Empirical evidence of the cost of producing toxic compounds in harmful microalgae is completely lacking. Yet costs are often assumed to be high, implying substantial ecological benefits with adaptive significance exist. To study potential fitness costs of toxin production, 16 strains including three species of the former Alexandrium tamarense species complex were grown under both carbon limitation and unlimited conditions. Growth rates, levels of intracellular paralytic shellfish toxins (PSTs), and effects of lytic compounds were measured to provide trade-off curves of toxicity for both PST and lytic toxicity under high light (300 μmol photons m-2 s-1) and under low light (i.e., carbon limited; 20 μmol photons m-2 s-1). Fitness costs in terms of reduced growth rates with increasing PST content were only evident under unlimited conditions, but not under carbon limitation, in which case PST production was positively correlated with growth. The cost of production of lytic compounds was detected both under carbon limitation and unlimited conditions, but only in strains producing PST. The results may direct future research in understanding the evolutionary role and ecological function of algal toxins. The intrinsic growth rate costs should be accounted for in relation to quantifying benefits such as grazer avoidance or toxin-mediated prey capture in natural food web settings.

Many species of the ciliate genus Strombidium can acquire functional chloroplasts from a wide range of algal prey and are thus classified as generalist non-constitutive mixotrophs. Little, however, is known about the influence of irradiance and prey availability on their ability to exploit the photosynthetic potential of the chloroplasts, and how this may explain their spatial and temporal distribution in nature. In this study, inorganic carbon uptake, growth, and ingestion rates were measured for S. cf. basimorphum under three different irradiances (10, 40, and 120 μmol photons m-2 s-1) when acclimated to three different prey densities (5 × 103, 1 × 104, and 4 × 104 cells mL-1), as well as when allowed to deplete the prey. After prey depletion, cultures survived without prey longest (∼6 days) at the medium irradiance treatment (40 μmol photons m-2 s-1), while ciliate density, inorganic carbon uptake rates, and cellular chl-a content declined fastest at the highest irradiance treatment. This indicates that the ciliates may be unable to maintain the chloroplasts functionally without replacement at high irradiances. Ingestion rates were not shown to be significantly influenced by irradiance. The maximum gross growth efficiency (GGE) in this study (1.1) was measured in cultures exposed to the medium test irradiance and lowest prey density treatment (5 × 103 cells mL-1). The relative contribution of inorganic carbon uptake to the ciliate carbon budget was also highest in this treatment (42%). A secondary GGE peak (0.99) occurred when cultures were exposed to the highest test irradiance and the medium prey density. These and other results suggest that S. cf. basimorphum, and other generalist non-constitutive mixotrophs, can flexibly exploit many different environmental conditions across the globe.

Some phagotrophic organisms can retain chloroplasts of their photosynthetic prey as so-called kleptochloroplasts and maintain their function for shorter or longer periods of time. Here we show for the first time that the dinoflagellate Dinophysis acuta takes control over "third-hand" chloroplasts obtained from its ciliate prey Mesodinium spp. that originally ingested the cryptophyte chloroplasts. With its kleptochloroplasts, D. acuta can synthesize photosynthetic as well as photoprotective pigments under long-term starvation in the light. Variable chlorophyll fluorescence measurements showed that the kleptochloroplasts were fully functional during 1 month of prey starvation, while the chlorophyll a-specific inorganic carbon uptake decreased within days of prey starvation under an irradiance of 100 μmol photons m(-2) s(-1). While D. acuta cells can regulate their pigmentation and function of kleptochloroplasts they apparently lose the ability to maintain high inorganic carbon fixation rates.

Anabaena variabilis is a filamentous cyanobacterium that is capable to differentiate specialized cells, the heterocysts and akinetes, to survive under different stress conditions. Under nitrogen limited condition, heterocysts provide the filament with nitrogen by fixing N2. Akinetes are spore-like dormant cells that allow survival during adverse environmental conditions. Both cell types are characterized by the presence of a thick multilayered envelope, including a glycolipid layer. While in the heterocyst this glycolipid layer is required for the maintenance of a microoxic environment and nitrogen fixation, its function in akinetes is completely unknown. Therefore, we constructed a mutant deficient in glycolipid synthesis and investigated the performance of heterocysts and akinetes in that mutant strain. We chose to delete the gene Ava_2595, which is homolog to the known hglB gene, encoding a putative polyketide synthase previously shown to be involved in heterocyst glycolipid synthesis in Anabaena sp. PCC 7120, a species which does not form akinetes. Under the respective conditions, the Ava_2595 null mutant strain formed aberrant heterocysts and akinete-like cells, in which the specific glycolipid layers were absent. This confirmed firstly that both cell types use a glycolipid of identical chemical composition in their special envelopes and, secondly, that HglB is essential for glycolipid synthesis in both types of differentiated cells. As a consequence, the mutant was not able to fix N2 and to grow under diazotrophic conditions. Furthermore, the akinetes lacking the glycolipids showed a severely reduced tolerance to stress conditions, but could germinate normally under standard conditions. This demonstrates the importance of the glycolipid layer for the ability of akinetes as spore-like dormant cells to withstand freezing, desiccation, oxidative stress and attack by lytic enzymes. Our study established the dual role of the glycolipid layer in fulfilling different functions in the evolutionary-related specialized cells of cyanobacteria. It also indicates the existence of a common pathway involving HglB for the synthesis of glycolipids in heterocysts and akinetes.

We have assessed how varying CO2 (180, 380, and 720 μatm) and growth light intensity (40 and 400 μmol photons m-2 s-1) affected Trichodesmium erythraeum IMS101 growth and photophysiology over free iron (Fe') concentrations between 20 and 9,600 pM. We found significant iron dependencies of growth rate and the initial slope and maximal relative PSII electron transport rates (rPm). Under iron-limiting concentrations, high-light increased growth rates and rPm; possibly indicating a lower allocation of resources to iron-containing photosynthetic proteins. Higher CO2 increased growth rates across all iron concentrations, enabled growth to occur at lower Fe' concentrations, increased rPm and lowered the iron half saturation constants for growth (Km). We attribute these CO2 responses to the operation of the CCM and the ATP spent/saved for CO2 uptake and transport at low and high CO2, respectively. It seems reasonable to conclude that T. erythraeum IMS101 can exhibit a high degree of phenotypic plasticity in response to CO2, light intensity and iron-limitation. These results are important given predictions of increased dissolved CO2 and water column stratification (i.e., higher light exposures) over the coming decades.

A novel thermophilic, microaerophilic, anoxygenic, and chlorophototrophic member of the phylum Acidobacteria, Chloracidobacterium thermophilum strain B(T), was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, Wyoming. C. thermophilum is strictly dependent on light and oxygen and grows optimally as a photoheterotroph at irradiance values between 20 and 50 μmol photons m(-2) s(-1). C. thermophilum is unable to synthesize branched-chain amino acids (AAs), l-lysine, and vitamin B12, which are required for growth. Although the organism lacks genes for autotrophic carbon fixation, bicarbonate is also required. Mixtures of other AAs and 2-oxoglutarate stimulate growth. As suggested from genomic sequence data, C. thermophilum requires a reduced sulfur source such as thioglycolate, cysteine, methionine, or thiosulfate. The organism can be grown in a defined medium at 51(∘)C (Topt; range 44-58(∘)C) in the pH range 5.5-9.5 (pHopt = ∼7.0). Using the defined growth medium and optimal conditions, it was possible to isolate new C. thermophilum strains directly from samples of hot spring mats in Yellowstone National Park, Wyoming. The new isolates differ from the type strain with respect to pigment composition, morphology in liquid culture, and temperature adaptation.

