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Ascidians are known to harbour bacterial symbionts in their tunics. In particular, the ascidian genus Lissoclinum can host abundant and diverse cyanobacterial associates. Here, we determined the di...

Patellamides are highly bioactive compounds found along with other cyanobactins in the symbiosis between didemnid ascidians and the enigmatic cyanobacterium

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that 'Paraprochloron' and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of 'Paraprochloron terpiosi' in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.

Terpios hoshinota is a ferocious, space-competing sponge that kills a variety of stony corals by overgrowth. Outbreaks of this species have led to intense coral reef damage and declines in living corals on the square kilometer scale in many geographical locations. Our large-scale 16S rRNA gene survey across three oceans revealed that the core microbiome of T. hoshinota included operational taxonomic units (OTUs) related to Prochloron, Endozoicomonas, Pseudospirillum, SAR116, Magnetospira, and Ruegeria. A Prochloron- related OTU was the most dominant cyanobacterium in T. hoshinota in the western Pacific Ocean, South China Sea, and Indian Ocean. The complete metagenome-assembled genome of the Prochloron-related cyanobacterium and our pigment analysis revealed that this bacterium had phycobiliproteins and phycobilins and lacked chlorophyll b, inconsistent with the iconic definition of Prochloron. Furthermore, the phylogenetic analyses based on 16S rRNA genes and 120 single-copy genes demonstrated that the bacterium was phylogenetically distinct to Prochloron, strongly suggesting that it should be a sister taxon to Prochloron; we therefore proposed this symbiotic cyanobacterium as a novel species under a new genus: Candidatus Paraprochloron terpiosii. With the recovery of the complete genome, we characterized the metabolic potentials of the novel cyanobacterium in carbon and nitrogen cycling and proposed a model for the interaction between Ca. Pp. terpiosi LD05 and T. hoshinota. In addition, comparative genomics analysis revealed that Ca. Paraprochloron and Prochloron showed distinct features in transporter systems and DNA replication.ImportanceThe finding that one species predominates cyanobacteria in T. hoshinota from different geographic locations indicates that this sponge and Ca. Pp. terpiosi LD05 share a tight relationship. This study builds the foundation for T. hoshinota’s microbiome and paves a way for understanding the ecosystem, invasion mechanism, and causes of outbreak of this coral-killing sponge. Also, the first Prochloron-related complete genome enables us to study this bacterium with molecular approaches in the future and broadens our knowledge of the evolution of symbiotic cyanobacteria.

Dinuclear Cu-II-patellamide complexes (patellamides are naturally occurring cyclic pseudo-octapeptides) are known to be efficient catalysts for hydrolysis reactions of biological importance, for example, those of phosphatase, carbonic anhydrase, and glycosidase. However, the biological role of patellamides is still unknown. Patellamides were originally extracted from the sea squirt Lissoclinum patella, but are now known to be ribosomally expressed by the blue-green algae Prochloron that live in symbiosis with L. patella. In a further step to unravel the metabolic significance of the patellamide complexes, the question as to whether these are also formed inside Prochloron cells is addressed. In this study, a biocompatible patellamide-fluorescent dye conjugate has been introduced into living Prochloron cells and, by means of flow cytometry and confocal microscopy, it is shown that Cu-II ions are coordinated to patellamides in vivo.

Background Marine sponges and their microbiomes contribute significantly to carbon and nutrient cycling in global reefs, processing and remineralizing dissolved and particulate organic matter. Lamellodysidea herbacea sponges obtain additional energy from abundant photosynthetic Hormoscilla cyanobacterial symbionts, which also produce polybrominated diphenyl ethers (PBDEs) chemically similar to anthropogenic pollutants of environmental concern. Potential contributions of non-Hormoscilla bacteria to Lamellodysidea microbiome metabolism and the synthesis and degradation of additional secondary metabolites are currently unknown. Results This study has determined relative abundance, taxonomic novelty, metabolic capacities, and secondary metabolite potential in 21 previously uncharacterized, uncultured Lamellodysidea-associated microbial populations by reconstructing near-complete metagenome-assembled genomes (MAGs) to complement 16S rRNA gene amplicon studies. Microbial community compositions aligned with sponge host subgroup phylogeny in 16 samples from four host clades collected from multiple sites in Guam over a 3-year period, including representatives of Alphaproteobacteria, Gammaproteobacteria, Oligoflexia, and Bacteroidetes as well as Cyanobacteria (Hormoscilla). Unexpectedly, microbiomes from one host clade also included Cyanobacteria from the prolific secondary metabolite-producer genus Prochloron, a common tunicate symbiont. Two novel Alphaproteobacteria MAGs encoded pathways diagnostic for methylotrophic metabolism as well as type III secretion systems, and have been provisionally assigned to a new order, designated Candidatus Methylospongiales. MAGs from other taxonomic groups encoded light-driven energy production pathways using not only chlorophyll, but also bacteriochlorophyll and proteorhodopsin. Diverse heterotrophic capabilities favoring aerobic versus anaerobic conditions included pathways for degrading chitin, eukaryotic extracellular matrix polymers, phosphonates, dimethylsulfoniopropionate, trimethylamine, and benzoate. Genetic evidence identified an aerobic catabolic pathway for halogenated aromatics that may enable endogenous PBDEs to be used as a carbon and energy source. Conclusions The reconstruction of high-quality MAGs from all microbial taxa comprising greater than 0.1% of the sponge microbiome enabled species-specific assignment of unique metabolic features that could not have been predicted from taxonomic data alone. This information will promote more representative models of marine invertebrate microbiome contributions to host bioenergetics, the identification of potential new sponge parasites and pathogens based on conserved metabolic and physiological markers, and a better understanding of biosynthetic and degradative pathways for secondary metabolites and halogenated compounds in sponge-associated microbiota.

