation process of the small subunit is still unknown. Recently a set of genes, putatively involved in the maturation process of the small subunit, was discovered in Nostoc sp. PCC 7120 and Nostoc punctiforme ATCC 29133. These five ORFs, encoding unknown proteins, are located in between the uptake hydrogenase structural genes and the hyp-genes were shown to be transcribed together with the hyp-genes in Nostoc PCC 7120. The ORFs upstream the hyp-genes can be found in the same genomic arrangement in other filamentous, nitrogen fixing cyanobacterial strains but are interestingly missing in strains incapable of nitrogen fixation. In this study we have further investigated the function of the ORFs upstream the hyp-genes by studying their transcription pattern after nitrogen depletion in the filamentous, nitrogen fixing strains Nostoc PCC 7120 and N. punctiforme. The transcription pattern were compared to the transcription pattern of hupS and hoxY, encoding the uptake and bidirectional hydrogenase small subunits, nifD, encoding a nitrogenase subunit and hypC and hypF, encoding the maturation process accessory proteins HypC and HypF. All the five ORFs upstream the hyp-genes, in both organisms, were upregulated after nitrogen step down in accordance with the transcription pattern for hupS, nifD, hypC and hypF which support the theory that these genes might be involved in the maturation of the small subunit.

    Place, publisher, year, edition, pages
    London: BioMed Central, 2011
    Keyword
    Nostoc sp.strain PCC 7120, Nostoc punctiforme ATCC29133, hyp, hydrogenase maturation
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-110865 (URN)10.1186/1756-0500-4-186 (DOI)
    Available from: 2009-11-27 Created: 2009-11-27 Last updated: 2017-12-12
    3. Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120
    Open this publication in new window or tab >>Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120
    2008 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 8, 69- p.Article in journal (Refereed) Published
    Abstract [en]

    Background: The maturation of hydrogenases into active enzymes is a complex process and e. g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N2fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hypgenes in Nostoc sp. strain PCC 7120. Results: RTPCRs demonstrated that the six hypgenes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from hypF and 445 bp upstream of hypC, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between hupSL, encoding the small and large subunits of the uptake hydrogenase, and the hypoperon, and two downstream ORFs from the hypgenes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifUlike protein. Orthologues of the four ORFs asr0689alr0692, with a highly conserved genomic arrangement positioned between hupSL, and the hyp genes are found in several other N2fixing cyanobacteria, but are absent in non N2fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended hypoperon, appearing between 11 and 79 times in the genome. Conclusion: This study demonstrated that five ORFs upstream of the hypgene cluster are cotranscribed with the hypgenes, and identified three TSPs in the extended hypgene cluster in Nostoc sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-110173 (URN)10.1186/1471-2180-8-69 (DOI)000256297500001 ()
    Available from: 2009-11-05 Created: 2009-11-05 Last updated: 2017-12-12
    4. Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120
    Open this publication in new window or tab >>Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120
    2009 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 9, 53- p.Article in journal (Refereed) Published
    Abstract [en]

    Background: The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively. Results: In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of hupW in Nostoc punctiforme ATCC 29133 and hupW and hoxW in Nostoc sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (hupW) and LexA (hoxW). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for[NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase. Conclusion: Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the[NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-129076 (URN)10.1186/1471-2180-9-53 (DOI)000265847000001 ()
    Available from: 2010-08-06 Created: 2010-08-05 Last updated: 2017-12-12
    5.
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    6. Metabolic Adaptations in a H-2 Producing Heterocyst-Forming Cyanobacterium: Potentials and Implications for Biological Engineering
    Open this publication in new window or tab >>Metabolic Adaptations in a H-2 Producing Heterocyst-Forming Cyanobacterium: Potentials and Implications for Biological Engineering
    Show others...
    2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 4, 1772-1784 p.Article in journal (Refereed) Published
    Abstract [en]

    Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the ability to fix atmospheric nitrogen and photoproduce hydrogen through the enzyme nitrogenase. The H-2 produced is reoxidized by an uptake hydrogenase. Inactivation of the uptake hydrogenase in N. punctiforme leads to increased H-2 release but unchanged rates of N-2 fixation, indicating redirected metabolism. System-wide understanding of the mechanisms of this metabolic redirection was obtained using complementary quantitative proteomic approaches, at both the filament and the heterocyst level. Of the total 1070 identified and quantified proteins, 239 were differentially expressed in the uptake hydrogenase mutant (NHM5) as compared to wild type. Our results indicate that the inactivation of uptake hydrogenase in N. punctiforme changes the overall metabolic equilibrium, affecting both oxygen reduction mechanisms in hetcrocysts as well as processes providing reducing equivalents for metabolic functions such as N-2 fixation. We identify specific metabolic processes used by NHM5 to maintain a high rate of N-2 fixation, and thereby potential targets for further improvement of nitrogenase based H-2 photogeneration. These targets include, but are not limited to, components of the oxygen scavenging capacity and cell envelope of heterocysts and proteins directly or indirectly involved in reduced carbon transport from vegetative cells to heterocysts.

    Place, publisher, year, edition, pages
    American Chemical Society (ACS), 2011
    Keyword
    iTRAQ, shotgun proteomics, Nostoc punctiforme ATCC 29133, uptake hydrogenase mutant, nitrogenase, dinitrogen fixation, heterocysts, NHMS, biotechnology
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-129227 (URN)10.1021/pr101055v (DOI)000288924000031 ()21284387 (PubMedID)
    Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12
  • 19.
    Holmqvist, Marie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindberg, Pia
    Agervald, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Transcript analysis of the extended hyp-operons in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 291332011In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 4, no 186Article in journal (Other academic)
    Abstract [en]

    The ability of cyanobacteria to capture solar energy, via oxygenic photosynthesis, and convert that energy to molecular hydrogen (H2) has made them an interesting group of organisms with potential as future energy producers. There are three types of enzymes directly involved in the cyanobacterial hydrogen metabolism; nitrogenases that produce H2 as a by-product when fixating atmospheric nitrogen, uptake hydrogenases that catalyze the oxidation of H2,thereby preventing energy losses from the cells, and bidirectional hydrogenases that has the capacity to both oxidize and reduce H2. Hydrogenases are complex metalloenzymes, and the insertion of ligands and correct folding of the proteins require assistance of accessory proteins, the Hyp proteins. Cyanobacterial hydrogenases are NiFe-type hydrogenases and consist of a large and a small subunit. Today, the maturation process of the large subunit has been uncovered to a large extent in cyanobacteria, mostly by analogy assumptions from studies done in other bacteria such as Escherichia coli but also from mutational analyses in cyanobacteria, while the maturation process of the small subunit is still unknown. Recently a set of genes, putatively involved in the maturation process of the small subunit, was discovered in Nostoc sp. PCC 7120 and Nostoc punctiforme ATCC 29133. These five ORFs, encoding unknown proteins, are located in between the uptake hydrogenase structural genes and the hyp-genes were shown to be transcribed together with the hyp-genes in Nostoc PCC 7120. The ORFs upstream the hyp-genes can be found in the same genomic arrangement in other filamentous, nitrogen fixing cyanobacterial strains but are interestingly missing in strains incapable of nitrogen fixation. In this study we have further investigated the function of the ORFs upstream the hyp-genes by studying their transcription pattern after nitrogen depletion in the filamentous, nitrogen fixing strains Nostoc PCC 7120 and N. punctiforme. The transcription pattern were compared to the transcription pattern of hupS and hoxY, encoding the uptake and bidirectional hydrogenase small subunits, nifD, encoding a nitrogenase subunit and hypC and hypF, encoding the maturation process accessory proteins HypC and HypF. All the five ORFs upstream the hyp-genes, in both organisms, were upregulated after nitrogen step down in accordance with the transcription pattern for hupS, nifD, hypC and hypF which support the theory that these genes might be involved in the maturation of the small subunit.

