Electron microscopy for imaging organelles in plants and algae.

Recent developments in both instrumentation and image analysis algorithms have allowed three-dimensional electron microscopy (3D-EM) to increase automated image collections through large tissue volumes using serial block-face scanning EM (SEM) and to achieve near-atomic resolution of macromolecular complexes using cryo-electron tomography (cryo-ET) and sub-tomogram averaging. In this review, we discuss applications of cryo-ET to cell biology research on plant and algal systems and the special opportunities they offer for understanding the organization of eukaryotic organelles with unprecedently resolution. However, one of the most challenging aspects for cryo-ET is sample preparation, especially for multicellular organisms. We also discuss correlative light and electron microscopy (CLEM) approaches that have been developed for ET at both room and cryogenic temperatures.

Myelin sheath and cyanobacterial thylakoids as concentric multilamellar structures with similar bioenergetic properties.

There is a surprisingly high morphological similarity between multilamellar concentric thylakoids in cyanobacteria and the myelin sheath that wraps the nerve axons. Thylakoids are multilamellar structures, which express photosystems I and II, cytochromes and ATP synthase necessary for the light-dependent reaction of photosynthesis. Myelin is a multilamellar structure that surrounds many axons in the nervous system and has long been believed to act simply as an insulator. However, it has been shown that myelin has a trophic role, conveying nutrients to the axons and producing ATP through oxidative phosphorylation. Therefore, it is tempting to presume that both membranous structures, although distant in the evolution tree, share not only a morphological but also a functional similarity, acting in feeding ATP synthesized by the ATP synthase to the centre of the multilamellar structure. Therefore, both molecular structures may represent a convergent evolution of life on Earth to fulfill fundamentally similar functions.

Structural insight into the mechanism of energy transfer in cyanobacterial phycobilisomes.

Phycobilisomes (PBS) are the major light-harvesting machineries for photosynthesis in cyanobacteria and red algae and they have a hierarchical structure of a core and peripheral rods, with both consisting of phycobiliproteins and linker proteins. Here we report the cryo-EM structures of PBS from two cyanobacterial species, Anabaena 7120 and Synechococcus 7002. Both PBS are hemidiscoidal in shape and share a common triangular core structure. While the Anabaena PBS has two additional hexamers in the core linked by the 4th linker domain of ApcE (LCM). The PBS structures predict that, compared with the PBS from red algae, the cyanobacterial PBS could have more direct routes for energy transfer to ApcD. Structure-based systematic mutagenesis analysis of the chromophore environment of ApcD and ApcF subunits reveals that aromatic residues are critical to excitation energy transfer (EET). The structures also suggest that the linker protein could actively participate in the process of EET in both rods and the cores. These results provide insights into the organization of chromophores and the mechanisms of EET within cyanobacterial PBS.

A unique photosystem I reaction center from a chlorophyll d-containing cyanobacterium Acaryochloris marina.

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c6 ) and the reduction of ferredoxin. This catalytic reaction is realized by a transmembrane electron transfer chain consisting of primary electron donor (a special chlorophyll (Chl) pair) and electron acceptors A0 , A1 , and three Fe4 S4 clusters, FX , FA , and FB . Here we report the PSI structure from a Chl d-dominated cyanobacterium Acaryochloris marina at 3.3 Å resolution obtained by single-particle cryo-electron microscopy. The A. marina PSI exists as a trimer with three identical monomers. Surprisingly, the structure reveals a unique composition of electron transfer chain in which the primary electron acceptor A0 is composed of two pheophytin a rather than Chl a found in any other well-known PSI structures. A novel subunit Psa27 is observed in the A. marina PSI structure. In addition, 77 Chls, 13 α-carotenes, two phylloquinones, three Fe-S clusters, two phosphatidyl glycerols, and one monogalactosyl-diglyceride were identified in each PSI monomer. Our results provide a structural basis for deciphering the mechanism of photosynthesis in a PSI complex with Chl d as the dominating pigments and absorbing far-red light.

Revealing new sponge-associated cyanobacterial diversity: Novel genera and species.

