Coral bleaching has devastating effects on coral survival; arid reef ecosystem ftindiort,i but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography tandem mass spectrometry to compare the effects of rapidly (33.S degrees C,';24 h) and gradually'. (30 and 33.5 degrees C, 12 Clays) elevated temperatures on. the proteome of the model symbiotic anemone Aiptasia; We identified 2133 proteins in Aiptasia, 136 of which were differentially abundant between treatments.; Thermal shock, but not acclimation, resulted in significant abundance changes in 104 proteins, including those involved in protein folding and synthesis, redox homeostasis, and:central metabolism. Nineteen abundant structural proteins. showed particularly reduced abundance, demonstrating proteostasis, disruption and phtential proteih synthesis inhibition. Heat shock induced antioxidant mechanisms and proteins involved in stabilizing nascent proteins, preventing. protein aggregation arid degrading damaged proteins, which is indicative of endoplasmic reticulum stress: Host proteostasis disruption occurred before either bleaching or symbiont photoinhibition was detected, suggesting host-derived reactive oxygen species production as the proximate cause of thermal damage. The pronounced abundance changes in endoplasmic reticulum proteins associated with proteostasis and protein turnover indicate that these processes ate essential in the cellular response of symbiotic cnidarians to severe thermal stress.

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-fight-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. s110041; also called pisJI) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A DeltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

Many photosynthetic microorganisms have evolved the ability to sense light quality and/or quantity and can steer themselves into optimal conditions within the environment. Phototaxis and gliding motility in unicellular cyanobacteria require type IV pili, which are multifunctional cell surface appendages. Screens for cells exhibiting aberrant motility uncovered several non-motile mutants as well as some that had lost positive phototaxis (consequently, they were negatively phototactic). Several negatively phototactic mutants mapped to the tax1 locus, which contains five chemotaxis-like genes. This locus includes a gene that encodes a putative photoreceptor (TaxD1) for positive phototaxis. A second chemotaxis-like cluster (tax3 locus) appears to be involved in pilus biogenesis. The biosynthesis and regulation of type IV pilus-based motility as well as the communication between the pilus motor and photosensory molecules appear to be complex and tightly regulated. Furthermore, the discovery that cyclic AMP and novel gene products are necessary for phototaxis/motility suggests that there might be additional levels of communication and signal processing.

Harvesting photosynthetic electrons (PEs) from plant or algal cells can be a highly efficient and environmentally friendly way of generating renewable energy. Recent work on nanoelectrode insertion into algal cells has demonstrated the possibility to directly extract PEs from living algal cells with high efficiencies. However, the instability of the inserted cells limits the practicality of this technology. Here, the impact of nanoelectrode insertion on intracellular extraction of PEs is characterized with the goal of stabilizing algal cells after nanoelectrode insertion. Using nanoelectrodes < 500 nm in diameter, algal cells remained stable for over one week after insertion and continued to provide PEs through direct extraction by the inserted nanoelectrodes. After nanoelectrode insertion, a photosynthetic current density of 6 mA.cm(-2), which is several fold higher than the current densities attained using approaches based on isolated thylakoid membranes or photosystem I complexes, was observed in the dark and during illumination at various light intensities.

The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms ( plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b(6)f complex (Cytb(6)f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb(6)f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb(6)f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb(6)f.

Genome sequences of two Synechococcus ecotypes inhabiting the octopus Spring microbial mat in Yellowstone National Park revealed the presence of all genes required for nitrogenase biosynthesis. We demonstrate that nif genes of the Synechococcus ecotypes are expressed in situ in a region of the mat that varies in temperature from 53.5 degrees C to 63.4 degrees C (average 60 degrees C) transcripts are only detected at the end of the day when the mat becomes anoxic. Nitrogenase activity in mat samples was also detected in the evening. Hitherto, N-2 fixation in hot spring mats was attributed either to filamentous cyanobacteria (not present at > 50 degrees C in these mats) or to heterotrophic bacteria. To explore how energy-generating processes of the Synechococcus ecotypes track natural light and O-2 conditions, we evaluated accumulation of transcripts encoding proteins involved in photosynthesis, respiration, and fermentation. Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes declined in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fell into two categories; some (Idh, pdhB, ald, and ackA) decreased in the evening, whereas others (PflB, pflA, adhE, and acs) increased at the end of the day and remained high into the night. Energy required for N-2 fixation during the night may be derived from fermentation pathways that become prominent as the mat becomes anoxic. In a broader context, our data suggest that there are critical regulatory switches in situ that are linked to the diel cycle and that these switches alter many metabolic processes within the microbial mat.

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non-toxic algae, including those of many green algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in 2-day-old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild-type zygospores contain knob-like structures (similar to 50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild-type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption in zygospore development. In agreement with the role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild-type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions.

Phytoplankton in nature must acclimate to a wide range of light conditions resulting from diel light cycles, ocean circulation and mixing, cloud cover, and the variable bio-optical characteristics of the water column. In this study, we used whole-genome cDNA microarrays to investigate the effects of a gradually fluctuating daily light cycle on gene expression in the cyanobacterium Synechocystis sp. strain PCC6803. From these data, we developed a conceptual framework depicting the diel regulation of metabolic pathways in the cell. The framework is focused on potential photoacclimation responses, including the regulation of the photosystems, cell division, and DNA replication. The mRNA abundance of genes involved in many metabolic pathways, and particularly those encoding proteins that function in photosynthesis and DNA replication, changed markedly over the course of the day. The levels of mRNA encoding polypeptides important for the formation of the light-harvesting apparatus, photosystems I and II, and cell division were found in high concentrations during the day. The transcript levels of many genes encoding enzymes involved in anabolic processes also increased considerably during the day. In contrast, transposon transcripts and mRNAs encoding proteins involved in DNA replication, cell wall synthesis, and respiratory activity were not found in high concentrations during the day. Although gradually varying light exposure induced significant changes in transcript accumulation within Synechocystis, the direction of these changes differed between our study and previous studies in which there was an abrupt transition between irradiances.