Marine Synechococcus has successfully adapted to environments with different light colors, which likely contributes to this genus being the second most abundant group of microorganisms worldwide. Populations of Synechococcus that grow in deep, blue ocean waters contain large amounts of the blue-light absorbing chromophore phycourobilin (PUB) in their light harvesting complexes (phycobilisomes). Here, we show that all Synechococcus strains adapted to blue light possess a gene called mpeU. MpeU is structurally similar to phycobilin lyases, enzymes that ligate chromophores to phycobiliproteins. Interruption of mpeU caused a reduction in PUB content, impaired phycobilisome assembly and reduced growth rate more strongly in blue than green light. When mpeU was reintroduced in the mpeU mutant background, the mpeU-less phenotype was complemented in terms of PUB content and phycobilisome content. Fluorescence spectra of mpeU mutant cells and purified phycobilisomes revealed red-shifted phycoerythrin emission peaks, likely indicating a defect in chromophore ligation to phycoerythrin-I (PE-I) or phycoerythrin-II (PE-II). Our results suggest that MpeU is a lyase-isomerase that attaches a phycoerythrobilin to a PEI or PEII subunit and isomerizes it to PUB. MpeU is therefore an important determinant in adaptation of Synechococcus spp. to capture photons in blue light environments throughout the world's oceans.

The extremophilic red alga Galdieria partita is a facultative heterotroph that occupies mostly low-light microhabitats. However, the exceptional detection of abundant populations of G. partita in sunlight-exposed soil raises the possibility that exogenous organic carbon sources protect cells from photo-oxidative damage. The present study aimed to identify the photoprotective process activated by exogenous glucose under photo-oxidative stress. We demonstrated that exogenous glucose mitigated the photo-oxidative damage of cells exposed to 300 μmol photons m-2 s-1 photosynthetic active radiation. Photosynthesis carbon assimilation scarcely contributed to the cell growth in the presence of glucose, but the photosynthetic apparatus was nevertheless maintained and protected by glucose in a concentration-dependent manner. Supplementation of glucose increased expression of the L-gulonolactone oxidase gene essential for ascorbic acid biosynthesis, whereas no enhanced expression of the genes involved in carotenoid or tocopherol biosynthesis was observed. Under the photo-oxidative stress condition, the ascorbic acid content was strongly enhanced by exogenous glucose. We propose that the biosynthesis of ascorbic acid is one of the major photoprotective processes induced by exogenous glucose. The elucidation of how ascorbic acid is involved in scavenging reactive oxygen species provides key insights into the photoprotective mechanism in red algae.

Synechococcus sp. strain PCC 7002 is a unicellular, euryhaline cyanobacterium. It is a model organism for studies of cyanobacterial metabolism and has great potential for biotechnological applications. It exhibits an exceptional tolerance of high-light irradiation and shows very rapid growth. The habitats from which this and closely related strains were isolated are subject to changes in several environmental factors, including light, nutrient supply, temperature, and salinity. In this study global transcriptome profiling via RNAseq has been used to perform a comparative and integrated study of global changes in cells grown at different temperatures, at different salinities, and under mixotrophic conditions, when a metabolizable organic carbon source was present. Furthermore, the transcriptomes were investigated for cells that were subjected to a heat shock and that were exposed to oxidative stress. Lower growth temperatures caused relatively minor changes of the transcriptome; the most prominent changes affected fatty acid desaturases. A heat shock caused severe changes of the transcriptome pattern; transcripts for genes associated with major metabolic pathways declined and those for different chaperones increased dramatically. Oxidative stress, however, left the transcript pattern almost unaffected. When grown at high salinity, Synechococcus sp. PCC 7002 had increased expression of genes involved in compatible solute biosynthesis and showed increased mRNA levels for several genes involved in electron transport. Transcripts of two adjacent genes dramatically increased upon growth at high salinity; the respective proteins are putatively involved in coping with oxidative stress and in triggering ion channels. Only minor changes were observed when cells were grown at low salinity or when the growth medium was supplemented with glycerol. However, the transcriptome data suggest that cells must acclimate to excess reducing equivalents when a reduced C-source is present.

