Transformation of the filamentous Cyanobacterium Phormidium lacuna: Electroporation versus Natural Transformation

Der Großteil der aktuellen, auf Kohlenstoff basierenden Waren wird entweder aus pflanzlichen Rohstoffen oder aus Erdöl gewonnen. Die damit verbundene Nachfrage nach Ressourcen steigt auf Grund der wachsenden Erdbevölkerung und den allgemeinen Bestrebungen zur Verbesserung des Lebensstandards. Im Gegensatz dazu sind die Verfügbarkeit von Agrarflächen und fossilen Energiereserven begrenzt. Außerdem wird der Anstieg von CO2 in der Atmosphäre seit dem Beginn der industriellen Revolution im 19. Jahrhundert als Hauptbeitrag des Menschen zur globalen Erwärmung angesehen. Es gibt einige Strategien um die Abhängigkeit von fossilen Kohlenstoffquellen zu reduzieren, wie etwa die Entwicklung von regenerativen Energien oder erhöhte Energieeffizienz. Der Einsatz von Cyanobakterien in der Biotechnologie ist eine weitere Strategie, die das Potential besitzt Landwirtschaft und Erdölindustrie als Quelle für kohlenstoffbasierte Produkte zu ersetzen. Produkte, die sich von Cyanobakterien oder Pflanzen ableiten, führen nicht zur Erhöhung des CO2 Gehalts in der Atmosphäre. Im Vergleich zu Landpflanzen ist die Kultivierung von Cyanobakterien nicht abhängig von fruchtbarem Land und viele Arten der Cyanobakterien weisen höhere Wachstumsraten als Pflanzen auf. Außerdem sind einige Cyanobakterien genetisch manipulierbar, wodurch die Synthese natürlicher Produkte verstärkt werden kann oder die rekombinante Produktion von neuen Stoffen möglich ist. Die mögliche Produktpalette reicht von preiswerten Verbindungen, wie Biokraftstoffen, bis hin zu hochwertigen Produkten, wie pharmazeutischen Wirkstoffen. Auch wenn es im Bereich der rekombinanten Biotechnologie mit Cyanobakterien schon kommerzielle Anwendung gibt, befindet sich das gesamte Feld dennoch in seinen Kinderschuhen. Während im Bereich der metabolischen Optimierung, der molekularen Werkzeuge und der Bioreaktorentwicklung in den letzten Jahren und Jahrzehnten deutliche Fortschritte gemacht wurden, ist die Etablierung von neuen Organismen nach wie vor eine Herausforderung. Die Forschung konzentriert sich größtenteils auf wenige, leicht transformierbare Modelorganismen der Ordnungen Synechococcales und Nostocales, da die Etablierung von Transformationsprotokollen für neue Stämme und Arten oft eine Herausforderung darstellt. Allerdings wird die rekombinante Biotechnologie mit Cyanobakterien ohne ein breites Spektrum an nutzbaren Stämmen möglicherweise hinter ihrem Potential zurück bleiben, da vorteilhafte Eigenschaften von vielversprechenden Kandidaten nicht genutzt werden können. Diese Arbeit setzt sich mit der beschriebenen Limitierung auseinander, indem ein verlässliches und effizientes Transformationsprotokoll für Phormidium lacuna etabliert wurde. Diese Art wurde kürzlich von unserer Arbeitsgruppe als vielversprechend für die cyanobakterielle Biotechnologie charakterisiert. Da die Gattung Phormidium bislang noch nicht für genetische Manipulation zugänglich war, war es das Ziel dieser Arbeit ein Transformationsprotokoll zu entwickeln und das Potential für die Biotechnologie an Hand der rekombinanten Ethanolsynthese zu charakterisieren. Die rekombinante Produktion von Ethanol ist gut charakterisiert innerhalb der cyanobakteriellen Biotechnologie und ermöglicht den Vergleich mit anderen Cyanobakterien hinsichtlich Produktivität. VII Bei der Etablierung eines Protokolls zur Elektroporation wurde deutlich, dass Phormidium lacuna natürlich transformierbar ist. Natürliche Transformation ist bislang nur für wenige Stämme der Cyanobakterien beschrieben und dies ist der erste Bericht für die Ordnung Oscillatoriales. Natürliche Transformation erlaubt verlässlichen und effizienten Gentransfer mittels homologer Rekombination in Phormidium lacuna. Die Integration des Selektionsmarkers kanR ins Genom von Phormidium lacuna vermittelt eine deutliche Resistenz gegenüber Kanamycin (bis zu 14,3 mg/ml). Die kanR Sequenz verteilt sich sehr schnell in allen Genomkopien: Nach zwei Kulturzyklen nach der ersten Kultivierung der entsprechenden Transformanten war die kanR Sequenz in allen Genomkopien einer Zelle vorhanden. Phormidium lacuna Transformaten für die rekombinante Ethanolproduktion wurden generiert indem die codierenden Sequenzen für die Enzyme Pyruvatdecarboxylase und Alkoholdehydrogenase ins Genom integriert wurden. Die Ethanolproduktion wurde über die Ethanolkonzentration im Kulturüberstand mittels Gaschromatographie nachgewiesen. Allerdings konnte keine erhöhte Ethanolproduktion von Transformanten im Vergleich zum Wildtyp von Phormidium lacuna nachgewiesen werden. Mögliche Gründe hierfür und geeignete Schritte in Richtung einer biotechnologischen Anwendung von Phormidium lacuna wurden diskutiert. Der unerwartete Fund der natürlichen Transformation für Phormidium lacuna ist möglicherweise ein Hinweis darauf, dass die Fähigkeit zu natürlicher Transformation weiter unter den Cyanobakterien verbreitet ist, als bislang angenommen wurde. Um diese Fragestellung zu adressieren wurden der Fund der natürlichen Transformation in dieser Arbeit und die weiteren Beispiele, die in der Literatur beschrieben sind, mit den Cyanobakterien im Allgemeinen verglichen. Basierend auf der Homologie zu Proteinen, die an der natürlichen Transformation beteiligt sind, wurden cyanobakterielle Stämme vorgeschlagen, die potentiell transformierbar sind. Der Fund der natürlichen Transformation in dieser Arbeit hat viele Implikationen. Erstens: Phormidium lacuna ist nun effizient natürlich transformierbar, was eine essentielle Voraussetzung für die weiteren Arbeitsschritte mit diesem Organismus in Bereich der rekombinanten Biotechnologie darstellt. Zweitens: Da Phormidium lacuna leicht transformierbar ist und homozygote Transformanten in kurzer Zeit generiert werden können, bietet sich dieses Bakterium als vielversprechender Modellorganismus der filamentösen Cyanobakterien ohne Heterocysten an. Drittens: Der Fund von natürlicher Transformation in einer neuen Ordnung der Cyanobakterien weiß vermutlich daraufhin, dass natürliche Kompetenz eine weit verbreitete Eigenschaft innerhalb der Cyanobakterien ist und dass möglicherweise deutlich mehr Stämme zugängliche für rekombinante Biotechnologie sind