  • 20.
    Huang, Hsin-Ho
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Camsund, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Heidorn, Thorsten
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 8, 2577-2593 p.Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like P-rnpB and variants of P-rbcL, and a version of the promoter P-trc with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

  • 21.
    Khetkorn, Wanthanee
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Incharoensakdi, Aran
    Enhanced biohydrogen production by the N-2-fixing cyanobacterium Anabaena siamensis strain TISTR 80122010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 23, 12767-12776 p.Article in journal (Refereed)
    Abstract [en]

    The efficiency of hydrogen production depends on several factors We focused on external conditions leading to enhanced hydrogen production when using the N-2 fixing cyanobacterium Anabaena siamensis TISTR 8012 a novel strain isolated from a rice paddy field in Thailand In this study we controlled key factors affecting hydrogen production such as cell age light intensity time of light incubation and source of carbon Our results showed an enhanced hydrogen production when cells at log phase were adapted under N-2 fixing condition using 0 5% fructose as carbon source and a continuous illumination of 200 mu E m(-2) s(-1) for 12 h under anaerobic incubation The maximum hydrogen production rate was 32 mu mol H-2 mg chl a(-1) h(-1) This rate was higher than that observed in the model organisms Anabaena PCC 7120 Nostoc punctiforme ATCC 29133 and Synechocystis PCC 6803 This higher production was likely caused by a higher nitrogenase activity since we observed an upregulation of nifD The production did not increase after 12 h which was probably due to an increased activity of the uptake hydrogenase as evidenced by an increased hupL transcript level Interestingly a proper adjustment of light conditions such as intensity and duration is important to minimize both the photodamage of the cells and the uptake hydrogenase activity Our results indicate that A siamensis TISTR 8012 has a high potential for hydrogen production with the ability to utilize sugars as substrate to produce hydrogen.

  • 22.
    Li, Xin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Sandh, Gustaf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Nenninger, Anja
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Muro-Pastor, Alicia M.
    Stensjo, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Differential transcriptional regulation of orthologous dps genes from two closely related heterocyst-forming cyanobacteria2015In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 362, no 6, fnv017Article in journal (Refereed)
    Abstract [en]

    In cyanobacteria, DNA-binding proteins from starved cells (Dps) play an important role in the cellular response to oxidative and nutritional stresses. In this study, we have characterized the cell-type specificity and the promoter regions of two orthologous dps genes, Npun_R5799 in Nostoc punctiforme and alr3808 in Anabaena sp. PCC 7120. A transcriptional start site (TSS), identical in location to the previously identified proximal TSS of alr3808, was identified for Npun_R5799 under both combined nitrogen supplemented and N-2-fixing growth conditions. However, only alr3808 was also transcribed from a second distal TSS. Sequence homologies suggest that the promoter region containing the distal TSS is not conserved upstream of orthologous genes among heterocyst-forming cyanobacteria. The analysis of promoter GFP-reporter strains showed a different role in governing cell-type specificity between the proximal and distal promoter of alr3808. We here confirmed the heterocyst specificity of the distal promoter of alr3808 and described a very early induction of its expression during proheterocyst differentiation. In contrast, the complete promoters of both genes were active in all cells. Even though Npun_R5799 and alr3808 are orthologs, the regulation of their respective expression differs, indicating distinctions in the function of these cyanobacterial Dps proteins depending on the strain and cell type.

  • 23.
    Llavero Pasquina, Marcel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Engineered light controlled cell development for enhanced hydrogen production in Nostoc punctiforme ATCC 291332016Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    The aim of this thesis is to enhance heterocyst-based hydrogen production inNostoc punctiforme ATCC 29133. We envision to do so by finely regulatingthe ratio of heterocyst in order to optimize the filament energy balance. Wehereby report the development of an optogenetic synthetic switch basedon the native PcpeC promoter. The optogenetic switch featured a 24-folddynamic range when measuring reporter sfGFP fluorescence. Such a geneticgate was conceived to artificially drive the expression of hetR, the masterregulator of heterocyst development. We achieved to induce enhancedheterocyst differentiation in the presence of ammonia only by changing thechromatic properties of the light source. Thus, the natural cell developmentregulation was substituted by effectively introducing a full person-drivencontrol over the process.