Synechocystis sp. PCC 6803 is a widely used model cyanobacterium whose genome has been well annotated. However, several additional small protein coding sequences (sORFs) have been recently identified, which might play important roles, for example in the regulation of cellular metabolism. Here, we analyzed the function of a sORF encoding a 44 amino acid peptide showing high similarity to the N-terminal part of aconitase (AcnB). The expression of the gene, which probably originated from a partial gene duplication of chromosomal acnB into the plasmid pSYSA, was verified and it was designated as acnSP. The protein-coding part of acnSP was inactivated by interposon mutagenesis. The obtained mutant displayed slower growth under photoautotrophic conditions with light exceeding 100 μmol photons m-2 s-1 and showed significant changes in the metabolome compared to wild type, including alterations in many metabolites associated to the tricarboxylic acid (TCA) cycle. To analyze a possible direct impact of AcnSP on aconitase, the recombinant Synechocystis enzyme was generated and biochemically characterized. Biochemical analysis revealed that addition of equimolar amounts of AcnSP resulted in an improved substrate affinity (lower K m) and lowered V max of aconitase. These results imply that AcnSP can regulate aconitase activity, thereby impacting the carbon flow into the oxidative branch of the cyanobacterial TCA cycle, which is mainly responsible for the synthesis of carbon skeletons needed for ammonia assimilation.

The chlorosomes of green sulfur bacteria (GSB) are mainly assembled from one of three types of bacteriochlorophylls (BChls), BChls c, d, and e. By analogy to the relationship between BChl c and BChl d (20-desmethyl-BChl c), a fourth type of BChl, BChl f (20-desmethyl-BChl e), should exist but has not yet been observed in nature. The bchU gene (bacteriochlorophyllide C-20 methyltransferase) of the brown-colored green sulfur bacterium Chlorobaculum limnaeum was inactivated by conjugative transfer from Eshcerichia coli and homologous recombination of a suicide plasmid carrying a portion of the bchU. The resulting bchU mutant was greenish brown in color and synthesized BChl f(F). The chlorosomes of the bchU mutant had similar size and polypeptide composition as those of the wild type (WT), but the Q(y) absorption band of the BChl f aggregates was blue-shifted 16 nm (705 nm vs. 721 nm for the WT). Fluorescence spectroscopy showed that energy transfer to the baseplate was much less efficient in chlorosomes containing BChl f than in WT chlorosomes containing BChl e. When cells were grown at high irradiance with tungsten or fluorescent light, the WT and bchU mutant had identical growth rates. However, the WT grew about 40% faster than the bchU mutant at low irradiance (10 μmol photons m(-2) s(-1)). Less efficient energy transfer from BChl f aggregates to BChl a in the baseplate, the much slower growth of the strain producing BChl f relative to the WT, and competition from other phototrophs, may explain why BChl f is not observed naturally.

The unicellular, euryhaline cyanobacterium Synechococcus sp. strain PCC 7002 is a model organism for laboratory-based studies of cyanobacterial metabolism and is a potential platform for biotechnological applications. Two of its most notable properties are its exceptional tolerance of high-light intensity and very rapid growth under optimal conditions. In this study, transcription profiling by RNAseq has been used to perform an integrated study of global changes in transcript levels in cells subjected to limitation for the major nutrients CO(2), nitrogen, sulfate, phosphate, and iron. Transcriptional patterns for cells grown on nitrate, ammonia, and urea were also studied. Nutrient limitation caused strong decreases of transcript levels of the genes encoding major metabolic pathways, especially for components of the photosynthetic apparatus, CO(2) fixation, and protein biosynthesis. Uptake mechanisms for the respective nutrients were strongly up-regulated. The transcription data further suggest that major changes in the composition of the NADH dehydrogenase complex occur upon nutrient limitation. Transcripts for flavoproteins increased strongly when CO(2) was limiting. Genes involved in protection from oxidative stress generally showed high, constitutive transcript levels, which possibly explains the high-light tolerance of this organism. The transcriptomes of cells grown with ammonia or urea as nitrogen source showed increased transcript levels for components of the CO(2) fixation machinery compared to cells grown with nitrate, but in general transcription differences in cells grown on different N-sources exhibited surprisingly minor differences.