Outer membrane proteins of Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. PCC 7120 (further referred to as Anabaena sp.) is a model system to study nitrogen fixation, cell differentiation, cell pattern formation and evolution of plastids. It is a multicellular photosynthetic microorganism consisting of two cell types, vegetative cells and nitrogen fixing heterocysts. This study focuses on the function and dynamics of the proteome of the Gram-negative outer membrane in Anabaena sp. with emphasis on cell differentiation and iron limitation. The newly developed methods for the membrane fractionation are presented, followed by analysis and comparison of the outer membrane proteomes of vegetative cells and heterocysts. The absence of major proteomic alterations in the outer membrane between two cell types, together with the presented data on GFP activity in mutant strains, experimentally support the previously proposed continuum of the outer membrane and the periplasm in Anabaena sp. filament. Also, somewhat different properties of the Anabaena sp. periplasm than in unicellular cyanobacteria are suggested. Furthermore, two common classes of the outer membrane -barrel proteins are analyzed closer. First, Alr2887 protein, as shown here, is a TolC homologue present in both cell types. Protein secretion through Alr2887 / TolC channel-tunnel is essential for the heterocysts maturation and the glycolipid layer formation. Furthermore, the inner membrane ABC transporter encoded by devBCA operon is proposed as component of the TolC efflux system in Anabaena sp. heterocysts. Second, phylogenetic analysis of the surprisingly abundant protein family of 24 TonB-dependent iron transporters in Anabaena sp. is presented. Five members of this family are detected in the outer membrane of vegetative cells under iron-repletion and two of them, All4026 and Alr0397, are explored closer. It is demonstrated that the function of these iron transporters is required for maintaining iron homeostasis of the filaments under iron-replete conditions. Consequently, their gene expression is constant and not enhanced by iron limitation. All4026 and Alr0397 have different specificity for siderophore substrates and in addition to iron transport, All4026 protein is capable of copper uptake and influence on copper homeostasis in Anabaena sp. as well