  • 24. Maneeruttanarungroj, Cherdsak
    et al.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Incharoensakdi, Aran
    A newly isolated green alga, Tetraspora sp. CU2551, from Thailand with efficient hydrogen production2010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 24, 13193-13199 p.Article in journal (Refereed)
    Abstract [en]

    A novel unicellular hydrogen-producing green alga was isolated from fresh water pond in Pathumthani province, Thailand. Under light microscope, this alga was identified as belonging to the genus Tetraspora. Phylogenetic analysis of 18S rRNA sequence revealed that the green alga, identified as Tetraspora sp. CU2551, is closely related to other unicellular green algal species. Tetraspora sp. CU2551 had the shortest doubling time when grown in Tris-acetate-phosphate (TAP) medium under a light intensity of 48-92 mu E/m(2)/s and a temperature of 36 C. Hydrogen production increased with increasing pH from 5.75 to 9.30; however, almost no production was observed at a pH of 5.25. Addition of 0.5 mM P-mercaptoethanol to the TAP medium stimulated hydrogen production about two-fold. During the hydrogen production phase, the use of TAP medium lacking both nitrogen and sulfur resulted in about 50% increase in the hydrogen production. This was in contrast to only a small increase in the production when either nitrogen or sulfur was omitted in TAP medium. The stimulation of hydrogen production by 0.5 mM beta-mercaptoethanol under nitrogen- and sulfur-deprived conditions occurred only when the cells were grown at a light intensity lower than 5 mu E/m(2)/s with no effects at higher intensities. Maximal calculated hydrogen production, 17.3-61.7 mu mol/mg Chl a/h, is a very high production rate compared to other green algae and makes Tetraspora sp. CU2551 an interesting model strain for photobiological hydrogen production.

  • 25.
    Moirangthem, Lakshmipyari Devi
    et al.
    Mizoram Univ, Dept Biotechnol, Aizawl 796004, Mizoram, India..
    Ibrahim, Kalibulla Syed
    Mizoram Univ, Dept Biotechnol, Aizawl 796004, Mizoram, India..
    Vanlalsangi, Rebecca
    Mizoram Univ, Dept Biotechnol, Aizawl 796004, Mizoram, India..
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Bhattacharya, Jyotirmoy
    Mizoram Univ, Dept Biotechnol, Aizawl 796004, Mizoram, India..
    Molecular Cloning and Biochemical Characterization of the Iron Superoxide Dismutase from the Cyanobacterium Nostoc punctiforme ATCC 29133 and Its Response to Methyl Viologen-Induced Oxidative Stress2015In: Molecular Biotechnology, ISSN 1073-6085, E-ISSN 1559-0305, Vol. 57, no 11-12, 1003-1009 p.Article in journal (Refereed)
    Abstract [en]

    Superoxide dismutase (SOD) detoxifies cell-toxic superoxide radicals and constitutes an important component of antioxidant machinery in aerobic organisms, including cyanobacteria. The iron-containing SOD (SodB) is one of the most abundant soluble proteins in the cytosol of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133, and therefore, we investigated its biochemical properties and response to oxidative stress. The putative SodB-encoding open reading frame Npun_R6491 was cloned and overexpressed in Escherichia coli as a C-terminally hexahistidine-tagged protein. The purified recombinant protein had a SodB specific activity of 2560 +/- 48 U/mg protein at pH 7.8 and was highly thermostable. The presence of a characteristic iron absorption peak at 350 nm, and its sensitivity to H2O2 and azide, confirmed that the SodB is an iron-containing SOD. Transcript level of SodB in nitrogen-fixing cultures of N. punctiforme decreased considerably (threefold) after exposure to an oxidative stress-generating herbicide methyl viologen for 4 h. Furthermore, in-gel SOD activity analysis of such cultures grown at increasing concentrations of methyl viologen also showed a loss of SodB activity. These results suggest that SodB is not the primary scavenger of superoxide radicals induced by methyl viologen in N. punctiforme.