We report extremely low-light-adapted anoxygenic photosynthesis in a thick microbial mat in Magical Blue Hole, Abaco Island, The Bahamas. Sulfur cycling was reduced by iron oxides and organic carbon limitation. The mat grows below the halocline/oxycline at 30 m depth on the walls of the flooded sinkhole. In situ irradiance at the mat surface on a sunny December day was between 0.021 and 0.084 μmol photons m-2 s-1, and UV light (<400 nm) was the most abundant part of the spectrum followed by green wavelengths (475-530 nm). We measured a light-dependent carbon uptake rate of 14.5 nmol C cm-2 d-1. A 16S rRNA clone library of the green surface mat layer was dominated (74%) by a cluster (>97% sequence identity) of clones affiliated with Prosthecochloris, a genus within the green sulfur bacteria (GSB), which are obligate anoxygenic phototrophs. Typical photopigments of brown-colored GSB, bacteriochlorophyll e and (β-)isorenieratene, were abundant in mat samples and their absorption properties are well-adapted to harvest light in the available green and possibly even UV-A spectra. Sulfide from the water column (3-6 μmol L-1) was the main source of sulfide to the mat as sulfate reduction rates in the mats were very low (undetectable-99.2 nmol cm-3 d-1). The anoxic water column was oligotrophic and low in dissolved organic carbon (175-228 μmol L-1). High concentrations of pyrite (FeS2; 1-47 μmol cm-3) together with low microbial process rates (sulfate reduction, CO2 fixation) indicate that the mats function as net sulfide sinks mainly by abiotic processes. We suggest that abundant Fe(III) (4.3-22.2 μmol cm-3) is the major source of oxidizing power in the mat, and that abiotic Fe-S-reactions play the main role in pyrite formation. Limitation of sulfate reduction by low organic carbon availability along with the presence of abundant sulfide-scavenging iron oxides considerably slowed down sulfur cycling in these mats.

Antimicrobial resistance is one of the biggest health and subsequent economic threat humanity faces. Next to massive global awareness campaigns, governments and NGOs alike stress the need for new innovative strategies to treat microbial infections. One of such innovative strategies is the photodynamic antimicrobial chemotherapy (PACT) in which the synergistic effects of photons and drugs are exploited. While many promising reports are available, PACT - and especially the drug-design part behind - is still in its infancy. Common best-practice rules, such as the EUCAST or CLSI protocols for classic antibiotics as well as high-throughput screenings, are missing, and this, in turn, hampers the identification of hit structures. Hit-like structures might come from synthetic approaches or from natural sources. They are identified via activity-guided synthesis or isolation strategies. As source for new antimicrobials, fungi are highly ranked. They share the same ecological niche with many other microbes and consequently established chemical strategies to combat with the others. Recently, in members of the Cortinariaceae, especially of the subgenus Dermocybe, photoactive metabolites were detected. To study their putative photoantimicrobial effect, a photoantimicrobial high-throughput screening (HTS) based on The European Committee on Antimicrobial Susceptibility Testing (EUCAST) was established. After validation, the established HTS was used to evaluate a sample set containing six colorful representatives from the genus Cortinarius (i.e., Cortinarius callisteus, C. rufo-olivaceus, C. traganus, C. trivialis, C. venetus, and C. xanthophyllus). The assay is built on a uniform, light-emitting diode (LED)-based light irradiation across a 96-well microtiter plate, which was achieved by a pioneering arrangement of the LEDs. The validation of the assay was accomplished with well-known photoactive drugs, so-called photosensitizers, utilizing six distinct emission wavelengths (λexc = 428, 478, 523, 598, or 640 nm) and three microbial strains (Candida albicans, Staphylococcus aureus, and Escherichia coli). Evaluating the extracts of six Cortinarius species revealed two highly promising species, i.e., C. rufo-olivaceus and C. xanthophyllus. Extracts from the latter were photoactive against the Gram-positive S. aureus (c = 7.5 μg/ml, H = 30 J/cm2, λ = 478 nm) and the fungus C. albicans (c = 75 μg/ml, H = 30 J/cm2, λ = 478 nm).

Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and analysis of sequence counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. k-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes, but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes.