Markéta Linhartová

Oxygenic photosynthesis generates ATP and NADPH via linear electron flow from water to NADP ⁺ , a process thought to require photosystem I (PSI) for reductant formation. Here we demonstrate that oxygenic photosynthesis can operate without PSI in the cyanobacterium Synechocystis sp. PCC 6803. Using genetic engineering and adaptive laboratory evolution, we obtained PSI-deficient lineages capable of photoautotrophic growth, inorganic carbon fixation, and light-driven oxygen evolution. PSI-independent photoautotrophy arose from co-mutations in at least two proteins, including the translation elongation factor G (FusA), and required a functional NDH-1 complex. We propose that the light-driven electron transport in the evolved strains is reorganised in two branches: one involving terminal oxidases to generate proton motive force, and a second in which reverse NDH-1 activity exploits this gradient to produce reductant. These findings uncover unexpected plasticity in the thylakoid electron transport network and prompt a reassessment of the canonical requirement for PSI in oxygenic photosynthesis.

Cable bacteria are filamentous sulphide-oxidising bacteria that transport electrons through conductive periplasmic fibres over centimetre-scale distances in redox-stratified sediments to respire oxygen. Here, we show that the freshwater cable bacterium Electronema aureum GS employs a versatile extracellular electron transfer (EET) system to respire insoluble electron acceptors under anoxic conditions, achieving growth rates comparable to aerobic respiration. Electrochemical and molecular analyses reveal that E. aureum GS performs both direct and riboflavin-mediated EET to electrodes, including at +600 mV vs. Ag/AgCl, an unusually high redox potential typically not accessed by electroactive bacteria. Two distinct cell-surface redox components were identified, which are metal-dependent, pH-sensitive, and heat-labile, consistent with outer-membrane-localised cytochromes. The redox shuttle riboflavin accumulated in bioelectrochemical systems with E. aureum GS, supporting mediated EET. These findings reveal a respiratory flexibility in E. aureum GS and highlight the role of EET in energy conservation in dynamic redox environments.

Oxygenic photosynthesis in cyanobacteria, algae, and plants fixes carbon dioxide and releases oxygen. This process depends on ATP and NADPH generated by light-driven reactions involving multi-protein complexes and mobile electron carriers. Central to these reactions are Photosystem II (PSII) and Photosystem I (PSI), which enable linear electron flow (LEF) from water to NADP ⁺ molecules ¹ . LEF both drives proton gradient formation for ATP synthesis and supports NADP ⁺ reduction via ferredoxin-mediated electron transfer from PSI ^2–4 . According to prevailing models, LEF cannot occur without PSI, as its absence would block NADP⁺ reduction. Here we show that oxygenic photosynthesis can take place independently of PSI in the cyanobacterium Synechocystis PCC 6803. Through adaptive laboratory evolution, we obtained PSI-deficient lineages capable of photoautotrophic growth, inorganic carbon fixation, and light-dependent oxygen evolution. PSI-independent photoautotrophy emerged following co-mutations in at least two proteins, including the translation elongation factor G (FusA), and was abolished when the NDH-1 complex was disrupted. These results suggest that NDH-1 can substitute for PSI by operating in reverse to transfer electrons from plastoquinone to ferredoxin. Our findings reveal an unanticipated plasticity in the thylakoid electron transport network of cyanobacteria and compel a revision of existing models of oxygenic photosynthesis.

Microbial sulfur cycling is typically divided into an oxidative and a reductive branch, with microbes driving either sulfide oxidation or sulfate reduction distinguished by their genomic setup. Paradoxically, filamentous cable bacteria perform electrogenic sulfide oxidation but contain genes indicative of sulfate reduction, including the reductive type of dissimilatory sulfite reductase (DsrAB), whereas they apparently lack the canonical sulfur transferase DsrEFH essential for sulfur oxidation. AlphaFold2 structure prediction of conserved cable bacteria proteins with unknown functions identified a protein complex resembling canonical DsrEFH (hereafter termed DsrEFH type II). In vitro characterization of heterologously expressed DsrEFH type II confirmed its sulfur transferase function and, together with site-directed mutagenesis, verified that the conserved cysteine, Cys67, is the active sulfur transfer residue. Genes encoding the novel DsrEFH type II were found in 985 prokaryotic genomes. They typically co-occurred with genes for reductive DsrAB in microbes characterized as sulfate reducers or sulfur disproportionators. This study not only fills an important gap in the sulfide oxidation pathway of cable bacteria, but also suggests that a wide range of sulfate reducing bacteria may be more metabolically versatile than currently understood, representing a major shift in the perception of this globally significant physiological group of microorganisms.

Cable bacteria are filamentous sulphide-oxidisers performing long-distance electron transfer in redox-stratified sediments by transporting electrons over centimetre-scale distances to reduce oxygen. Here, we show that the freshwater cable bacterium Electronema aureum GS can respire insoluble electron acceptors under anoxic conditions via a versatile extracellular electron transfer (EET) system, supporting growth rates comparable to those under aerobic conditions. Using electrochemical and molecular biology analyses, we demonstrate that E. aureum GS engages in both direct and mediated electron transfer to electrodes, including at +600 mV vs. Ag/AgCl—an unusually high redox potential typically not accessed by electroactive bacteria. Two distinct cell-surface redox components were identified, which are metal-dependent, pH-sensitive, and heat-labile, consistent with outer-membrane-localised cytochromes. Moreover, the redox shuttle riboflavin accumulated extracellularly and enhanced current production in bioelectrochemical systems, indicating a role for soluble mediators in cable bacteria. Together, these findings reveal a previously unrecognised respiratory flexibility in E. aureum GS and highlight EET as a key alternative strategy for energy conservation in fluctuating redox environments.

Summary: An increasing number of small proteins has been identified in the genomes of well‐annotated organisms, including the model cyanobacterium Synechocystis sp. PCC 6803. We describe a newly assigned protein comprising 37 amino acids that is encoded upstream of the superoxide dismutase SodB encoding gene. To clarify the role of SliP4, we analyzed a Synechocystis sliP4 mutant and a strain containing a fully active, Flag‐tagged variant of SliP4 (SliP4.f). The initial hypothesis that this small protein might be functionally related to SodB could not be supported. Instead, we provide evidence that it fulfills important functions related to the organization of photosynthetic complexes. Therefore, we named it a small light‐induced protein of 4 kDa, SliP4. This protein is strongly induced under high‐light conditions. The lack of SliP4 causes a light‐sensitive phenotype due to impaired cyclic electron flow and state transitions. Interestingly, SliP4.f was co‐isolated with NDH1 complex and both photosystems. The interaction between SliP4.f and all three types of complexes was further confirmed by additional pulldowns and 2D‐electrophoreses. We propose that the dimeric SliP4 serves as a molecular glue promoting the aggregation of thylakoid complexes, which contributes to different electron transfer modes and energy dissipation under stress conditions.

Synechocystis sp. PCC 6803 is a model cyanobacterium, glucose‐tolerant substrains of which are commonly used as laboratory strains. In recent years, it has become evident that ‘wild‐type’ strains used in different laboratories show some differences in their phenotypes. We report here the chromosome sequence of our Synechocystis sp. PCC 6803 substrain, named substrain GT‐T. The chromosome sequence of GT‐T was compared to those of two other commonly used laboratory substrains, GT‐S and PCC‐M. We identified 11 specific mutations in the GT‐T substrain, whose physiological consequences are discussed. We also provide an update on evolutionary relationships between different Synechocystis sp. PCC 6803 substrains.