In plants, chlorophyll (Chl) a and b are interconvertible by the action of three enzymes—chlorophyllide a oxygenase, Chl b reductase (CBR) and 7-hydroxymethyl chlorophyll a reductase (HCAR). These reactions are collectively referred to as the Chl cycle. In plants, this cyclic pathway ubiquitously exists and plays essential roles in acclimation to different light conditions at various developmental stages. By contrast, only a limited number of cyanobacteria species produce Chl b, and these include Prochlorococcus, Prochloron, Prochlorothrix and Acaryochloris. In this study, we investigated a possible existence of the Chl cycle in Chl b synthesizing cyanobacteria by testing in vitro enzymatic activities of CBR and HCAR homologs from Prochlorothrix hollandica and Acaryochloris RCC1774. All of these proteins show respective CBR and HCAR activity in vitro, indicating that both cyanobacteria possess the potential to complete the Chl cycle. It is also found that CBR and HCAR orthologs are distributed only in the Chl b-containing cyanobacteria that habitat shallow seas or freshwater, where light conditions change dynamically, whereas they are not found in Prochlorococcus species that usually habitat environments with fixed lighting. Taken together, our results implicate a possibility that the Chl cycle functions for light acclimation in Chl b-containing cyanobacteria.

The patellamides (cyclic pseudo-octapeptides) are produced by Prochloron, a symbiont of the ascidians, marine invertebrate filter feeders. These pseudo-octapeptides are present in the cytoplasm and a possible natural function of putative metal complexes of these compounds is hydrolase activity, however the true biological role is still unknown. The dinuclear CuII complexes of synthetic patellamide derivatives have been shown in in vitro experiments to be efficient hydrolase model catalysts. Many hydrolase enzymes, specifically phosphatases and carboanhydrases, are ZnII-based enzymes and therefore, we have studied the ZnII and mixed ZnII/CuII solution chemistry of a series of synthetic patellamide derivatives, including solution structural and computational work, with the special focus on model phosphatase chemistry with bis-(2,4-dinitrophenyl)phosphate (BDNPP) as the substrate. The ZnII complexes of a series of ligands are shown to form complexes of similar structure and stability compared to the well-studied CuII analogues and the phosphatase reactivities are also similar. Since the complex stabilities and phosphatase activities are generally a little lower compared to those of CuII and since the concentration of ZnII in Prochloron cells is slightly smaller, we conclude that the CuII complexes of the patellamides are more likely to be of biological importance.

Among coral reef ascidians inhabiting Manado Bay, North Sulawesi, Lissoclinum patella and Didemnum molle were found housing symbiotic microbe in their tunics. These tunicates benefit from the association by feeding directly on the microbial cells, and in turn the microbe were facilitated to produce functional compounds such as anticancer cyclic peptides. Preliminary screening for these compounds were conducted by cytotoxicity assays against human blood cells. The symbiotic microbe was molecularly identified as Prochloron didemni. Microbial samples were obtained by squeezing colonial tissues from the two newly collected host ascidians. Crude extracts were prepared by extraction in 80% methanol, and followed by in vitro cytotoxic assayed against human blood cells using May-grunwald-Giemsa method. The extract of the D. molle derived Prochloron showed more cytotoxic effects than the one of the L. patella. However, the cultivation of Prochloron cells originated from D. molle was not established compared to those of L. patella. Therefore for further production of anticancer potential compounds, cultivation of the L. patella derived Prochloron is being developed.

Microbial symbionts in humans, animals, and plants are unparalleled sources of compounds for drug discovery. However, methods are needed to access natural biodiversity and make it amenable to pharmaceutical and biotechnological development. Here, we employ a novel approach marrying traditional natural products chemistry with modern metagenomics and synthetic biology in Escherichia coli to discover, synthesize, and analogue divamides, anti‐HIV molecules from rare coral reef animals. We demonstrate that divamides are produced by Prochloron symbiotic bacteria, which have eluded cultivation for >40 years. This represents a new paradigm of direct compound discovery from a true symbiotic relationship. We show that the divamides arise from a diversitygenerating biosynthetic pathway, which was exploited to generate derivatives for mechanism of action studies. The synthesis of closely related, yet functionally distinct compounds by a single biosynthetic pathway provides the first direct evidence supporting the "speculative metabolism" hypothesis of chemical diversity.

Summary The cyanobacterium Prochloron didemni is primarily found in symbiotic relationships with various marine hosts such as ascidians and sponges. Prochloron remains to be successfully cultivated outside of its host, which reflects a lack of knowledge of its unique ecophysiological requirements. We investigated the microenvironment and diversity of Prochloron inhabiting the upper, exposed surface of didemnid ascidians, providing the first insights into this microhabitat. The pH and O2 concentration in this Prochloron biofilm changes dynamically with irradiance, where photosynthetic activity measurements showed low light adaptation (Ek ∼80 ± 7 μmol photons m−2 s−1) but high light tolerance. Surface Prochloron cells exhibited a different fine structure to Prochloron cells from cloacal cavities in other ascidians, the principle difference being a central area of many vacuoles dissected by single thylakoids in the surface Prochloron. Cyanobacterial 16S rDNA pyro-sequencing of the biofilm community on four ascidians resulted in 433 operational taxonomic units (OTUs) where on average −85% (65–99%) of all sequence reads, represented by 136 OTUs, were identified as Prochloron via blast search. All of the major Prochloron-OTUs clustered into independent, highly supported phylotypes separate from sequences reported for internal Prochloron, suggesting a hitherto unexplored genetic variability among Prochloron colonizing the outer surface of didemnids.

