O-Methyltransferase is shared between the pentose phosphate and shikimate pathways and is essential for mycosporine-like amino acid biosynthesis in Anabaena variabilis ATCC 29413.

The parent core structure of mycosporine-like amino acids (MAAs) is 4-deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7-phosphate by the enzymes 2-epi-5-epivaliolone synthase (EVS) and O-methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway-specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in-frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV-induced MAA biosynthesis.

2-epi-5-epi-Valiolone synthase activity is essential for maintaining phycobilisome composition in the cyanobacterium Anabaena variabilis ATCC 29413 when grown in the presence of a carbon source.

The cyclase 2-epi-5-epi-valiolone synthase (EVS) is reported to be a key enzyme for biosynthesis of the mycosporine-like amino acid shinorine in the cyanobacterium Anabaena variabilis ATCC 29413. Subsequently, we demonstrated that an in-frame complete deletion of the EVS gene had little effect on in vivo production of shinorine. Complete segregation of the EVS gene deletion mutant proved difficult and was achieved only when the mutant was grown in the dark and in a medium supplemented with fructose. The segregated mutant showed a striking colour change from native blue-green to pale yellow-green, corresponding to substantial loss of the photosynthetic pigment phycocyanin, as evinced by combinations of absorbance and emission spectra. Transcriptional analysis of the mutant grown in the presence of fructose under dark or light conditions revealed downregulation of the cpcA gene that encodes the alpha subunit of phycocyanin, whereas the gene encoding nblA, a protease chaperone essential for phycobilisome degradation, was not expressed. We propose that the substrate of EVS (sedoheptulose 7-phosphate) or possibly lack of its EVS-downstream products, represses transcription of cpcA to exert a hitherto unknown control over photosynthesis in this cyanobacterium. The significance of this finding is enhanced by phylogenetic analyses revealing horizontal gene transfer of the EVS gene of cyanobacteria to fungi and dinoflagellates. It is also conceivable that the EVS gene has been transferred from dinoflagellates, as evident in the host genome of symbiotic corals. A role of EVS in regulating sedoheptulose 7-phosphate concentrations in the photophysiology of coral symbiosis is yet to be determined.

A profile of an endosymbiont-enriched fraction of the coral Stylophora pistillata reveals proteins relevant to microbial-host interactions.

This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal "bleaching" stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure.

Redundant pathways of sunscreen biosynthesis in a cyanobacterium.

Route of the sun block: according to empirical evidence, sun-screening mycosporine-like amino acids (MAAs) in Eukarya originate from the shikimic acid pathway, whereas in cyanobacteria, biosynthesis of the MAA shinorine reportedly occurs through the pentose phosphate pathway. However, gene deletion shows that the cyanobacterium Anabaena variabilis ATCC 29143 does not biosynthesise shinorine exclusively by this route.

Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching.

BACKGROUND: The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH) from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues. METHODOLOGY/PRINCIPAL FINDINGS: A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs), which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a 'shared metabolic adaptation' between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca(2+)-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis. CONCLUSIONS/SIGNIFICANCE: Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.

Global genome analysis of the shikimic acid pathway reveals greater gene loss in host-associated than in free-living bacteria.

BACKGROUND: A central tenet in biochemistry for over 50 years has held that microorganisms, plants and, more recently, certain apicomplexan parasites synthesize essential aromatic compounds via elaboration of a complete shikimic acid pathway, whereas metazoans lacking this pathway require a dietary source of these compounds. The large number of sequenced bacterial and archaean genomes now available for comparative genomic analyses allows the fundamentals of this contention to be tested in prokaryotes. Using Hidden Markov Model profiles (HMM profiles) to identify all known enzymes of the pathway, we report the presence of genes encoding shikimate pathway enzymes in the hypothetical proteomes constructed from the genomes of 488 sequenced prokaryotes. RESULTS: Amongst free-living prokaryotes most Bacteria possess, as expected, genes encoding a complete shikimic acid pathway, whereas of the culturable Archaea, only one was found to have a complete complement of recognisable enzymes in its predicted proteome. It may be that in the Archaea, the primary amino-acid sequences of enzymes of the pathway are highly divergent and so are not detected by HMM profiles. Alternatively, structurally unrelated (non-orthologous) proteins might be performing the same biochemical functions as those encoding recognized genes of the shikimate pathway. Most surprisingly, 30% of host-associated (mutualistic, commensal and pathogenic) bacteria likewise do not possess a complete shikimic acid pathway. Many of these microbes show some degree of genome reduction, suggesting that these host-associated bacteria might sequester essential aromatic compounds from a parasitised host, as a 'shared metabolic adaptation' in mutualistic symbiosis, or obtain them from other consorts having the complete biosynthetic pathway. The HMM results gave 84% agreement when compared against data in the highly curated BioCyc reference database of genomes and metabolic pathways. CONCLUSIONS: These results challenge the conventional belief that the shikimic acid pathway is universal and essential in prokaryotes. The possibilities that non-orthologous enzymes catalyse reactions in this pathway (especially in the Archaea), or that there exist specific uptake mechanisms for the acquisition of shikimate intermediates or essential pathway products, warrant further examination to better understand the precise metabolic attributes of host-beneficial and pathogenic bacteria.

