The trimeric organisation of photosystem I is not necessary for the iron-stress induced CP43′ protein to functionally associate with this reaction centre

AbstractA mutant of Synechocystis PCC 6803 lacking the PsaL subunit of photosystem I (PSI) has been grown in iron-deficient media to induce the expression of the isiA gene, which encodes the chlorophyll a-binding protein CP43′. The purpose of this was to establish whether or not the formation of an 18-mer CP43′-PSI supercomplex reported for wild type Synechocystis cells [Nature 412 (2001) 743–745] was dependent on the trimeric conformation of the cyanobacterial PSI reaction centre. Structural characterisation by electron microscopy and single particle image analysis has revealed that the PsaL-mutant does not form trimers of PSI. However, despite this, CP43′ was found to associate with the PSI monomer. The PSI monomer bound six or seven copies of CP43′ along one edge of the PSI monomer and can be compared with one segment of the trimeric 18-mer CP43′-PSI supercomplex. We therefore conclude that the trimeric nature of cyanobacterial PSI is not required for the assembly of the CP43′ antenna system under iron-deficient conditions

Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide

Interest in the exploitation of microalgae for biotechnological applications has increased over the last decade, and microalgae are now viewed as offering a sustainable alternative to traditionally used host chassis. A number of recombinant proteins have been expressed in genetically modified algal strains, with the green alga Chlamydomonas reinhardtii being a particularly popular host strain. While nuclear transformation is possible with this organism, chloroplast transformation offers more reliable expression, and several proteins have been expressed in the stroma. Here, we present the first utilisation of the thylakoid lumen for recombinant protein production in microalgae. A bacterial export signal peptide was used to efficiently translocate two recombinant proteins, a fluorescent reporter protein (pHRed) and a biopharmaceutical model substrate (scFv) into the thylakoid lumen. This approach expands the algal chloroplast genetic toolkit and offers a means of expressing proteins that are difficult to express in the stroma for reasons of toxicity, stability or a requirement for disulphide bonding

Thermodynamic limits on oxygenic photosynthesis around M-dwarf stars: Generalized models and strategies for optimization

We explore the feasibility and potential characteristics of photosynthetic light-harvesting on exo-planets orbiting in the habitable zone of low mass stars ($< 1$ M$_{\odot}$). As stellar temperature, $T_{s}$, decreases, the irradiance maximum red-shifts out of the $400 \textrm{nm} \leq \lambda < 750$ nm range of wavelengths that can be utilized by \emph{oxygenic} photosynthesis on Earth. However, limited irradiance in this region does not preclude oxygenic photosynthesis and Earth's plants, algae and cyanobacteria all possess very efficient \emph{light-harvesting antennae} that facilitate photosynthesis in very low light. Here we construct general models of photosynthetic light-harvesting structures to determine how an oxygenic photosystem would perform in different irradiant spectral fluxes. We illustrate that the process of light-harvesting, capturing energy over a large antenna and concentrating it into a small \emph{reaction centre}, must overcome a fundamental \emph{entropic barrier}. We show that a plant-like antenna cannot be adapted to the light from stars of $T_{s}<3400$ K, as increasing antenna size offers diminishing returns on light-harvesting. This can be overcome if one introduces a slight \emph{enthalpic gradient}, to the antenna. Interestingly, this strategy appears to have been adopted by Earth's oxygenic cyanobacteria, and we conclude that \emph{bacterial} oxygenic photosynthesis is feasible around even the lowest mass M-dwarf stars.Comment: 5 Figures, submitted to Astrobiology and awaiting return of revie

Probing the biogenesis pathway and dynamics of thylakoid membranes

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery. Cyanobacterial thylakoid membranes host the molecular machinery for the light-dependent reactions of photosynthesis and respiratory electron flow. Here, the authors show that newly synthesized thylakoids emerge between the plasma membrane and pre-existing thylakoids and describe the time-dependent assembly process of photosynthetic complexes

Cyanobacteria in motion

This work was supported by a grant from the DFG (WI 2014/7-1) to A.W

Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment