The genes encoding the alpha and beta subunits of allophycocyanin, phycocyanin and phycoerythrin from the red alga Aglaothamnion neglectum were isolated and characterized. While the operons containing the different phycobiliprotein genes are dispersed on the plastid genome, the genes encoding the alpha and beta subunits for each phycobiliprotein are contiguous. The beta subunit gene is 5' for both the phycocyanin and phycoerythrin operons, while the alpha subunit gene is 5' for the allophycocyanin operon. The amino acid sequences of A. neglectum phycobiliproteins, as deduced from the nucleotide sequences of the genes, are 65-85% identical to analogous proteins from other red algae and cyanobacteria. The conserved nature of the plastid-encoded red algal and cyanobacterial phycobiliprotein genes supports the proposed origin of red algal plastids from cyanobacterial endosymbionts.

We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximate to 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta(2) adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells ( Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.

Metabolite exchange is fundamental to the viability of the cnidarian-Symbiodiniaceae symbiosis and survival of coral reefs. Coral holobiont tolerance to environmental change might be achieved through changes in Symbiodiniaceae species composition, but differences in the metabolites supplied by different Symbiodiniaceae species could influence holobiont fitness. Using C-13 stable-isotope labelling coupled to gas chromatography-mass spectrometry, we characterized newly fixed carbon fate in the model cnidarian Exaiptasia pallida (Aiptasia) when experimentally colonized with either native Breviolum minutum or non-native Durusdinium trenchii Relative to anemones containing B. minutum, D. trenchii-colonized hosts exhibited a 4.5-fold reduction in C-13-labelled glucose and reduced abundance and diversity of C-13-labelled carbohydrates and lipogenesis precursors, indicating symbiont species-specific modifications to carbohydrate availability and lipid storage. Mapping carbon fate also revealed significant alterations to host molecular signalling pathways. In particular, D. trenchii-colonized hosts exhibited a 40-fold reduction in C-13-labelled scyllo-inositol, a potential interpartner signalling molecule in symbiosis specificity. C-13-labelling also highlighted differential antioxidant- and ammonium-producing pathway activities, suggesting physiological responses to different symbiont species. Such differences in symbiont metabolite contribution and host utilization may limit the proliferation of stress-driven symbioses; this contributes valuable information towards future scenarios that select in favour of less-competent symbionts in response to environmental change.

Thermophilic cyanobacteria of the genus Synechococcus are major contributors to photosynthetic carbon fixation in the photic zone of microbial mats in Octopus Spring, Yellowstone National Park. Synechococcus OS-B' was characterized with regard to the ability to acclimate to a range of different light irradiances; it grows well at 25 to 200 mu mol photons m(-2) s(-1) but dies when the irradiance is increased to 400 mu mol photons m(-2) s(-1). At 200, mu mol photons m(-2) s(-1) (high light [HL]), we noted several responses that had previously been associated with HL acclimation of cyanobacteria, including cell bleaching, reduced levels of phycobilisomes and chlorophyll, and elevated levels of a specific carotenoid. Synechococcus OS-B' synthesizes the carotenoids zeaxanthin and P,P-carotene and a novel myxol-anhydrohexoside. Interestingly, 77-K fluorescence emission spectra suggest that Synechococcus OS-B' accumulates very small amounts of photosystem 11 relative to that of photosystem 1. This ratio further decreased at higher growth irradiances, which may reflect potential photodamage following exposure to HL. We also noted that HL caused reduced levels of transcripts encoding phycobilisome components, particularly that for CpcH, a 20.5-kDa rod linker polypeptide. There was enhanced transcript abundance of genes encoding terminal oxidases, superoxide dismutase, tocopherol cyclase, and phytoene desaturase. Genes encoding the photosystem II D1:1 and D1:2 isoforms (psbAI and psbAII/psbAIII, respectively) were also regulated according to the light regimen. The results are discussed in the context of how Synechococcus OS-B' may cope with high light irradiances in the high-temperature environment of the microbial mat.