Cyanobacteria are often reported as abundant components of the sponge microbiome; however their diversity below the phylum level is still underestimated. Aiming to broaden our knowledge of sponge-cyanobacteria association, we isolated cyanobacterial strains from Aegean Sea sponges in previous research, which revealed high degree of novel cyanobacterial diversity. Herein, we aim to further characterize sponge-associated cyanobacteria and re-evaluate their classification based on an extensive polyphasic approach, i.e. a combination of molecular, morphological and ecological data. This approach resulted in the description of five new genera (Rhodoploca, Cymatolege, Metis, Aegeococcus, and Thalassoporum) and seven new species (R. sivonenia, C. spiroidea, C. isodiametrica, M. fasciculata, A. anagnostidisi, A. thureti, T. komareki) inside the order Synechococcales, and a new pleurocapsalean species (Xenococcus spongiosum). X. spongiosum is a baeocyte-producing species that shares some morphological features with other Xenococcus species, but has distinct phylogenetic and ecological identity. Rhodoploca, Cymatolege, Metis and Thalassoporum are novel well supported linages of filamentous cyanobacteria that possess distinct characters compared to their sister taxa. Aegeococcus is a novel monophyletic linage of Synechococcus-like cyanobacteria exhibiting a unique ecology, as sponge-dweller. The considerable number of novel taxa characterized in this study highlights the importance of employing polyphasic culture-dependent approaches in order to reveal the true cyanobacterial diversity associated with sponges.

Detrimental effect of UV-B radiation on growth, photosynthetic pigments, metabolites and ultrastructure of some cyanobacteria and freshwater chlorophyta.

BACKGROUND: Global warming directly influencing ozone layer depletion, which eventually is increasing ultraviolet radiation penetration having far-reaching impacts on living biota. This particularly influences the primary producer microalgae which are the basic unit of food webs in the aquatic habitats. Therefore, it is necessary to concentrate the research at this micro-level to understand the harmful impact of increased UV-B radiation ever before. Consequently, the present attempt aimed to focus on the influence of UV-B on growth criteria, photosynthetic pigments, some metabolites, and ultrastructure of the freshwater cyanobacteria, Planktothrix cryptovaginata (Microcoleaceae), Nostoc carneum (Nostocaceae), Microcystis aeruginosa (Microcystaceae), the Chlorophyte Scenedesmus acutus (Scenedesmaceae), and the marine Cyanobacterium Microcystis (Microcystaceae). METHODS: The cultures of investigated algae were subjected directly to different duration periods (1, 3, 5, and 7 h) of artificial UV-B in addition to unirradiated control culture and allowed to grow for 10 days, after which the algal samples were analyzed for growth, photosynthetic activities, primary metabolities and cellular ultrastructure. RESULTS: A remarkable inhibitory influence of UV-B was observed on growth criteria (measured as optical density and dry weight) and photosynthetic pigments of P. cryptovaginata, N. carneum, M. aeruginosa, S. acutus, and marine Microcystis. Where increasing the exposure time of UV-B was accompanied by increased inhibition. The variation in carbohydrate and protein contents under UV stress was based on the exposure periods and the algal species. The variation in algal ultrastructure by UV-B stress was noticed by an Electron Microscope. Cells damage and lysis, cell wall and cell membrane ruptured and release of intracellular substances, loss of cell inclusion, plasmolysis and necrosis, or apoptosis of the algal cells were observed by exposure to 7 h of UV-B. CONCLUSION: Exposure to UV-B has a marked harmful impact on the growth, pigments, and metabolic activity, as well as the cellular ultrastructure of some cyanobacteria and chlorophytes.

Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP.

The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria. The 35 kDa OCP is structurally and functionally modular, consisting of an N-terminal effector domain (NTD) and a C-terminal regulatory domain (CTD); a carotenoid spans the two domains. The CTD is a member of the ubiquitous Nuclear Transport Factor-2 (NTF2) superfamily (pfam02136). With the increasing availability of cyanobacterial genomes, bioinformatic analysis has revealed the existence of a new family of proteins, homologs to the CTD, the C-terminal domain-like carotenoid proteins (CCPs). Here we purify holo-CCP2 directly from cyanobacteria and establish that it natively binds canthaxanthin (CAN). We use small-angle X-ray scattering (SAXS) to characterize the structure of this carotenoprotein in two distinct oligomeric states. A single carotenoid molecule spans the two CCPs in the dimer. Our analysis with X-ray footprinting-mass spectrometry (XFMS) identifies critical residues for carotenoid binding that likely contribute to the extreme red shift (ca. 80 nm) of the absorption maximum of the carotenoid bound by the CCP2 dimer and a further 10 nm shift in the tetramer form. These data provide the first structural description of carotenoid binding by a protein consisting of only an NTF2 domain.