Cyclic pseudo-peptides, derived from marine metabolites found in the genus Lissoclinum bistratum and Lissoclinum patella and produced by their symbiont Prochloron have attracted scientific interest in the last two decades. Their structural properties and solution dynamics were analyzed in detail, elaborate synthetic procedures for the natural products and synthetic derivatives were developed, the biosynthetic pathways were studied and it now is possible to produce them biosynthetically. A major focus in the last decade was on their CuII – more recently also on the ZnII – coordination chemistry, as a number of studies have indicated that dinuclear CuII and ZnII complexes of cyclic peptides may be involved in the ascidians’ metabolism. Solution equilibria of various derivatives of the synthetic ligands in presence of CuII were studied thoroughly and the solution structures were determined by EPR spectroscopy and spectra simulation in combination with molecular mechanics and DFT calculations. Recent in vitro studies indicate that the dicopper(II) complexes are phosphatase, glycosidase, lactamase and very efficient carbonic anhydrase model systems. First in vivo studies with a patellamide derivative containing an appended fluorescence tag suggest that CuII is coordinated to the patellamides in the Prochloron cells.

The photosymbiotic ascidian Diplosoma simile is characterized by a wide global distribution. The current study provides the first record of D. simile in the Red Sea, with remarks on its spatial distribution and phylogeny. Partial COI and 18S rDNA sequences of D. simile from the Red Sea revealed high similarity to those from the West Pacific, indicating a much broader range than previously recorded. In the Red Sea D. simile is common on natural coral reefs at depths ranging from 1–35 m. Diplosoma simile colonies were found to overgrow and would appear to suffocate several species of live stony corals, though this does not appear to cause mass coral mortality in the Red Sea, unlike that recorded in other locations.

Diversity-generating metabolism leads to the evolution of many different chemicals in living organisms. Here, by examining a marine symbiosis, we provide a precise evolutionary model of how nature generates a family of novel chemicals, the cyanobactins. We show that tunicates and their symbiotic Prochloron cyanobacteria share congruent phylogenies, indicating that Prochloron phylogeny is related to host phylogeny and not to external habitat or geography. We observe that Prochloron exchanges discrete functional genetic modules for cyanobactin secondary metabolite biosynthesis in an otherwise conserved genetic background. The module exchange leads to gain or loss of discrete chemical functional groups. Because the underlying enzymes exhibit broad substrate tolerance, discrete exchange of substrates and enzymes between Prochloron strains leads to the rapid generation of chemical novelty. These results have implications in choosing biochemical pathways and enzymes for engineered or combinatorial biosynthesis. IMPORTANCE While most biosynthetic pathways lead to one or a few products, a subset of pathways are diversity generating and are capable of producing thousands to millions of derivatives. This property is highly useful in biotechnology since it enables biochemical or synthetic biological methods to create desired chemicals. A fundamental question has been how nature itself creates this chemical diversity. Here, by examining the symbiosis between coral reef animals and bacteria, we describe the genetic basis of chemical variation with unprecedented precision. New compounds from the cyanobactin family are created by either varying the substrate or importing needed enzymatic functions from other organisms or via both mechanisms. This natural process matches successful laboratory strategies to engineer the biosynthesis of new chemicals and teaches a new strategy to direct biosynthesis.

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca . E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

The marine cyanobacterium Prochloron is a unique photosynthetic organism that lives in obligate symbiosis with colonial ascidians. We compared Prochloron harbored in four different host species and cultured Prochlorothrix by means of spectroscopic measurements, including time-resolved fluorescence, to investigate host-induced differences in light-harvesting strategies between the cyanobacteria. The light-harvesting efficiency of photosystems including antenna Pcb, PS II–PS I connection, and pigment status, especially that of PS I Red Chls, were different among the four samples. We also discuss relationships between these observed characteristics and the light conditions, to which Prochloron cells are exposed, influenced by distribution pattern in the host colonies, presence or absence of tunic spicules, and microenvironments within the ascidians' habitat.

Among all photosynthetic and non-photosynthetic prokaryotes, only cyanobacterial species belonging to the genera Acaryochloris and Prochlorococcus have been reported to synthesize α-carotene. We reviewed the carotenoids, including their chirality, in unusual cyanobacteria containing diverse Chls. Predominantly Chl d-containing Acaryochloris (two strains) and divinyl-Chl a and divinyl-Chl b-containing Prochlorococcus (three strains) contained β-carotene and zeaxanthin as well as α-carotene, whereas Chl b-containing Prochlorothrix (one strain) and Prochloron (three isolates) contained only β-carotene and zeaxanthin but no α-carotene as in other cyanobacteria. Thus, the capability to synthesize α-carotene seemed to have been acquired only by Acaryochloris and Prochlorococcus. In addition, we unexpectedly found that α-carotene in both cyanobacteria had the opposite chirality at C-6': (6'S)-chirality in Acaryochloris and normal (6'R)-chirality in Prochlorococcus, as reported in some green algae and land plants. The results represent the first evidence for the natural occurrence and biosynthesis of (6'S)-α-carotene. All the zeaxanthins in these species were of the usual (3R,3'R)-chirality. Therefore, based on the identification of the carotenoids and genome sequence data, we propose a biosynthetic pathway for the carotenoids, particularly α-carotene, including the participating genes and enzymes.

Vertical transmission of cyanobacterial symbionts occurs in didemnid ascidians harboring Prochloron as an obligate symbiont; the photosymbionts are transferred from the parental ascidian colony to the offspring in various ways depending on host species. Although several didemnids harbor non-Prochloron cyanobacteria in their tunics, few studies have reported the processes of vertical transmission in these didemnids. Here we describe the histological processes of the transmission of cyanobacteria in two didemnids, Trididemnum nubilum harboring Synechocystis and T. clinides harboring three cyanobacterial species. In both species, the photosymbionts in the tunic of the parent colony were apparently captured by the tunic cells of the host and transferred to the embryos brooded in the tunic. The symbiont cells were then incorporated into the inner tunic of the embryo. This mode of transmission is essentially the same as that of T. miniatum harboring Prochloron in the tunic, although there are some differences among species in the timing of the release of the symbionts from the tunic cells. We suggest that the similar modes of vertical transmission are an example of convergent evolution caused by constraints in the distribution patterns of symbiont cells in the host colony.

