Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B', that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/ adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. H-1-N-15 two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.

The acquisition of thermally tolerant algal symbionts by corals has been proposed as a natural or assisted mechanism of increasing coral reef resilience to anthropogenic climate change, but the cell-level processes determining the performance of new symbiotic associations are poorly understood. We used liquid chromatography-mass spectrometry to investigate the effects of an experimentally induced symbiosis on the host proteome of the model sea anemone Exaiptasia pallida. Aposymbiotic specimens were colonised by either the homologous dinoflagellate symbiont (Breviolum minutum) or a thermally tolerant, ecologically invasive heterologous symbiont (Durusdinium trenchii). Anemones containing D. trenchii exhibited minimal expression of Niemann-Pick C2 proteins, which have predicted biochemical roles in sterol transport and cell recognition, and glutamine synthetases, which are thought to be involved in nitrogen assimilation and recycling between partners. D. trenchii-colonised anemones had higher expression of methionine-synthesising betaine-homocysteine S-methyltransferases and proteins with predicted oxidative stress response functions. Multiple lysosome-associated proteins were less abundant in both symbiotic treatments compared with the aposymbiotic treatment. The differentially abundant proteins are predicted to represent pathways that may be involved in nutrient transport or resource allocation between partners. These results provide targets for specific experiments to elucidate the mechanisms underpinning compensatory physiology in the coral-dinoflagellate symbiosis.

The genomes of two closely related thermophilic cyanobacterial isolates, designated Synechococcus isolate OS-A and Synechococcus isolate OS-B', from the microbial mats of Octopus Spring (Yellowstone National Park) have been sequenced. An extensive suite of genes that are controlled by phosphate levels constitute the putative Pho regulon in these cyanobacteria. We examined physiological responses of an axenic OS-B' isolate as well as transcript abundances of Pho regulon genes as the cells acclimated to phosphorus-limiting conditions. Upon imposition of phosphorus deprivation, OS-B' stopped dividing after three to four doublings, and absorbance spectra measurements indicated that the cells had lost most of their phycobiliproteins and chlorophyll a. Alkaline phosphatase activity peaked and remained high after 48 h of phosphorus starvation, and there was an accumulation of transcripts from putative Pho regulon genes. Interestingly, the genome of Synechococcus isolate OS-B' harbors a cluster of phn genes that are not present in OS-A isolates. The proteins encoded by the phn genes function in the transport and metabolism of phosphonates, which could serve as an alternative phosphorus source when exogenous phosphate is low. The phn genes were upregulated within a day of eliminating the source of phosphate from the medium. However, the ability of OS-B' to utilize methylphosphonate as a sole phosphorus source occurred only after an extensive period of exposure to the substrate. Once acclimated, the cells grew rapidly in fresh medium with methylphosphonate as the only source of phosphorus. The possible implications of these results are discussed with respect to the ecophysiology of the microbial mats.

The relationship between cnidarians and their micro-algal symbionts is crucial for normal animal function and the formation of coral reefs. We used the sea anemone Exaiptasia pallida (Aiptasia) as a model cnidarian-dinoflagellate system to determine the effects of white, blue and red light on photo-movement. In white light, phototropism and phototaxis of Aiptasia were dependent on the presence of symbionts; anemones with symbionts bent and moved toward the light, whereas aposymbiotic anemones (lacking algal symbionts) moved, but without strong directionality. Phototaxis and phototropism also occurred in blue light, but to a lesser extent than in white light, with no apparent response to red light. Phototactic behavior was also sensitive to the specific anemone-symbiont pairing. The ability to sense and move in response to light would presumably allow for selection of favorable habitats. Overall, this study demonstrates that the algal symbiont is required for photo-movement of the host and that the extent of movement is influenced by the different anemone-symbiont associations.

CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B9) as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B9 genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B9, but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as "viritopes'' to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities.

Polyphosphate, an energy-rich polymer conserved in all kingdoms of life, is integral to many cellular stress responses, including nutrient deprivation, and yet, the mechanisms that underlie its biological roles are not well understood. In this work, we elucidate the physiological function of this polymer in the acclimation of the model alga Chlamydomonas reinhardtii to nutrient deprivation. Our data reveal that polyphosphate synthesis is vital to control cellular adenosine 5'-triphosphate homeostasis and maintain both respiratory and photosynthetic electron transport upon sulfur deprivation. Using both genetic and pharmacological approaches, we show that electron flow in the energy-generating organelles is essential to induce and sustain acclimation to sulfur deprivation at the transcriptional level. These previously unidentified links among polyphosphate synthesis, photosynthetic and respiratory electron flow, and the acclimation of cells to nutrient deprivation could unveil the mechanism by which polyphosphate helps organisms cope with a myriad of stress conditions in a fluctuating environment.

Hosting different symbiont species can affect inter-partner nutritional fluxes within the cnidarian-dinoflagellate symbiosis. Using nanoscale secondary ion mass spectrometry (NanoSIMS), we measured the spatial incorporation of photosynthetically fixed(13)C and heterotrophically derived(15)N into host and symbiont cells of the model symbiotic cnidarian Aiptasia (Exaiptasia pallida) when colonized with its native symbiontBreviolum minutumor the non-nativeDurusdinium trenchii.Breviolum minutumexhibited high photosynthetic carbon assimilationpercell and translocation to host tissue throughout symbiosis establishment, whereasD. trenchiiassimilated significantly less carbon, but obtained more host nitrogen. These findings suggest thatD. trenchiihas less potential to provide photosynthetically fixed carbon to the host despite obtaining considerable amounts of heterotrophically derived nitrogen. These sub-cellular events help explain previous observations that demonstrate differential effects ofD. trenchiicompared toB. minutumon the host transcriptome, proteome, metabolome and host growth and asexual reproduction. Together, these differential effects suggest that the non-native host-symbiont pairing is sub-optimal with respect to the host's nutritional benefits under normal environmental conditions. This contributes to our understanding of the ways in which metabolic integration impacts the benefits of a symbiotic association, and the potential evolution of novel host-symbiont pairings.

Synechococcus sp. represents an ecologically diverse group of cyanobacteria found in numerous environments, including hot-spring microbial mats, where they are spatially distributed along thermal, light and oxygen gradients. These thermophiles engage in photosynthesis and aerobic respiration during the day, but switch to fermentative metabolism and nitrogen fixation at night. The genome of Synechococcus OS-B', isolated from Octopus Spring (Yellowstone National Park) contains a phn gene cluster encoding a phosphonate (Phn) transporter and a C-P lyase. A closely related isolate, Synechococcus OS-A, lacks this cluster, but contains genes encoding putative phosphonatases (Phnases) that appear to be active only in the presence of the Phn substrate. Both isolates grow well on several different Phns as a sole phosphorus (P) source. Interestingly, Synechococcus OS-B' can use the organic carbon backbones of Phns for heterotrophic growth in the dark, whereas in the light this strain releases organic carbon from Phn as ethane or methane (depending on the specific Phn available); Synechococcus OS-A has neither of these capabilities. These differences in metabolic strategies for assimilating the P and C of Phn by two closely related Synechococcus spp. are suggestive of niche-specific constraints in the evolution of nutrient assimilation pathways and syntrophic relationships among the microbial populations of the hotspring mats. Thus, it is critical to evaluate levels of various P sources, including Phn, in thermally active habitats and the potential importance of these compounds in the biogeochemical cycling of P and C (some Phn compounds also contain N) in diverse terrestrial environments. The ISME Journal (2011) 5, 141-149; doi:10.1038/ismej.2010.96; published online 15 July 2010