Phosphorus Feast and Famine in Cyanobacteria: Is Luxury Uptake of the Nutrient Just a Consequence of Acclimation to Its Shortage?

To cope with fluctuating phosphorus (P) availability, cyanobacteria developed diverse acclimations, including luxury P uptake (LPU)-taking up P in excess of the current metabolic demand. LPU is underexplored, despite its importance for nutrient-driven rearrangements in aquatic ecosystems. We studied the LPU after the refeeding of P-deprived cyanobacterium Nostoc sp. PCC 7118 with inorganic phosphate (Pi), including the kinetics of Pi uptake, turnover of polyphosphate, cell ultrastructure, and gene expression. The P-deprived cells deployed acclimations to P shortage (reduction of photosynthetic apparatus and mobilization of cell P reserves). The P-starved cells capable of LPU exhibited a biphasic kinetic of the Pi uptake and polyphosphate formation. The first (fast) phase (1-2 h after Pi refeeding) occurred independently of light and temperature. It was accompanied by a transient accumulation of polyphosphate, still upregulated genes encoding high-affinity Pi transporters, and an ATP-dependent polyphosphate kinase. During the second (slow) phase, recovery from P starvation was accompanied by the downregulation of these genes. Our study revealed no specific acclimation to ample P conditions in Nostoc sp. PCC 7118. We conclude that the observed LPU phenomenon does not likely result from the activation of a mechanism specific for ample P conditions. On the contrary, it stems from slow disengagement of the low-P responses after the abrupt transition from low-P to ample P conditions.

Formation of prolamellar-body-like ultrastructures in etiolated cyanobacterial cells overexpressing light-dependent protochlorophyllide oxidoreductase in Leptolyngbya boryana.

Protochlorophyllide (Pchlide) reduction is the penultimate step of chlorophyll (Chl) biosynthesis, and is catalyzed by two evolutionarily unrelated enzymes: dark-operative Pchlide oxidoreductase (DPOR) and light-dependent Pchlide oxidoreductase (LPOR). Because LPOR is the sole Pchlide reductase in angiosperms, dark-grown seedlings of angiosperms become etiolated. LPOR exists as a ternary complex of Pchlide-NADPH-LPOR to form paracrystalline prolamellar bodies (PLBs) in etioplasts. Because LPOR is distributed ubiquitously across oxygenic phototrophs including cyanobacteria, it would be important to determine whether cyanobacterial LPOR has the ability to form PLBs. We isolated a DPOR-less transformant ΔchlL/LPORox, carrying a plasmid to overexpress cyanobacterial LPOR in the cyanobacterium Leptolyngbya boryana. The transformant did not produce Chl in the dark and became etiolated with an accumulation of Pchlide and LPOR. Novel PLB-like ultrastructures were observed in etiolated cells, which disappeared during the early stage of the light-dependent greening process. However, the rate of Chl production in the greening process of ΔchlL/LPORox was almost the same as that observed in the control cells, which carried an empty vector. An in vitro LPOR assay of extracts of dark-grown ΔchlL/LPORox cells suggested that the PLB-like structures are deficient in NADPH. Low-temperature fluorescence emission spectra of membrane fractions of the etiolated cells indicated the absence of the photoactive form of Pchlide, which was consistent with the inefficiency of the greening process. Cyanobacterial LPOR exhibited an intrinsic ability to form PLB-like ultrastructures in the presence of the co-accumulation of Pchlide; however, the PLB-like structure differed from the authentic PLB regarding NADPH deficiency.

Acclimation process of the chlorophyll d-bearing cyanobacterium Acaryochloris marina to an orange light environment revealed by transcriptomic analysis and electron microscopic observation.