Genome mutation and physiology of Synechocystis sp. PCC 6803 wild types and pH-sensitive Photosystem II mutants

The model cyanobacterium Synechocystis sp. PCC 6803 is widely used in studies of photosynthesis, environmental sensing, and stress-response. Its capacity for straightforward genetic engineering and the early publication of its genome sequence meant that substrains of this organism have been dispersed widely among laboratories, particularly for the purposes of investigating the function of the water-splitting enzyme of photosynthesis, Photosystem II (PS II). Recently, advances in genome sequencing technology have revealed genomic divergence among these substrains, with a largely unknown level of resultant phenotypic variation. In this study, the capacity for Synechocystis sp. PCC 6803 wild types to undergo genomic change was analysed by assembly of the genome sequence of the ‘GT-O1’ and ‘GT-O2’ substrains in use at the University of Otago. In the GT-O1 substrain, a possible instance of active genome transposition processes involving a Tc1/mariner-type transposase-encoding gene was observed, and in the GT-O2 substrain a mutation detected in chlH was associated with a reduction in chlorophyll biosynthesis. It is suggested that long-term culture conditions induce genomic changes with major functional consequences in some wild-type substrains, in spite of theoretically ideal laboratory growth conditions. However, phenotypic analysis suggested that the GT-O1 substrain is comparable to other substrains of Synechocystis sp. PCC 6803 held overseas. The capacity for genome mutation in response to gene deletions affecting PS II was also analysed. Some strains carrying mutations in the extrinsic proteins or domains of PS II display an enigmatic pH 7.5-sensitive phenotype, but pH 7.5-growth of a ∆PsbO:∆PsbU strain could be rescued by genome mutations that apparently affect PS II-independent cellular processes. Assembly of the genome of a pH-insensitive ∆PsbO:∆PsbU pseudorevertant identified a mutation in pmgA that appears to affect carbon uptake, and accordingly CO2 enrichment rescued growth of some pH-sensitive PS II mutants, including the ∆PsbO:∆PsbU strain. To further investigate the effect of external pH on the membrane-embedded PS II complex, analysis of a pH-sensitive strain lacking PsbV and carrying a mutation in Loop E of the PS II core antenna CP47 protein revealed that mutations in the vicinity of the redox-active tyrosine YD appear to alter PS II redox equilibria. In a CP47 E364Q:∆PsbV mutant, the stability of YD+QA- charge pairs in PS II and possibly the capacity of YD to maintain charge equilibrium with the PS II oxygen-evolving complex was altered, likely contributing to pH-sensitivity. This suggests that pH affects PS II directly and indirectly, due to a complex interplay of pH effects on electron transport, carbon uptake, pH homeostasis, and PS II redox equilibria

Structural characterisation of Histidine Kinase 2

PhDTwo-component systems (TCS) are the predominant signal transduction pathways in prokaryotes, being present also in eukaryotic organisms, such as algae, fungi and yeast, and higher plants. TCSs play an important role in environmental signal perception and response, essentially implementing adaptation to the surrounding environment. Histidine Kinase 2 (Hik2) in cyanobacteria is a typical sensor histidine kinase, one component of a TCS, and has been identified to be a homologue protein of Arabidopsis Chloroplast Sensor Kinase (CSK). Previous research has elucidated Hik2 to regulate photosynthetic gene transcription with two response regulators, Rre1 and RppA via phosphorylation. A typical histidine kinase contains a variable sensor domain and a conserved kinase domain. It usually functions as a homodimer. This thesis describes the structural characterisation of Hik2, probing particularly its discovered oligomeric states. Results obtained from size exclusion chromatography, native-PAGE, chemical cross-linking analyses and mass spectrometry, amongst others, have shown a variety of Hik2 structural populations exist, further validated by negative stain transmission electron microscopy coupled to single particle analysis. Hik2 protein exists predominantly as a hexamer in low salt conditions, and adding NaCl dissociates hexamers into tetramers, critical for the autophosphorylation activity of Hik2. Thus, a model is proposed for the constitution change of Hik2 oligomers when salt concentration differs. In addition, the sensor domain is typically responsible for detecting environmental input, however, it is not yet clear how Hik2 and CSK sense signals. In this thesis, the structures of Hik2 and CSK sensor domains were analysed and discussed, to aid our understanding of their mechanism of signal perception and transduction.China Scholarship Council and Queen Mary University of Londo

Characterisation of the slr1212 genomic region of the freshwater cyanobacterium Synechocystis sp. PCC 6803

Synechocystis sp. pee 6803 is a unicellular, freshwater cyanobacterium. Its dependence upon light to support its photoautotrophic lifestyle increases the importance of environmental sensing mechanisms to be able to maximise lightharvesting whilst avoiding the harmful effects of light-mediated cell damage. The sequencing of the Synechocystis sp. pee 6803 genome in 1996 now allows the identification of genes that encode putative proteins with roles in sensing the environment. Two such open reading frames, slr1212 and slrI213, were identified from the genome following computer analysis of the protein sequences. These two proteins encode a putative two-component signal transduction system with a role in sensing the environment. SIr1212 possesses homology to (i) the binding domain of ethylene receptors of higher plants, (ii) P AS/P AC domains, potentially involved in ligand binding and protein dimerisation, (iii) GAF domains, which contain the chromophore binding region of plant phytochromes, and (iv) histidine kinases of two-component signal transduction systems. SIr1213 possesses homology to characterised response regulators, and contains a helix-turn-helix DNA binding motif. This study set out to characterise a physiological role for these enigmatic proteins by analysing interposon mutants. Single and double interposon mutants were generated in these open reading frames. Growth of these mutants was unaffected in different light qualities, but SIr1212 was shown to be involved in the acclimation of the Synechocystis sp. pee 6803 cells to high light irradiance as analysed by 77K fluorescence spectroscopy, which also indicated possible structural alterations in PSI reaction centres ofORF slr1212 mutants. Using laser photo acoustic spectroscopy, it was shown that Synechocystis sp. pee 6803 could release ethylene following incubation with Ace suggesting a possible ethylene biosynthetic route, though genome analysis revealed no obvious homologues of ACC oxidase, an enzyme required for conversion of Ace to ethylene in vascular plants. It is hypothesised that an ethylene signalling mechanism may be present that regulates cell responses to non-specific stress. Site-directed mutagenesis of SIr1213 that caused constitutive activation of the protein had a lethal effect in Synechocystis sp. pee 6803 cells. The mutation consisted of a substitution of the conserved aspartate residue with glutamate, thus mimicking the phosphorylated state of the protein. In summary, SIr 1212 has a role in the acclimation of cells to high light irradiance and binds ethylene, and may act in conjunction with Slr1213 to modulate these responses