  • 26.
    Ow, Saw Yen
    et al.
    Department of Chemical and Process Engineering, The University of Sheffield.
    Cardona, Tanai
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Taton, Arnaud
    Virginia Commonwealth University.
    Magnuson, Ann
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Wright, Philip C
    Department of Chemical and Process Engineering, The University of Sheffield.
    Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-Plex isobaric peptide tags2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 4, 1615-1628 p.Article in journal (Refereed)
    Abstract [en]

    The filamentous cyanobacterium Nostoc sp. strain PCC 7120 is capable of fixing atmospheric nitrogen. The labile nature of the core process requires the terminal differentiation of vegetative cells to form heterocysts, specialized cells with altered cellular and metabolic infrastructure to mediate the N2-fixing process. We present an investigation targeting the cellular proteomic expression of the heterocysts compared to vegetative cells of a population cultured under N2-fixing conditions. New 8-plex iTRAQ reagents were used on enriched replicate heterocyst and vegetative cells, and replicate N2-fixing and non-N2-fixing filaments to achieve accurate measurements. With this approach, we successfully identified 506 proteins, where 402 had confident quantifications. Observations provided by purified heterocyst analysis enabled the elucidation of the dominant metabolic processes between the respective cell types, while emphasis on the filaments enabled an overall comparison. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production.

  • 27.
    Ow, Saw Yen
    et al.
    Department of Chemical and Process Engineering, The University of Sheffield.
    Noirel, Josselin
    Department of Chemical and Process Engineering, The University of Sheffield.
    Cardona, Tanai
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Taton, Arnaud
    Virginia Commonwealth University.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Wright, Philip C
    Department of Chemical and Process Engineering, The University of Sheffield.
    Quantitative overview of N2 fixation in Nostoc punctiforme ATCC 29133 through cellular enrichments and iTRAQ shotgun proteomics2009In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 1, 187-198 p.Article in journal (Refereed)
    Abstract [en]

    Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the capacity to fix atmospheric N2. Its ability to mediate this process is similar to that described for Nostoc sp. PCC 7120, where vegetative cells differentiate into heterocysts. Quantitative proteomic investigations at both the filament level and the heterocyst level are presented using isobaric tagging technology (iTRAQ), with 721 proteins at the 95% confidence interval quantified across both studies. Observations from both experiments yielded findings confirmatory of both transcriptional studies, and published Nostoc sp. PCC 7120 iTRAQ data. N. punctiforme exhibits similar metabolic trends, though changes in a number of metabolic pathways are less pronounced than in Nostoc sp. PCC 7120. Results also suggest a number of proteins that may benefit from future investigations. These include ATP dependent Zn-proteases, N-reserve degraders and also redox balance proteins. Complementary proteomic data sets from both organisms present key precursor knowledge that is important for future cyanobacterial biohydrogen research.

  • 28.
    Skjånes, Kari
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lopes Pinto, Fernando
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Evidence for transcription of three genes with characteristics of hydrogenases in the green alga Chlamydomonas noctigama2010In: International journal of hydrogen energy, ISSN 0360-3199, E-ISSN 1879-3487, Vol. 35, no 3, 1074-1088 p.Article in journal (Refereed)
    Abstract [en]

    Some green algae have shown the ability to produce hydrogen under anaerobic conditions. The production of hydrogen in green algae is catalyzed by hydrogenases, which are small monomeric enzymes with high conversion efficiency and high oxygen sensitivity. Most green algae analyzed to date where hydrogenase genes are detected, have been shown to contain two distinct hydrogenases. However, very little is known about which functions the two different enzymes represent. There are also many unknowns within the mechanisms behind hydrogen production as to the roles hydrogenases play under different conditions, and consequently also about the potential for optimization of a hydrogen production process which could be found in this respect. This study focuses on the possibility for the presence of more than two hydrogenases in a single green alga. A large number of degenerate primers were designed and used to produce 3′-RACE products, which in turn were used to design gene specific primers used for PCR and 5′-RACE reactions. The sequences were aligned with known algal hydrogenases to identify products which had homology to these. Products where homology was identified were then explored further. A high number of clones from each band were sequenced to identify products with similar lengths which would not show up as separate bands on a gel. Sequences found to have homology with algal hydrogenases were translated into putative amino acid sequences and analyzed further to obtain detailed information about the presence of specific amino acids with known functions in the enzyme. This information was used to evaluate the likelihood of these transcripts coding for true hydrogenases, versus hydrogenase-like or narf-like proteins. We here present evidence showing that Chlamydomonas noctigama is able to transcribe three genes which share a significant number of characteristics with other known algal FeFe-hydrogenases. The three genes have been annotated HYDA1, HYDA2 and HYDA3.

1 - 28 of 28
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