Acaryochloris spp. are unique cyanobacteria which contain chlorophyll d as the predominant pigment. The phylogenetic diversity of Acaryochloris spp. associated with 7 Prochloron- or Synechocystis-containing didemnid ascidians and 1 Synechococcus-containing sponge obtained from the coast of the Republic of Palau was analyzed; we established a PCR primer set designed to selectively amplify the partial 16S rRNA gene of Acaryochloris spp. even in DNA samples containing a large amount of other cyanobacterial and algal DNAs. Polymerase chain reaction-denaturing gradient gel electrophoresis with this primer set enabled detection of the phyogenetic diversity of Acaryochloris spp. All the ascidian and sponge samples contained Acaryochloris spp. Fourteen phylotypes that were highly homologous (98–100%) with A. marina MBIC11017 were detected, while only 2 phylotypes were detected with our previously developed method for detecting cyanobacteria. The results also revealed that many uncultured phylotypes of Acaryochloris spp. were associated with those didemnid ascidians, since a clonal culture of only 1 phylotype has been established thus far. No specific relationship was found among the Acaryochloris phylotypes and the genera of the ascidians even when sample localities were identical; therefore, these invertebrates may provide a favorable habitat for Acaryochloris spp. rather than hosts showing any specific symbiotic relationships.

The solution structure of the leader sequence of the patellamide precursor peptide was analysed by using CD and determined with NOE-restrained molecular dynamics calculations. This leader sequence is highly conserved in the precursor peptides of some other cyanobactins harbouring heterocycles, and is assumed to play a role in targeting the precursor peptide to the post-translational machinery. The sequence was observed to form an alpha-helix spanning residues 13-28 with a hydrophobic surface on one side of the helix. This hydrophobic surface is proposed to be the site of the initial binding with modifying enzymes.

The external surfaces of marine animals are colonized by a wide variety of epibionts. Here, we study the phototrophic epibiotic community attached to the colonial ascidian Cystodytes dellechiajei collected in the Mediterranean Sea. Epifluorescence microscopy analysis showed abundant filamentous cyanobacteria on the upper and basal parts of the ascidian that displayed autofluorescence, as well as some unicellular cyanobacteria, diatoms, and structures, which could belong to microscopic rhodophyte algae. In addition, high-performance liquid chromatography of the photosynthetic pigments confirmed that the phototrophic epibionts possess chlorophyll (Chl) d, as well as Chl a, b, and c, which enable them to use far-red light for photosynthesis in that peculiar microenvironment. Furthermore, laser scanning confocal microscopy showed the presence of a few small patches of cells on the basal part of the ascidian displaying fluorescence between 700 and 750 nm after excitement with a 635-nm red laser, typically within the range of Chl d. Denaturing gradient gel electrophoresis of the 16S rRNA gene polymerase chain reaction amplified using specific primers for Cyanobacteria detected sequences related with the genera Planktothricoides, Synechococcus, Phormidium, and Myxosarcina, as well as sequences of chloroplasts of diatoms and rhodophyte algae. Remarkably, only the sequences related to the filamentous cyanobacteria Planktothricoides spp. and some chloroplast sequences were found in almost all specimens collected under different macroecological conditions and geographical areas, suggesting thus certain specificity in the epibiotic association. On the other hand, Prochloron spp. and Acaryochloris marina, typically associated to tropical ascidians, were not detected by denaturing gradient gel electrophoresis. However, given the low abundance of cells displaying Chl d in C. dellechiajei and the fact that molecular fingerprinting techniques not always recover low abundance groups, the presence of these cyanobacteria cannot be ruled out. Nevertheless, our data indicate that tropical ascidians and C. dellechiajei differ in their phototrophic communities, although Chl d-containing cells are present in both microenvironments.

Lissoclinum punctatum is a colonial ascidian that harbors the symbiotic prokaryotic alga Prochloron in its tunic and in the peribranchial and common cloacal cavities. Most symbiotic cells in the tunic are intracellular (tunic phycocytes), while those in the cavities are extracellular. We found that neither gametes nor embryos brooded in the tunic were associated with photosymbionts. We determined that algal cells attach to posterior parts of the trunk of hatching larvae swimming in the common cloacal cavity. No symbiont cells were found intracellularly in larval tissues. Thus, extracellular Prochloron cells in the cloacal cavities were transferred to the larvae, but intracellular photosymbionts in the tunic were not. The intracellular symbiosis must be reestablished in each generation after larval settlement.

A collection of ascidians from the Wessel Islands (off north-eastern Arnhem Land) confirms the Indo-West Pacific affinities of the Australian tropical ascidian fauna. The collection, dominated by species of the family Didemnidae previously observed to be diverse in tropical waters, includes several of the species with obligate 'Prochloron' symbioses.The collection also contains three large solitary species ('Polycarpa aurita', 'P. papillata' and 'Microcosmus helleri') known to have a pan-tropical range. The geographic range and life history strategies of the majority of species in this collection support the view that gene flow in the Indo-West Pacific and through the straits to the north of the Australian continent that connect the two oceans is not constrained by either a short free-swimming larval life, or internal fertilisation in the fixed, colonial organisms that dominate the tropical fauna. In colonial species at least, population maintenance may be a more significant selective advantage than gene flow through larval dispersal.

