A novel screening protocol for the isolation of hydrogen producing Chlamydomonas reinhardtii strains

<p>Abstract</p> <p>Background</p> <p>Sealed <it>Chlamydomonas reinhardtii </it>cultures evolve significant amounts of hydrogen gas under conditions of sulfur depletion. However, the eukaryotic green alga goes through drastic metabolic changes during this nutritional stress resulting in cell growth inhibition and eventually cell death. This study aimed at isolating <it>C. reinhardtii </it>transformants which produce hydrogen under normal growth conditions to allow a continuous hydrogen metabolism without the stressful impact of nutrient deprivation.</p> <p>Results</p> <p>To achieve a steady photobiological hydrogen production, a screening protocol was designed to identify <it>C. reinhardtii </it>DNA insertional mutagenesis transformants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). The screening protocol entails a new and fast method for mutant strain selection altered in their oxygen production/consumption balance. Out of 9000 transformants, four strains with P/R ratios varying from virtually zero to three were isolated. Strain <it>apr</it>1 was found to have a slightly higher respiration rate and a significantly lower photosynthesis rate than the wild type. Sealed cultures of <it>apr</it>1 became anaerobic in normal growth medium (TAP) under moderate light conditions and induced [FeFe]-hydrogenase activity, yet without significant hydrogen gas evolution. However, Calvin-Benson cycle inactivation of anaerobically adapted <it>apr</it>1 cells in the light led to a 2-3-fold higher <it>in vivo </it>hydrogen production than previously reported for the sulfur-deprived <it>C. reinhardtii </it>wild type.</p> <p>Conclusion</p> <p>Attenuated P/R capacity ratio in microalgal mutants constitutes a platform for achieving steady state photobiological hydrogen production. Using this platform, algal hydrogen metabolism can be analyzed without applying nutritional stress. Furthermore, these strains promise to be useful for biotechnological hydrogen generation, since high <it>in vivo </it>hydrogen production rates are achievable under normal growth conditions, when the photosynthesis to respiration capacity ratio is lowered in parallel to down regulated assimilative pathways.</p

Chlorophyll Fluorescence Video Imaging: A Versatile Tool for Identifying Factors Related to Photosynthesis

Measurements of chlorophyll fluorescence provide an elegant and non-invasive means of probing the dynamics of photosynthesis. Advances in video imaging of chlorophyll fluorescence have now made it possible to study photosynthesis at all levels from individual cells to entire crop populations. Since the technology delivers quantitative data, is easily scaled up and can be readily combined with other approaches, it has become a powerful phenotyping tool for the identification of factors relevant to photosynthesis. Here, we review genetic chlorophyll fluorescence-based screens of libraries of Arabidopsis and Chlamydomonas mutants, discuss its application to high-throughput phenotyping in quantitative genetics and highlight potential future developments

Introduction of the Carotenoid Biosynthesis alpha-Branch Into Synechocystis sp. PCC 6803 for Lutein Production

Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous β-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD730 (1.03 ± 0.47 mmol mol–1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system

The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.Peer Reviewe

The Arabidopsis Protein CONSERVED ONLY IN THE GREEN LINEAGE160 Promotes the Assembly of the Membranous Part of the Chloroplast ATP Synthase

The chloroplast F(1)F(o)-ATP synthase/ATPase (cpATPase) couples ATP synthesis to the light-driven electrochemical proton gradient. The cpATPase is a multiprotein complex and consists of a membrane-spanning protein channel (comprising subunit types a, b, b′, and c) and a peripheral domain (subunits α, β, γ, δ, and ε). We report the characterization of the Arabidopsis (Arabidopsis thaliana) CONSERVED ONLY IN THE GREEN LINEAGE160 (AtCGL160) protein (AtCGL160), conserved in green algae and plants. AtCGL160 is an integral thylakoid protein, and its carboxyl-terminal portion is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins that are thought to function in ATP synthase assembly. Plants without AtCGL160 display an increase in xanthophyll cycle activity and energy-dependent nonphotochemical quenching. These photosynthetic perturbations can be attributed to a severe reduction in cpATPase levels that result in increased acidification of the thylakoid lumen. AtCGL160 is not an integral cpATPase component but is specifically required for the efficient incorporation of the c-subunit into the cpATPase. AtCGL160, as well as a chimeric protein containing the amino-terminal part of AtCGL160 and Synechocystis sp. PCC6803 Atp1, physically interact with the c-subunit. We conclude that AtCGL160 and Atp1 facilitate the assembly of the membranous part of the cpATPase in their hosts, but loss of their functions provokes a unique compensatory response in each organism