The cyanobacterium Acaryochloris marina MBIC 11017 (A. marina 11017) possesses chlorophyll d (Chl. d) peaking at 698 nm as photosystem reaction center pigments, instead of chlorophyll a (Chl. a) peaking at 665 nm. About 95% of the total chlorophylls is Chl. d in A. marina 11017. In addition, A. marina 11017 possesses phycobilisome (PBS) supercomplex to harvest orange light and to transfer the absorbing energy to the photosystems. In this context, A. marina 11017 utilizes both far-red and orange light as the photosynthetic energy source. In the present study, we incubated A. marina 11017 cells under monochromatic orange and far-red light conditions and performed transcriptional and morphological studies by RNA-seq analysis and electron microscopy. Cellular absorption spectra, transcriptomic profiles, and microscopic observations demonstrated that PBS was highly accumulated under an orange light condition relative to a far-red light condition. Notably, transcription of one cpcBA operon encoding the phycobiliprotein of the phycocyanin was up-regulated under the orange light condition, but another operon was constitutively expressed under both conditions, indicating functional diversification of these two operons for light harvesting. Taking the other observations into consideration, we could illustrate the photoacclimation processes of A. marina 11017 in response to orange and far-red light conditions in detail.

Characterization and Biosynthesis of Lipids in Paulinella micropora MYN1: Evidence for Efficient Integration of Chromatophores into Cellular Lipid Metabolism.

The chromatophores found in the cells of photosynthetic Paulinella species, once believed to be endosymbiotic cyanobacteria, are photosynthetic organelles that are distinct from chloroplasts. The chromatophore genome is similar to the genomes of α-cyanobacteria and encodes about 1,000 genes. Therefore, the chromatophore is an intriguing model of organelle formation. In this study, we analyzed the lipids of Paulinella micropora MYN1 to verify that this organism is a composite of cyanobacterial descendants and a heterotrophic protist. We detected glycolipids and phospholipids, as well as a betaine lipid diacylglyceryl-3-O-carboxyhydroxymethylcholine, previously detected in many marine algae. Cholesterol was the only sterol component detected, suggesting that the host cell is similar to animal cells. The glycolipids, presumably present in the chromatophores, contained mainly C16 fatty acids, whereas other classes of lipids, presumably present in the other compartments, were abundant in C20 and C22 polyunsaturated fatty acids. This suggests that chromatophores are metabolically distinct from the rest of the cell. Metabolic studies using isotopically labeled substrates showed that different fatty acids are synthesized in the chromatophore and the cytosol, which is consistent with the presence of both type I and type II fatty acid synthases, supposedly present in the cytosol and the chromatophore, respectively. Nevertheless, rapid labeling of the fatty acids in triacylglycerol and phosphatidylcholine by photosynthetically fixed carbon suggested that the chromatophores efficiently provide metabolites to the host. The metabolic and ultrastructural evidence suggests that chromatophores are tightly integrated into the whole cellular metabolism.

Light stress in green and red Planktothrix strains: The orange carotenoid protein and its related photoprotective mechanism.

Photosynthetic organisms need to sense and respond to fluctuating environmental conditions, to perform efficient photosynthesis and avoid the formation of harmful reactive oxygen species. Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome, the extramembranal light-harvesting antenna. This mechanism is triggered by the photoactive orange carotenoid protein (OCP). In this study, we characterized OCP and the related photoprotective mechanism in non-stressed and light-stressed cells of three different strains of Planktothrix that can form impressive blooms. In addition to changing lake ecosystemic functions and biodiversity, Planktothrix blooms can have adverse effects on human and animal health as they produce toxins (e.g., microcystins). Three Planktothrix strains were selected: two green strains, PCC 10110 (microcystin producer) and PCC 7805 (non-microcystin producer), and one red strain, PCC 7821. The green strains colonize shallow lakes with higher light intensities while red strains proliferate in deep lakes. Our study allowed us to conclude that there is a correlation between the ecological niche in which these strains proliferate and the rates of induction and recovery of OCP-related photoprotection. However, differences in the resistance to prolonged high-light stress were correlated to a better replacement of damaged D1 protein and not to differences in OCP photoprotection. Finally, microcystins do not seem to be involved in photoprotection as was previously suggested.

Formation and Preservation of Microbial Palisade Fabric in Silica Deposits from El Tatio, Chile.

Palisade fabric is a ubiquitous texture of silica sinter found in low temperature (<40°C) regimes of hot spring environments, and it is formed when populations of filamentous microorganisms act as templates for silica polymerization. Although it is known that postdepositional processes such as biological degradation and dewatering can strongly affect preservation of these fabrics, the impact of extreme aridity has so far not been studied in detail. Here, we report a detailed analysis of recently silicified palisade fabrics from a geyser in El Tatio, Chile, tracing the progressive degradation of microorganisms within the silica matrix. This is complemented by heating experiments of natural sinter samples to assess the role of diagenesis. Sheathed cyanobacteria, identified as Leptolyngbya sp., were found to be incorporated into silica sinter by irregular cycles of wetting, evaporation, and mineral precipitation. Transmission electron microscopy analyses revealed that nanometer-sized silica particles are filling the pore space within individual cyanobacterial sheaths, giving rise to their structural rigidity to sustain a palisade fabric framework. Diagenesis experiments further show that the sheaths of the filaments are preferentially preserved relative to the trichomes, and that the amount of water present within the sinter is an important factor for overall preservation during burial. This study confirms that palisade fabrics are efficiently generated in a highly evaporative geothermal field, and that these biosignatures can be most effectively preserved under dry diagenetic conditions.

Ultrastructural modeling of small angle scattering from photosynthetic membranes.

The last decade has seen a range of studies using non-invasive neutron and X-ray techniques to probe the ultrastructure of a variety of photosynthetic membrane systems. A common denominator in this work is the lack of an explicitly formulated underlying structural model, ultimately leading to ambiguity in the data interpretation. Here we formulate and implement a full mathematical model of the scattering from a stacked double bilayer membrane system taking instrumental resolution and polydispersity into account. We validate our model by direct simulation of scattering patterns from 3D structural models. Most importantly, we demonstrate that the full scattering curves from three structurally typical cyanobacterial thylakoid membrane systems measured in vivo can all be described within this framework. The model provides realistic estimates of key structural parameters in the thylakoid membrane, in particular the overall stacking distance and how this is divided between membranes, lumen and cytoplasmic liquid. Finally, from fitted scattering length densities it becomes clear that the protein content in the inner lumen has to be lower than in the outer cytoplasmic liquid and we extract the first quantitative measure of the luminal protein content in a living cyanobacteria.

Aggregated Cell Masses Provide Protection against Space Extremes and a Microhabitat for Hitchhiking Co-Inhabitants.

The European Space Agency's EXPOSE facility, located on the outside of the International Space Station, was used to investigate the survival of cell aggregates of a cyanobacterium, Gloeocapsa sp., in space and simulated martian conditions for 531 days in low Earth orbit as part of the "Biofilm Organisms Surfing Space" (BOSS) experiments. Postflight analysis showed that the cell aggregates of the organism conferred protection against space conditions compared to planktonic cells. These cell aggregates, which consisted of groups of metabolically inactive cells that do not form structured layered biofilms, demonstrated that disordered "primitive" aggregates of sheathed cells can provide protection against environmental stress such as UV radiation. Furthermore, the experiment demonstrated that the cyanobacterial cell aggregates provided a microhabitat for a smaller bacterial co-cultured species that also survived in space. This observation shows that viable cells can "hitchhike" through space within the confines of larger protecting cells or cell aggregates, with implications for planetary protection, human health, and other space microbiology applications.

Cellular localization of tolyporphins, unusual tetrapyrroles, in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy.

The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole macrocycles known as tolyporphins. Almost all known tolyporphins (A-M except K) contain a dioxobacteriochlorin chromophore and exhibit an absorption spectrum somewhat similar to that of chlorophyll a. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under various growth conditions (media, illumination, culture age). Cultures grown in nitrate-depleted media (BG-110 vs. nitrate-rich, BG-11) are known to increase the production of tolyporphins by orders of magnitude (rivaling that of chlorophyll a) over a period of 30-45 days. Multivariate curve resolution (MCR) was applied to an image set containing images from each condition to obtain pure component spectra of the endogenous pigments. The relative abundances of these components were then calculated for individual pixels in each image in the entire set, and 3D-volume renderings were obtained. At 30 days in media with or without nitrate, the chlorophyll a and phycobilisomes (combined phycocyanin and phycobilin components) co-localize in the filament outer cytoplasmic region. Tolyporphins localize in a distinct peripheral pattern in cells grown in BG-110 versus a diffuse pattern (mimicking the chlorophyll a localization) upon growth in BG-11. In BG-110, distinct puncta of tolyporphins were commonly found at the septa between cells and at the end of filaments. This work quantifies the relative abundance and envelope localization of tolyporphins in single cells, and illustrates the ability to identify novel tetrapyrroles in the presence of chlorophyll a in a photosynthetic microorganism within a non-axenic culture.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM).