Photosynthetic organisms often experience extreme light conditions that can cause hyper-reduction of the chloroplast electron transport chain, resulting in oxidative damage. Accumulating evidence suggests that mitochondrial respiration and chloroplast photosynthesis are coupled when cells are absorbing high levels of excitation energy. This coupling helps protect the cells from hyper-reduction of photosynthetic electron carriers and diminishes the production of reactive oxygen species (ROS). To examine this cooperative protection, here we characterized Chlamydomonas reinhardtii mutants lacking the mitochondrial alternative terminal respiratory oxidases, CrAOX1 and CrAOX2. Using fluorescent fusion proteins, we experimentally demonstrated that both enzymes localize to mitochondria. We also observed that the mutant strains were more sensitive than WT cells to high light under mixotrophic and photoautotrophic conditions, with the aox1 strain being more sensitive than aox2. Additionally, the lack of CrAOX1 increased ROS accumulation, especially in very high light, and damaged the photosynthetic machinery, ultimately resulting in cell death. These findings indicate that the Chlamydomonas AOX proteins can participate in acclimation of C. reinhardtii cells to excess absorbed light energy. They suggest that when photosynthetic electron carriers are highly reduced, a chloroplast-mitochondria coupling allows safe dissipation of photosynthetically derived electrons via the reduction of O-2 through AOX (especially AOX1)-dependent mitochondrial respiration.

Vascular plants contain abundant, light-harvesting complexes in the thylakoid membrane that are non-covalently associated with chlorophylls and carotenoids. These light-harvesting chlorophyll a/b binding (LHC) proteins are members of an extended CAB/ELIP/HLIP superfamily of distantly related polypeptides, which have between one and four transmembrane helices (TMH). This superfamily includes the single TMH, high-light-inducible proteins (Hlips), found in cyanobacteria that are induced by various stress conditions, including high light, and are considered ancestral to the LHC proteins. The roles of, and evolutionary relationships between, these superfamily members are of particular interest, since they function in both light harvesting and photoprotection and may have evolved through tandem gene duplication and fusion events. We have investigated the Hlips (hli gene family) in the thermophilic unicellular cyanobacterium Synechococcus OS-B'. The five hli genes present on the genome of Synechococcus OS-B' are relatively similar, but transcript analyses indicate that there are different patterns of transcript accumulation when the cells are exposed to various growth conditions, suggesting that different Hlips may have specific functions. Hlip5 has an additional TMH at the N-terminus as a result of a novel fusion event. This additional TMH is very similar to a conserved hypothetical, single membrane-spanning polypeptide present in most cyanobacteria. The evolutionary significance of these results is discussed.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized(1,2). Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.

A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.

The high energy requirement associated with cell disruption has remained a major barrier to the commercial production of microalgal biofuels and bioproducts. Autolysis takes advantage of microalgal cells' biologically driven self-lysing properties and represents an environmentally sustainable mean of improving the recovery of intracellular products and the energy economics of biomass processing. This study demonstrated for the first time the induction of autolytic cell-wall thinning in Nannochloropsis. Autolysis was induced using a low-cost and low-energy non-invasive incubation treatment which stored highly concentrated microalgal slurry (0.11-0.29 mg biomass per mg slurry) in darkness at 38 degrees C for 24 h. Under thermally coupled dark-anoxia incubation, the Nannochloropsis cells were deprived of oxygen and activated anaerobic metabolism that depleted intracellular sugar reserves to sustain basal metabolism (total sugar content of the biomass decreased from 0.174 +/- 0.029 to 0.124 +/- 0.027 mg mg-1 biomass). The auto-fermentation pathways consumed the polysaccharide reserves in the cell wall and resulted in a 50% reduction in the thickness of the cellulose layer of the cell wall (from 27.8 +/- 9.4 nm to 12.6 +/- 5.5 nm). This wall thinning effect structurally weakened the cells, significantly increased the percentage of cells that could subsequently be ruptured with a range of cell-disruption technologies, such as high-pressure homogenisation (HPH), alkaline treatment or acidic treatment (from 36.8 +/- 11.9% for untreated slurry to 74.1 +/- 14.2% for incubated slurry), and ultimately led to a 2-fold increase in the amount of lipid recovered with a low-solvent biphasic extraction system (from 22.2 +/- 13.5 wt% of lipid for the untreated slurry to 48.6 +/- 17.6 wt% of lipid for the incubated slurry).

Nitrogen fixation, a prokaryotic, O-2-inhibited process that reduces N-2 gas to biomass, is of paramount importance in biogeochemical cycling of nitrogen. We analyzed the levels of nif transcripts of Synechococcus ecotypes, NifH subunit and nitrogenase activity over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park. The results showed a rise in nif transcripts in the evening, with a subsequent decline over the course of the night. In contrast, immunological data demonstrated that the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but O-2 consumption by respiration still exceeded the rate of O-2 evolution. Once the irradiance increased to the point at which the mat became oxic, the nitrogenase activity was strongly inhibited. Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis- and respiration-related transcripts accumulating during the day and late afternoon, respectively. These results are discussed with respect to the energetics and regulation of N-2 fixation in hot spring mats and factors that can markedly influence the extent of N-2 fixation over the diel cycle.