Functional characterization of AtTPK3 potassium channel of Arabidopsis thaliana

My Ph.D. project has focused on the characterization of TPK3, a putative channel selective for potassium (K+) with a predicted chloroplast localization in higher plants, from biochemical, physiological and electrophysiological point of view. This protein belongs to the TPK channel family (from Tandem-Pore K+ channels) and displays amino acid sequence homology with another K+ channel studied in our laboratory, called SynK (Zanetti et al., 2010). SynK shows thylakoid localization in Cyanobacteria. The SynK channel has been shown to be critical for photosynthetic performances in Cyanobacteria, given the photosensitive phenotype displayed by the mutants lacking the SynK protein. Given the homology, we hypothesized that similarly, TPK3 might be involved in the regulation of photosynthetic processes in higher plants. So far, no information is available about the properties of TPK3, nor about its physiological roles, neither about its possible involvement in photosynthesis; the work presented in this thesis had the aim of clarifying some important aspects of the functions of TPK3. Following subcellular localization studies carried out using biochemistry and confocal microscopy techniques, the TPK3 channel was expressed in E. coli cells for subsequent electrophysiological characterization in a planar lipid bilayer setup in order to prove its function as K+ channel. The unavailability of commercial mutants for tpk3 gene required setting up of a silencing procedure via RNA interference of the messenger for the protein, in order to analyze the possible physiological roles of TPK3. The resulting silenced plants have been studied under different growth conditions to determine changes in physiology of the plants including their photosynthetic parameters. In parallel with the TPK3 project, the most important part of my Ph.D., I also followed two other major areas of research: one concerning the study of the functions of two members of plant Glutamate Receptors (GluRs) and the other one concerning the characterization of the plant homologous of the recently identified MCU (Mitochondrial Calcium Uniporter) of mammals. This thesis also includes a manuscript (Checchetto et al., 2012) to which I contributed with the heterologous expression of a calcium-activated K+ channel, SynCaK, of Cyanobacteria

Ecophysiological Investigation of the Cyanobacterium Synechococcus for Potential Biomedical Application

Cyanobacteria are important primary producers in marine and other aqueous ecosystems. Members of the genus Synechococcus are globally distributed and exhibit high potential for acclimatisation and adaptation to diverse environmental conditions. The inter-disciplinary research project Endosymbiont (University of Bremen) proposes to utilize Synechococcus for the establishment of novel biomedical therapies based upon survival and growth under human physiological conditions. The main objective of the project is to successfully introduce living cyanobacterial cells into human keratinocytes (epidermal skin cells) in a quasi-stable functional coexistence. Such photosynthetic, endosymbiotic cells would then be able to produce oxygen and consequently promote wound healing in tissues with impaired perfusion. In this work, one marine and one freshwater strain of Synechococcus were characterised with respect to their short-term growth and tolerance to different culturing conditions,such as temperature, pH and salinity ranges mimicking certain aspects of the cytosol of human keratinocytes. The marine strain Synechococcus sp. RCC2384 (Red Sea) was not able to grow at salinities lower than 100% of the artificial seawater medium. The freshwater strain Synechococcus sp. PCC7942 showed sufficient tolerance to selected osmotic conditions, with growth rates between 2.4 ± 0.64 day-1 (0% salinity), 1.7 ± 0.23 (10%),2.6 ± 0.51 (20%) and 0.84 ± 0.3 day-1 (30%) during initial exponential growth at 30 °C. The pH that the medium was initially adjusted to had no effect on the actual pH measured in the cultures presumably due to the reduced carbonate buffer system in medium of lower salinity. However, the pH at time t0 had significant effects on the subsequent growth rates (t0 – t1), and the pigment signal strength at t1. This indicated a pH sensitivity regarding growth and physiological health that could not be fully evaluated for targeted pH values in this work. Nevertheless, a more acidic pH at t0 led to higher growth rates and lower pigment fluorescence when normalised to cell concentrations. The osmotic condition likely had an indirect effect on both parameters by widening the possible pH range. Due to the adaptability shown here for Synechococcus sp. PCC7942 for osmotic concentration and pH range from below pH 7.0 up to pH 10.0, the strain emerges as the ideal candidate for potential future medical application