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Day and Night: Metabolic Profiles and Evolutionary Relationships of Six Axenic Non-Marine Cyanobacteria.

Cyanobacteria are dominant primary producers of various ecosystems and they colonize marine as well as freshwater and terrestrial habitats. On the basis of their oxygenic photosynthesis they are known to synthesize a high number of secondary metabolites, which makes them promising for biotechnological applications. State-of-the-art sequencing and analytical techniques and the availability of several axenic strains offer new opportunities for the understanding of the hidden metabolic potential of cyanobacteria beyond those of single model organisms. Here, we report comprehensive genomic and metabolic analyses of five non-marine cyanobacteria, that is, Nostoc sp. DSM 107007, Anabaena variabilis DSM 107003, Calothrix desertica DSM 106972, Chroococcidiopsis cubana DSM 107010, Chlorogloeopsis sp. PCC 6912, and the reference strain Synechocystis sp. PCC 6803. Five strains that are prevalently belonging to the order Nostocales represent the phylogenetic depth of clade B1, a morphologically highly diverse sister lineage of clade B2 that includes strain PCC 6803. Genome sequencing, light and scanning electron microscopy revealed the characteristics and axenicity of the analyzed strains. Phylogenetic comparisons showed the limits of the 16S rRNA gene for the classification of cyanobacteria, but documented the applicability of a multilocus sequence alignment analysis based on 43 conserved protein markers. The analysis of metabolites of the core carbon metabolism showed parts of highly conserved metabolic pathways as well as lineage specific pathways such as the glyoxylate shunt, which was acquired by cyanobacteria at least twice via horizontal gene transfer. Major metabolic changes were observed when we compared alterations between day and night samples. Furthermore, our results showed metabolic potential of cyanobacteria beyond Synechocystis sp. PCC 6803 as model organism and may encourage the cyanobacterial community to broaden their research to related organisms with higher metabolic activity in the desired pathways.

Suppressing Cyanobacteria with Hydrogen Peroxide Is More Effective at High Light Intensities.

Hydrogen peroxide (H2O2) can be used as an emergency method to selectively suppress cyanobacterial blooms in lakes and drinking water reservoirs. However, it is largely unknown how environmental parameters alter the effectiveness of H2O2 treatments. In this study, the toxic cyanobacterial strain Microcystis aeruginosa PCC 7806 was treated with a range of H2O2 concentrations (0 to 10 mg/L), while being exposed to different light intensities and light colors. H2O2 treatments caused a stronger decline of the photosynthetic yield in high light than in low light or in the dark, and also a stronger decline in orange than in blue light. Our results are consistent with the hypothesis that H2O2 causes major damage at photosystem II (PSII) and interferes with PSII repair, which makes cells more sensitive to photoinhibition. Furthermore, H2O2 treatments caused a decrease in cell size and an increase in extracellular microcystin concentrations, indicative of leakage from disrupted cells. Our findings imply that even low H2O2 concentrations of 1-2 mg/L can be highly effective, if cyanobacteria are exposed to high light intensities. We therefore recommend performing lake treatments during sunny days, when a low H2O2 dosage is sufficient to suppress cyanobacteria, and may help to minimize impacts on non-target organisms.

Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria.

Carboxysomes are protein-based bacterial organelles encapsulating key enzymes of the Calvin-Benson-Bassham cycle. Previous work has implicated a ParA-like protein (hereafter McdA) as important for spatially organizing carboxysomes along the longitudinal axis of the model cyanobacterium Synechococcus elongatus PCC 7942. Yet, how self-organization of McdA emerges and contributes to carboxysome positioning is unknown. Here, we identify a small protein, termed McdB that localizes to carboxysomes and drives emergent oscillatory patterning of McdA on the nucleoid. Our results demonstrate that McdB directly stimulates McdA ATPase activity and its release from DNA, driving carboxysome-dependent depletion of McdA locally on the nucleoid and promoting directed motion of carboxysomes towards increased concentrations of McdA. We propose that McdA and McdB are a previously unknown class of self-organizing proteins that utilize a Brownian-ratchet mechanism to position carboxysomes in cyanobacteria, rather than a cytoskeletal system. These results have broader implications for understanding spatial organization of protein mega-complexes and organelles in bacteria.