Enzymes of the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins.

The shikimic acid pathway is responsible for the biosynthesis of many aromatic compounds by a broad range of organisms, including bacteria, fungi, plants, and some protozoans. Animals are considered to lack this pathway, as evinced by their dietary requirement for shikimate-derived aromatic amino acids. We challenge the universality of this traditional view in this report of genes encoding enzymes for the shikimate pathway in an animal, the starlet sea anemone Nematostella vectensis. Molecular evidence establishes horizontal transfer of ancestral genes of the shikimic acid pathway into the N. vectensis genome from both bacterial and eukaryotic (dinoflagellate) donors. Bioinformatic analysis also reveals four genes that are closely related to those of Tenacibaculum sp. MED152, raising speculation for the existence of a previously unsuspected bacterial symbiont. Indeed, the genome of the holobiont (i.e., the entity consisting of the host and its symbionts) comprises a high content of Tenacibaculum-like gene orthologs, including a 16S rRNA sequence that establishes the phylogenetic position of this associate to be within the family Flavobacteriaceae. These results provide a complementary view for the biogenesis of shikimate-related metabolites in marine Cnidaria as a "shared metabolic adaptation" between the partners.

Effects of temperature and UV radiation increases on the photosynthetic efficiency in four scleractinian coral species.

Experiments were performed on coral species containing clade A (Stylophora pistillata, Montipora aequituberculata) or clade C (Acropora sp., Pavona cactus) zooxanthellae. The photosynthetic efficiency (F(v)/F(m)) of the corals was first assessed during a short-term increase in temperature (from 27 degrees C to 29 degrees C, 32 degrees C, and 34 degrees C) and acute exposure to UV radiation (20.5 W m(-2) UVA and 1.2 W m(-2) UVB) alone or in combination. Increasing temperature to 34 degrees C significantly decreased the F(v)/F(m) in S. pistillata and M. aequituberculata. Increased UV radiation alone significantly decreased the F(v)/F(m) of all coral species, even at 27 degrees C. There was a combined effect of temperature and UV radiation, which reduced F(v)/F(m) in all corals by 25% to 40%. During a long-term exposure to UV radiation (17 days) the F(v)/F(m) was significantly reduced after 3 days' exposure in all species, which did not recover their initial values, even after 17 days. By this time, all corals had synthesized mycosporine-like amino acids (MAAs). The concentration and diversity of MAAs differed among species, being higher for corals containing clade A zooxanthellae. Prolonged exposure to UV radiation at the nonstressful temperature of 27 degrees C conferred protection against independent, thermally induced photoinhibition in all four species.

Mycosporine-like amino acids in the zooxanthella-ciliate symbiosis Maristentor dinoferus.

Coral reef organisms living in mutualistic symbioses with phototrophic dinoflagellates are widespread in shallow UV-transparent waters. Maristentor dinoferus is a recently discovered species of marine benthic ciliate that hosts symbiotic dinoflagellates of the genus Symbiodinium. In this study, we tested this ciliate for the occurrence of mycosporine-like amino acids, a family of secondary metabolites that minimize damage from exposure to solar UV radiation by direct screening. Using high-performance liquid chromatography and liquid chromatography coupled to mass spectrometry, five mycosporine-like amino acids (shinorine, palythenic acid, palythine, mycosporine-2-glycine, and porphyra-334) were identified in aqueous methanolic extracts of the symbiosis. This is the first report of mycosporine-like amino acids in a marine ciliate.