Arabidopsis CSP41 proteins form multimeric complexes that bind and stabilize distinct plastid transcripts

The spinach CSP41 protein has been shown to bind and cleave chloroplast RNA in vitro. Arabidopsis thaliana, like other photosynthetic eukaryotes, encodes two copies of this protein. Several functions have been described for CSP41 proteins in Arabidopsis, including roles in chloroplast rRNA metabolism and transcription. CSP41a and CSP41b interact physically, but it is not clear whether they have distinct functions. It is shown here that CSP41b, but not CSP41a, is an essential and major component of a specific subset of RNA-binding complexes that form in the dark and disassemble in the light. RNA immunoprecipitation and hybridization to gene chips (RIP-chip) experiments indicated that CSP41 complexes can contain chloroplast mRNAs coding for photosynthetic proteins and rRNAs (16S and 23S), but no tRNAs or mRNAs for ribosomal proteins. Leaves of plants lacking CSP41b showed decreased steady-state levels of CSP41 target RNAs, as well as decreased plastid transcription and translation rates. Representative target RNAs were less stable when incubated with broken chloroplasts devoid of CSP41 complexes, indicating that CSP41 proteins can stabilize target RNAs. Therefore, it is proposed that (i) CSP41 complexes may serve to stabilize non-translated target mRNAs and precursor rRNAs during the night when the translational machinery is less active in a manner responsive to the redox state of the chloroplast, and (ii) that the defects in translation and transcription in CSP41 protein-less mutants are secondary effects of the decreased transcript stability

Untersuchung der photobiologischen Wasserstoffproduktion eines Phosphatase defizienten Chlamydomonas reinhardtii\textit {Chlamydomonas reinhardtii} Stamms

Die einzellige, eukaryotische Grünalge $\textit {Chlamydomonas reinhardtii}$ besitzt die bemerkenswerte Fähigkeit, unter anaeroben Schwefelmangelbedingungen photobiologisch Wasserstoff zu produzieren. Jedoch stellt Schwefelmangel eine physiologische Stresssituation mit einer Beeinträchtigung der zellulären Stoffwechselprozesse dar. In der Doktorarbeit wurde die Wasserstoffbildung unter Normalbedingungen über die Isolierung von Mutanten mit einem verminderten Photosynthese/Respirationsverhältnis ($\it apr$ Mutanten) angestrebt. Für eine der isolierten Mutanten ($\it apr1$) konnte Wasserstoff parallel zur Inhibition des Calvin-Zyklus produziert werden. Molekularbiologische Untersuchungen ergaben, dass der $\it apr1$ Phänotyp durch ein deletiertes Phosphatasegen verursacht wird. Darüber hinaus erbrachte der Phosphoproteomvergleich zwischen Wildtyp und $\it apr1$ einen Hinweis, dass die Phosphatase als potentieller Interaktionspartner der Magnesium-Chelatase eine Rolle in der Regulation der Chlorophyllsynthese spielt.The unicellular, eukaryotic green alga $\textit {Chlamydomonas reinhardtii}$ is able to generate hydrogen under anaerobic, sulphur-deprived conditions. However, sulphur deprivation constitutes a physiological stress situation resulting in the disturbance of the cellular metabolism. The aim of the thesis was to isolate attenuated photosynthesis to respiration ratio mutants ($\it apr$) for the production of hydrogen under normal growth conditions. With one of the mutant strain ($\it apr1$) hydrogen can be generated in parallel to down regulated Calvin-Benson cycle reactions. Biomolecular studies revealed that the $\it apr1$ phenotype is caused by a deleted phosphatase gene. Moreover, phosphoproteome comparison between the wild type and $\it apr1$ provided information that the phosphatase is a potential interaction partner of the Mg-chelatase. Thus, the phosphatase might play a regulatory role in the chlorophyll biosynthesis pathway

A single vector-based strategy for marker-less gene replacement in Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids