Cyanobacteria are important contributors to primary production in the open oceans. Over the past decade, various photosynthesis-related genes have been found in viruses that infect cyanobacteria (cyanophages). Although photosystem II (PSII) genes are common in both cultured cyanophages and environmental samples , viral photosystem I (vPSI) genes have so far only been detected in environmental samples . Here, we have used a targeted strategy to isolate a cyanophage from the tropical Pacific Ocean that carries a PSI gene cassette with seven distinct PSI genes (psaJF, C, A, B, K, E, D) as well as two PSII genes (psbA, D). This cyanophage, P-TIM68, belongs to the T4-like myoviruses, has a prolate capsid, a long contractile tail and infects Prochlorococcus sp. strain MIT9515. Phage photosynthesis genes from both photosystems are expressed during infection, and the resultant proteins are incorporated into membranes of the infected host. Moreover, photosynthetic capacity in the cell is maintained throughout the infection cycle with enhancement of cyclic electron flow around PSI. Analysis of metagenomic data from the Tara Oceans expedition shows that phages carrying PSI gene cassettes are abundant in the tropical Pacific Ocean, composing up to 28% of T4-like cyanomyophages. They are also present in the tropical Indian and Atlantic Oceans. P-TIM68 populations, specifically, compose on average 22% of the PSI-gene-cassette carrying phages. Our results suggest that cyanophages carrying PSI and PSII genes are likely to maintain and even manipulate photosynthesis during infection of their Prochlorococcus hosts in the tropical oceans.

The initial steps of oxygenic photosynthetic electron transfer occur within photosystem II, an intricate pigment/protein transmembrane complex. Light-driven electron transfer occurs within a multistep pathway that is efficiently insulated from competing electron transfer pathways. The heart of the electron transfer system, composed of six linearly coupled redox active cofactors that enable electron transfer from water to the secondary quinone acceptor Q B , is mainly embedded within two proteins called D1 and D2. We have identified a site in silico, poised in the vicinity of the Q A intermediate quinone acceptor, which could serve as a potential binding site for redox active proteins. Here we show that modification of Lysine 238 of the D1 protein to glutamic acid (Glu) in the cyanobacterium Synechocystis sp. PCC 6803, results in a strain that grows photautotrophically. The Glu thylakoid membranes are able to perform light-dependent reduction of exogenous cytochrome c with water as the electron donor. Cytochrome c photoreduction by the Glu mutant was also shown to significantly protect the D1 protein from photodamage when isolated thylakoid membranes were illuminated. We have therefore engineered a novel electron transfer pathway from water to a soluble protein electron carrier without harming the normal function of photosystem II. cyanobacteria | energy conversion | proinhibition | photosynthesis | protein engineering P hotosynthesis is the major source of useful chemical energy in the biosphere. All photosynthetic processes require efficient electron transfer (ET) pathways that are utilized for proton gradient formation (to be used for the production of ATP) and/or accumulation of reducing equivalents (1, 2). Light energy, absorbed by light-harvesting antenna complexes, is transferred to photochemical reaction centers (RC), initiating charge separation in specific chlorophyll molecules bound to the RC proteins. Following charge separation, electrons are transferred sequentially to a series of acceptor molecules, each with a redox potential determined by its immediate environment (3, 4). The source of electron replenishment differs according to the reaction center type. For instance in purple non-sulfur bacteria, electrons are cycled back to the oxidized reaction center by a soluble cytochrome c (cyt c) type protein (5, 6). Oxygenic photosynthetic organisms (cyanobacteria, red and green algae and plants) contain two photosystems: photosystem I (PSI) and photosystem II (PSII) that work linearly (1, 7), and the source of electrons is water. One common facet of all ET pathways is the requirement for insulation of the redox active cofactors from potentially reducing/oxidizing molecules within the RC or in the surrounding media. Insulation provides the system with maximal ET rates and efficiencies and also prevents damage to the RC.PSII has a redox potential of up to 1.2 V (8, 9), required to abstract electrons from water (Fig. 1A). The photoexcited P 680 reaction center chlorophyll a primary donor transfers electron...

Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum, preventing the accumulation of anisodamine and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel, we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein. We also address the enantiomeric preference of DiH6H based on molecular modeling.

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