Lissoclinum timorense is a colonial ascidian that harbors the prokaryotic alga Prochloron . The algal photosymbionts adhere to the lamellae of the tunic on the posterior half of the trunk of larvae, which aggregate in the common cloacal cavity of the mother colony. Bead-adhesion tests demonstrated that the lamellae are adhesive, whereas the anterior half of the larval trunk is not. The anterior half is covered with a thin layer of outer tunic, which probably prevents Prochloron cells from attaching and interfering with sensory receptors and adhesive organs. The larval structures and the mode of algal transmission between generations are very similar to those of the Prochloron -harboring ascidian Didemnum molle . Molecular phylogenetic analyses have suggested that photosymbiosis was independently established in each genus, and thus the apparent similarity in the larvae probably resulted from convergence. The distribution pattern of photosymbionts is probably more determinative of algal transmission than phylogenetic constraints.

Prochloron is an oxygenic photosynthetic bacterium that lives in obligate symbiosis with didemnid ascidians, such as Diplosoma spp., Lissoclinum spp. and Trididemnum spp. This study investigated the genetic diversity of the genus Prochloron by constructing a phylogenetic tree based on the 16S rRNA gene sequences of 27 isolates from 11 species of didemnid ascidians collected from Japan, Australia and the USA. The 27 isolates formed three phylogenetic groups: 22 of the samples were identified to be closely related members of Prochloron. Two samples, isolated from Trididemnum nubilum and Trididemnum clinides, were found to belong to the species Synechocystis trididemni, the closest relative of Prochloron. Three isolates formed a separate group from both Prochloron sp. and S. trididemni, potentially indicating a new symbiotic phylotype. Genomic polymorphism analysis, employing cyanobacterium-specific highly iterative palindrome 1 repeats, could not delineate the isolates further. For the Prochloron sp. isolates, the phylogenetic outcome was independent of host species and geographic origin of the sample indicating a low level of host specificity, low genetic variation within the taxon and possibly a lack of a host-symbiont relationship during reproductive dispersal. This study contributes significantly to the understanding of Prochloron diversity and phylogeny, and implications for the evolutionary relationship of prochlorophytes, cyanobacteria and chloroplasts are also discussed.

Prochloron, a blue-green algae belonging to ancient prokaryotes, produces, like other cyanobacteria, cyclic pseudo-peptides, which are also found in its obligate symbiont ascidiae (Lissoclinum patellum). Although research has focused for some time on the putative metabolic function of these cyclic peptides, to date it is still not understood. Their role might be connected to the increased concentrations of divalent metal ions, especially Cu(II), found in ascidiae. Dinuclear copper(II) complexes of cyclic pseudo-peptides revealed a broad hydrolytic capacity, including carboanhydrase and phosphatase activity. This study reports their β-lactamase as well as α- and β-glycosidase activity with kcat=(11.34±0.91)ˑ10(-4)s(-1) for β-lactamase, kcat=(1.55±0.13)ˑ10(-4)s(-1) for α-glycosidase and kcat=(1.22±0.09)ˑ10(-4)s(-1) for β-glycosidase activity.

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd. The cyanobacterium Prochloron didemni is primarily found in symbiotic relationships with various marine hosts such as ascidians and sponges. Prochloron remains to be successfully cultivated outside of its host, which reflects a lack of knowledge of its unique ecophysiological requirements. We investigated the microenvironment and diversity of Prochloron inhabiting the upper, exposed surface of didemnid ascidians, providing the first insights into this microhabitat. The pH and O2 concentration in this Prochloron biofilm changes dynamically with irradiance, where photosynthetic activity measurements showed low light adaptation (Ek ∼80±7μmol photons m-2s-1) but high light tolerance. Surface Prochloron cells exhibited a different fine structure to Prochloron cells from cloacal cavities in other ascidians, the principle difference being a central area of many vacuoles dissected by single thylakoids in the surface Prochloron. Cyanobacterial 16S rDNA pyro-sequencing of the biofilm community on four ascidians resulted in 433 operational taxonomic units (OTUs) where on average -85% (65-99%) of all sequence reads, represented by 136 OTUs, were identified as Prochloron via blast search. All of the major Prochloron-OTUs clustered into independent, highly supported phylotypes separate from sequences reported for internal Prochloron, suggesting a hitherto unexplored genetic variability among Prochloron colonizing the outer surface of didemnids.

Chl b is a major photosynthetic pigment of peripheral antenna complexes in chlorophytes and prochlorophytes. Chl b is synthesized by chlorophyllide a oxygenase (CAO), an enzyme that has been identified from higher plants, moss, green algae and two groups of prochlorophytes, Prochlorothrix and Prochloron. Based on these results, we previously proposed the hypothesis that all of the Chl b synthesis genes have a common origin. However, the CAO gene is not found in whole genome sequences of Prochlorococcus although a gene which is distantly related to CAO was reported. If Prochlorococcus employs a different enzyme, a Chl synthesis gene should have evolved several times on the different phylogenetic lineages of Prochlorococcus and other Chl b-containing organisms. To examine these hypotheses, we identified a Prochlorococcus Chl b synthesis gene by using a combination of bioinformatics and molecular genetics techniques. We first identified Prochlorococcus-specific genes by comparing the whole genome sequences of Prochlorococcus marinus MED4, MIT9313 and SS120 with Synechococcus sp. WH8102. Synechococcus is closely related to Prochlorococcus phylogenetically, but it does not contain a Chl b synthesis gene. By examining the sequences of Prochlorococcus-specific genes, we found a candidate for the Chl b synthesis gene and introduced it into Synechocystis sp. PCC6803. The transformant cells accumulated Chl b, indicating that the gene product catalyzes Chl b synthesis. In this study, we discuss the evolution of CAO based upon the molecular phylogenetic studies we performed.

A large family of cytotoxic cyclic peptides exemplified by the patellamides has been isolated from ascidians harboring the obligate cyanobacterial symbionts Prochloron spp.. Genome sequence analysis of these symbionts has revealed that Prochloron spp. synthesize patellamides by a ribosomal pathway. To understand how this pathway evolved to produce a suite of related metabolites, we analyzed 46 prochloron-containing ascidians from the tropical Pacific Ocean for the presence of patellamide biosynthetic genes and taxonomic markers. Here, we show that Prochloron spp. generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contains a single pathway, and mixtures of symbionts within ascidians lead to the accumulation of chemical libraries. We used this information to engineer the production of a new cyclic peptide in Escherichia coli, thereby demonstrating the power of comparative analysis of closely related symbiotic pathways to direct the genetic synthesis of new molecules.

The major ultrastructural and pigment characteristics of three chlorophyll b-containing photosynthetic prokaryote genera, Prochloron, Prochlorothrix, and Prochlorococcus, are summarized. Aspects of their ecology are reviewed, as are also the major findings of comparisons among the three genera, and between them and the cyanobacteria, based on analyses of nucleotide sequences. The information summarized is discussed to assess how closely the three genera are related and whether they form a natural taxonomic grouping within the cyanobacteria. It is concluded that, although much of the evidence points to substantial differences among the three genera, the convenience of keeping them together as a group, namely, Oxychlorobacteria (formerly Prochlorophyta), outweighs certain inconsistencies revealed by analysis of a range of objective criteria. Current views on the possible significance of the group to considerations of the evolution of green chloroplasts are also presented.

The contents of mycosporine-like amino acids (MAAs) were compared in the two color morphs (dark-gray and brown colonies) of the tropical ascidian Didemnum molle (Herdman, 1886), which harbors the photosymbiotic prokaryote Prochloron. The colonies of each color morph were exclusively distributed in shallow reef lagoons at the different sites. Spectroscopic and chromatographic analyses showed that the Prochloron cell density and MAA concentration in the dark-gray colonies were an estimated 1.4 and 2.4 times higher, respectively, than in the brown colonies. The significant difference in MAA contents between the color morphs was primarily due to the difference in shinorine contents (p < 0.01, Mann–Whitney U-test). The high concentration of MAAs in the dark-gray colonies may provide better conditions for Prochloron cells, compared to the brown colonies with lower MAA concentrations.

In the tropics, certain didemnid ascidians harbor the prokaryotic photosymbiont Prochloron. To date, this photosymbiosis has been found in four didemnid genera that include non-symbiotic species. Here, we report the molecular phylogeny of symbiotic and non-symbiotic didemnids based on their 18S rDNA sequences. The data cover all four genera containing symbiotic species and one other genus comprised of only non-symbiotic species. Near-complete nucleotide sequences of 18S rDNAs were determined for four non-didemnid species and 52 didemnid samples (five genera), including 48 photosymbiotic samples collected from the Ryukyu Archipelago, the Great Barrier Reef, Hawaii, and Bali. Our phylogenetic trees indicated a monophyletic origin of the family Didemnidae, as well as each of the didemnid genera. The results strongly support the hypothesis that establishment of the ascidian-Prochloron symbiosis occurred independently in the Didemnidae lineage at least once in each of the genera that possess symbiotic species.

In obligate symbioses between didemnid ascidians and prokaryotic algae (cyanobacteria), the larvae of the host ascidian usually possess Prochloron cells that are acquired from their mother colony. The process of vertical transmission of the photosymbionts has been histologically described in several species harboring Prochloron in the common cloacal cavity, where Prochloron cells are attached to the hairy surface of the tunic of the larvae during or just before larval spawning. Since the process has never been described in species harboring the photosymbionts in the tunic of the colony, here we describe the histological and ultrastructural process of vertical transmission in a photosymbiotic didemnid, Trididemnum miniatum. No photosymbionts were associated with gametes of the ascidian. Prochloron cells appeared in the epithelial pouch enclosing the embryo and were then incorporated in the tunic of embryos in late embryonic stages. Although Prochloron cells in the tunic of the colony were rarely associated with the host cells, some amoeboid-shaped tunic cells containing Prochloron cells were occasionally found around the epithelial pouch. Therefore, the host cell is thought to be a vehicle transporting the photosymbionts in the tunic of the colony to the tunic of the embryos. The photosymbiont cells were directly embedded in the tunic of the larval trunk. They were not contained in the host cells, as are those in the tunic of the colony. These observations revealed that the mechanism of vertical transmission in T. miniatum is very different from that in didemnids harboring Prochloron in the common cloacal cavity of the colonies, such as Trididemnum cyclops. The mechanisms of symbiont transmission are diverse even within the genus Trididemnum.

The seasonality of sexual reproduction was studied in two Prochloron-bearing ascidians, Trididemnum cyclops and Lissoclinum bistratum, on a subtropical coral reef off Okinawajima Island, Japan. These colonial ascidians had testes and/or eggs/embryos from spring to summer. Embryos with tails occurred in summer. Whereas many photosymbiotic didemnids are thought to be sexually mature throughout the year in the tropics, sexual reproduction of the same species in subtropical waters may be limited to spring and summer. The subtropical winter may be too cold for gonad formation. A histological study of sexually mature colonies showed no Prochloron cells attached to ascidian larvae in the pre-hatch stage.

Prochloronspp. are obligate cyanobacterial symbionts of many didemnid family ascidians. It has been proposed that the cyclic peptides of the patellamide class found in didemnid extracts are synthesized byProchloronspp., but studies in which host and symbiont cells are separated and chemically analyzed to identify the biosynthetic source have yielded inconclusive results. As part of theProchloron didemnisequencing project, we identified patellamide biosynthetic genes and confirmed their function by heterologous expression of the whole pathway inEscherichia coli. The primary sequence of patellamides A and C is encoded on a single ORF that resembles a precursor peptide. We propose that this prepatellamide is heterocyclized to form thiazole and oxazoline rings, and the peptide is cleaved to yield the two cyclic patellamides, A and C. This work represents the full sequencing and functional expression of a marine natural-product pathway from an obligate symbiont. In addition, a related cluster was identified inTrichodesmium erythraeumIMS101, an important bloom-forming cyanobacterium.

Two new species of didemnid ascidians, Diplosoma ooru sp. nov. and Diplosoma simileguwa sp. nov., are described from coral reefs on Okinawajima (Ryukyu Archipelago, Japan). These two species form green colonies, having a symbiotic association with a prokaryotic alga Prochloron sp. The former species was found at the reef edges in the subtidal zone and the latter was found in a shallow reef lagoon. In these species, the colonies are thinner and the zooids are smaller than those of any other Prochloron-bearing Diplosoma species so far described. Moreover, each of the present new species has a unique combination of stigmatic numbers: 5 stigmata in the first and third rows, 6 in the second row, and 4 in the fourth in D. ooru; 4 stigmata in the first and third rows, 5 in the second row, and 3 in the fourth in D. simileguwa. In both of the new species, the retractor muscle emerges from the underside of the thorax. Larval morphology of D. ooru is also described.

Cyanophyte-like prokaryotes are widely presumed to be the progenitors of eukaryote plastids. A few rare protistan species bearing cyanophyte-like cyanelles may represent intermediate stages in the evolution of true organelles. Cyanophyte DNA disposition in the cell, so far as is known from electron microscopy, seems uniform within the group and distinctly different from the several known arrangements of DNA in plastids. Therefore a survey of representative cyanophytes and protistan cyanelles was undertaken to determine whether forms reminiscent of plastids could be found. DNA-specific fluorochromes were utilized, along with epifluorescent microscopy, to study the DNA arrangement in situ in whole cells. Only the endospore (baeocyte)-forming Cyanophyta contained more than one, centrally located DNA skein per cell, and then only for the period just preceding visible baeocyte formation. Such forms might, with modification, presage the “scattered nucleoid” DNA disposition found in plastids of several groups, including Rhodophytes, Cryptophytes, Chlorophytes and higher plants. The DNA arrangement in cyanelles of two protists, Cyanophora and Glaucocystis, appear different from each other and possibly related to, respectively, the cyanophytes Gloeobacter and Synechococcus. Cyanelles of the third protist, Glaucosphaera, like the cells of the unique prokaryote Prochloron, appear to have multiple sites of DNA, somewhat similar to those of the “scattered nucleoid” line of plastid evolution. No obvious precursor of the “ring nucleoid” or other types of plastid DNA conformation was found.

Summary Stromatolites have been present on Earth, at various levels of distribution and diversity, for more than 3 billion years. Today, the best examples of stromatolites forming in hypersaline marine environments are in Hamelin Pool at Shark Bay, Western Australia. Despite their evolutionary significance, little is known about their associated microbial communities. Using a polyphasic approach of culture-dependent and culture-independent methods, we report the discovery of a wide range of microorganisms associated with these biosedimentary structures. There are no comparable reports combining these methodologies in the survey of cyanobacteria, bacteria, and archaea in marine stromatolites. The community was characterized by organisms of the cyanobacterial genera Synechococcus, Xenococcus, Microcoleus, Leptolyngbya, Plectonema, Symploca, Cyanothece, Pleurocapsa and Nostoc. We also report the discovery of potentially free-living Prochloron. The other eubacterial isolates and clones clustered into seven phylogenetic groups: OP9, OP10, Marine A group, Proteobacteria, Low G+C Gram-positive, Planctomycetes and Acidobacteria. We also demonstrate the presence of sequences corresponding to members of halophilic archaea of the divisions Euryarchaeota and Crenarchaeota and methanogenic archaea of the order Methanosarcinales. This is the first report of such archaeal diversity from this environment. This study provides a better understanding of the microbial community associated with these living rocks.

pcb genes, encoding proteins binding light-harvesting chlorophylls, were cloned and sequenced from the Chl d-containing cyanobacterium, Acaryochloris marina, and the Chl b-containing cyanobacterium, Prochloron didemni. Both organisms contained two tandem pcb genes. Peptide fingerprinting confirmed the expression of one of the A. marina pcb genes. Phylogenetic tree reconstruction using distance-matrix and maximum-likelihood methods indicated a single origin of the pcb gene family, whether occurring in Chl b-containing or Chl d-containing organisms. This may indicate widespread lateral transfer of the Pcb protein-based light-harvesting system.

To characterize the ultraviolet (UV) light-absorbing function of the ascidian tunic, the light absorption spectrum was compared in 22 ascidian species collected from tropical and temperate waters. Non-photosymbiotic ascidians (17 species) had transparent tunics transmitting both visible and UV light, or pigmented or opaque tunics equally absorb both UV and visible light. However, a prominent absorption peak around 320 nm was exclusively found in the colonial ascidians hosting the algal symbiont Prochloron sp., and this absorption peak corresponded to absorption of UV-A (320–400 nm) and UV-B (280–320 nm). The UV-absorbing substances were extracted with methanol. They were mycosporine-like amino acids (MAAs): mycosporine-glycine, palythine, shinorine, and porphyra-334. The MAAs are thought to be synthesized by the shikimic acid pathway that does not occur in animals. Since the isolated Prochloron cells contain MAAs, the symbionts are the most possible candidates as the source of the MAAs in the tunic. In Diplosoma virens, the composition of MAAs was different between isolated Prochloron cells and colony residue from which Prochloron cells were extracted.