The Symbiotic Anthozoan: A Physiological Chimera between Alga and Animal.

The symbiotic life style involves mutual ecological, physiological, structural, and molecular adaptations between the partners. In the symbiotic association between anthozoans and photosynthetic dinoflagellates (Symbiodinium spp., also called zooxanthellae), the presence of the endosymbiont in the animal cells has constrained the host in several ways. It adopts behaviors that optimize photosynthesis of the zooxanthellae. The animal partner has had to evolve the ability to absorb and concentrate dissolved inorganic carbon from seawater in order to supply the symbiont's photosynthesis. Exposing itself to sunlight to illuminate its symbionts sufficiently also subjects the host to damaging solar ultraviolet radiation. Protection against this is provided by biochemical sunscreens, including mycosporine-like amino acids, themselves produced by the symbiont and translocated to the host. Moreover, to protect itself against oxygen produced during algal photosynthesis, the cnidarian host has developed certain antioxidant defenses that are unique among animals. Finally, living in nutrient-poor waters, the animal partner has developed several mechanisms for nitrogen assimilation and conservation such as the ability to absorb inorganic nitrogen, highly unusual for a metazoan. These facts suggest a parallel evolution of symbiotic cnidarians and plants, in which the animal host has adopted characteristics usually associated with phototrophic organisms.

Mycosporine-like amino acid content in four species of sea anemones in the genus Anthopleura reflects phylogenetic but not environmental or symbiotic relationships.

We examine the occurrence of UV-absorbing, mycosporine-like amino acids (MAAs) in four sympatric species of sea anemones in the genus Anthopleura, all collected from intertidal habitats on the Pacific Coast of temperate North America. We compare patterns of MAAs in A. elegantissima of several types: specimens having predominately zooxanthellae (dinoflagellates comprising at least two species) or zoochlorellae as symbionts; those containing algal endosymbionts of both kinds, and naturally occurring aposymbiotic specimens that lack the endosymbionts typically found in most specimens. We also compare MAAs in zooxanthellate specimens of A. sola and A. xanthogrammica, and specimens from the asymbiotic species A. artemisia. Our findings indicate that the complements of the four major MAAs in these species of Anthopleura (mycosporine-taurine, shinorine, porphyra-334, and mycosporine-2 glycine) broadly reflect phylogenetic differences among the anemones rather than the taxon of endosymbionts, presence or absence of symbionts, or environmental factors. An exception, however, occurs in A. elegantissima, where mycosporine-2 glycine increases in concentration with the density of zooxanthellae. Our evidence also shows that A. elegantissima can accumulate MAAs from its food, which may explain the occasional occurrence of minor MAAs in some individuals.

Mycosporine-like amino acids and related Gadusols: biosynthesis, acumulation, and UV-protective functions in aquatic organisms.

Organisms living in clear, shallow water are exposed to the damaging wavelengths of solar ultraviolet radiation (UVR) coincident with the longer wavelengths of photosynthetically available radiation (PAR) also necessary for vision. With the general exception of bacteria, taxonomically diverse marine and freshwater organisms have evolved the capacity to synthesize or accumulate UV-absorbing mycosporine-like amino acids (MAAs), presumably for protection against environmental UVR. This review highlights the evidence for this UV-protective role while also considering other attributed functions, including reproductive and osmotic regulation and vision. Probing the regulation and biosynthesis of MAAs provides insight to the physiological evolution and utility of UV protection and of biochemically associated antioxidant defenses.

Effects of age on gene expression during estrogen-induced synaptic sprouting in the female rat.

Age and estrogen treatment influenced fiber outgrowth and compensatory neuronal sprouting after unilateral entorhinal cortex lesions (ECL) which model Alzheimer disease-like deafferentation in the dentate gyrus of the hippocampus. In young F344 rats (3 months old), ovariectomy (OVX) decreased reactive fiber outgrowth by 60%. Sprouting in middle-aged rats (18 months old) was reduced in intact females; no further reduction was caused by OVX. Several astrocyte mRNAs were measured in the dentate gyrus of young and middle-aged female rats in three different estrogen states (sham OVX, OVX, or OVX + estradiol) 1 week after ECL. Glial fibrillary acidic protein (GFAP) mRNA was twofold greater in middle-aged rats than young, although both ages showed threefold increases in response to ECL. In prior studies GFAP was found to be decreased by estradiol treatment 3-4 days after ECL; in this study GFAP mRNA had returned to sham OVX levels in young rats by 7 days post-ECL. Surprisingly, estradiol treatment increased GFAP mRNA levels by 25% above OVX in middle-aged rats. Apolipoprotein E (apoE) mRNA was decreased 20% by age in the dentate, although both age groups showed a 25% increase in apoE mRNA in response to ECL. Apolipoprotein J (apoJ) mRNA was increased 20% in the dentate gyrus of middle-aged rats, and both age groups responded to ECL with a 65% increase in apoJ mRNA. The estrogen state did not alter levels of either apolipoprotein mRNA in the deafferented dentate. The data suggest that the estrogen-induced decrease of GFAP in response to lesions does not persist at 7 days post-ECL during sprouting. Overall effects of age on the dentate gyrus include elevated GFAP mRNA and decreased apoE mRNA. The cortical wound site showed consistent enhancement of GFAP mRNA in both age groups by estradiol above sham OVX and greater responses in middle-aged rats.

Sunscreens, oxidative stress and antioxidant functions in marine organisms of the Great Barrier Reef.

An overview of the biochemical photophysiology of tropical, reef-building corals is presented with a discussion on the biosynthetic relationship between natural UV-absorbing sunscreens and certain antioxidant functions in marine organisms. Our studies reveal that marine organisms, including 'UV-extremophilic' bacteria, are a rich source of novel antioxidants having potential for the development of commercial and biomedical applications. Novel sunscreening agents derived from tropical marine organisms of the Great Barrier Reef are in development. New marine-derived antioxidants are being isolated for testing as chemopreventatives in a variety of oxidatively degenerative diseases.

Free radicals and chemiluminescence as products of the spontaneous oxidation of sulfide in seawater, and their biological implications.

The discovery of symbioses between marine invertebrates and sulfide-oxidizing bacteria at deep-sea hydrothermal vents and in other high-sulfide marine environments has stimulated research into the adaptations of metazoans to potentially toxic concentrations of sulfide. Most of these studies have focused on a particular action of sulfide--its disruption of aerobic metabolism by the inhibition of mitochondrial respiration--and on the adaptations of sulfide-tolerant animals to avoid this toxic effect (1). We propose that sulfidic environments impose another, hitherto over-looked type of toxicity: exposure to free radicals of oxygen, which may be produced during the spontaneous oxidation of sulfide, thus imposing an oxidative stress. Here we present evidence that oxygen- and sulfur-centered free radicals are produced during the oxidation of sulfide in seawater, and we propose a reaction pathway for sulfide oxidation that is consistent with our observations. We also show that chemiluminescence at visible wavelengths occurs during sulfide oxidation, providing a possible mechanism for the unexplained light emission from hydrothermal vents (2, 3).

Ultraviolet radiation-absorbing mycosporine-like amino acids (MAAs) are acquired from their diet by medaka fish (Oryzias latipes) but not by SKH-1 hairless mice.

To assess whether vertebrates can acquire, from their diet, ultraviolet radiation-absorbing mycosporine-like amino acids (MAAs), medaka fish and hairless mice were maintained for 150 and 130 days, respectively, on diets either including Mastocarpus stellatus (rich in MAAs) or the same diets without this red alga. In medaka, the MAAs palythine and asterina-330, present in trace quantities in the diet with added M. stellatus, were present in significantly greater quantities in the eyes of fish fed this diet than in the eyes of control fish. Only traces of MAAs were present in the skin of medaka fed the diet containing MAAs. Shinorine, the principal MAA in M. stellatus, was not found in any tissues of medaka, which raises questions about the specificity of transport of MAAs. In hairless mice, no dietary MAAs were found in the tissues of the eyes, skin, or liver after maintenance on the experimental diet. Low concentrations of shinorine were present only in the tissues of the small and large intestines. These results indicate that MAAs are acquired from their diet and translocated to superficial tissues by teleost fish, but that mammals may be incapable of such. Thus, dietary supplementation with MAAs may be useful in aquacultured species of fish, but MAAs as 'dietary sunscreens' may not be an option for mammals, including humans. Nevertheless, our demonstration of the uptake of shinorine by human skin cancer cells in culture raises evolutionary questions regarding the organ specificity of the capacity for the cellular transport of MAAs.