Prochlorophytes are a class of cyanobacteria that do not use phycobiliproteins as light-harvesting systems, but contain chlorophyll (Chl) a / b -binding Pcb proteins. Recently it was shown that Pcb proteins form an 18-subunit light-harvesting antenna ring around the photosystem I (PSI) trimeric reaction center complex of the prochlorophyte Prochlorococcus marinus SS120. Here we have investigated whether the symbiotic prochlorophyte Prochloron didemni also contains the same supermolecular complex. Using cells isolated directly from its ascidian host, we found no evidence for the presence of the Pcb–PSI supercomplex. Instead we have identified and characterized a supercomplex composed of photosystem II (PSII) and Pcb proteins. We show that 10-Pcb subunits associate with the PSII dimeric reaction center core to form a giant complex having an estimated M r of 1,500 kDa with dimensions of 210 × 290 Å. Five-Pcb subunits flank each long side of the dimer and assuming each binds 13 Chl molecules, increase the antenna size of PSII by ≈200%. Fluorescence emission studies indicate that energy transfer occurs efficiently from the Pcb antenna. Modeling using the x-ray structure of cyanobacterial PSII suggests that energy transfer to the PSII reaction center is via the Chls bound to the CP47 and CP43 proteins.

Marine sponges frequently contain a complex mixture of bacteria, fungi, unicellular algae and cyanobacteria. Epifluorescent microscopy showed that Mycale (Carmia) hentscheli contained coccoid cyanobacteria. The 16S rRNA gene was amplified, fragments cloned and analysed using amplified rRNA gene restriction analysis. The nearly complete 16S rRNA gene of distinct clones was sequenced and aligned using ARB. The phylogenetic analysis indicated the presence of four closely related clones which have a high (8%) sequence divergence from known cyanobacteria, Cyanobacterium stanieri being the closest, followed by Prochloron sp. and Synechocystis sp. All belong to the order Chroococcales. The lack of non-molecular evidence prevents us from proposing a new genus.

The cyanobacterium Prochloron didemni is primarily found in symbiotic relationships with various marine hosts such as ascidians and sponges. Prochloron remains to be successfully cultivated outside of its host, which reflects a lack of knowledge of its unique ecophysiological requirements. We investigated the microenvironment and diversity of Prochloron inhabiting the upper, exposed surface of didemnid ascidians, providing the first insights into this microhabitat. The pH and O2 concentration in this Prochloron biofilm changes dynamically with irradiance, where photosynthetic activity measurements showed low light adaptation (Ek ∼ 80 ± 7 μmol photons m(-2) s(-1)) but high light tolerance. Surface Prochloron cells exhibited a different fine structure to Prochloron cells from cloacal cavities in other ascidians, the principle difference being a central area of many vacuoles dissected by single thylakoids in the surface Prochloron. Cyanobacterial 16S rDNA pyro-sequencing of the biofilm community on four ascidians resulted in 433 operational taxonomic units (OTUs) where on average -85% (65-99%) of all sequence reads, represented by 136 OTUs, were identified as Prochloron via blast search. All of the major Prochloron-OTUs clustered into independent, highly supported phylotypes separate from sequences reported for internal Prochloron, suggesting a hitherto unexplored genetic variability among Prochloron colonizing the outer surface of didemnids.

The embryos of Diplosoma similis are brooded within the thick walled tunic of the colony in isolation from the symbiotic algae Prochloron sp., which are in the cloacal cavity of the parent colony. Prior to the spawning, the plant rake, a tassel-like structure, protrudes from the postero-dorsal end of the larval trunk and extends into the cloacal cavity. The algal cells in the cloacal cavity adhere to the plant rake. When the larvae are spawned, the trunk tunic extends posteriorly and forms a pouch entirely covering the plant rake. The algal cells are packed in the pouch (algal pouch) enveloping the basal part of the tail. The cell density of the algae in the pouch is much higher than that in the colony, suggesting that the plant rake functions for gathering and concentrating the symbionts into the algal pouch. In the course of metamorphosis, the algal pouch expands and turns into the cloacal cavity of the young colony. The high density of algal cells in the pouch would ensure that the young colon...

Six species of compound ascidians found in a Fijian seagrass bed dominated by Syringodium isoetifolium were divided into two groups, each occupying somewhat different microhabitats provided by the seagrass. Didemnum molle and Lissoclinum bistratum, both with algal symbiont Prochloron sp., were abundant in high light microhabitats. D. molle was mostly attach to seagrass blades (maximum colony density: 980m-2), while L. bistratum occurred both on seagrass and sediment surfaces in places with sparse seagrass cover (maximum colony density: 11, 500m-2). Trididemnum clinides also had the symbiont Prochloron sp., but it mostly occupied dark microhabitats such as the sheaths of the seagrass. The other 3 species, Didemnum cuculiferum, D. sp. cf. albopunctatum and Trididemnum discrepans, lacked algal symbionts and were rare, all occupying dark places such as seagrass sheaths in areas with dense seagrass cover. Sympatric ascidians, thus, co-exist in seagrass beds and show a different microhabitat use. Ascidians were not distributed evenly over the area of the seagrass bed, but were concentrated in an area between 30 and 84m from the shore, independent of the distribution of seagrass biomass. In dense seagrass patches, light intensities varied greatly between the top and the basal part of the seagrass, and persistence and stability of seagrass as an attachment substrate were also different between leaf blades and sheaths. Populations of D. molle on the seagrasses included many smaller colonies. There were no colonies as large as those in the population on the more stable nearby rock substrates. The small size of the seagrass blades (1.5mm in diameter), their short lifetime (1.5mo) and their lower persistence and stability as an attachment substrate may explain the small size of the colonies on the seagrass.

[8-vinyl]-Protochlorophyllide a1The nomenclature for chlorophylls is somewhat confusing: [8-vinyl]-protochlorophyllide was known as Mg-2,4-divinyl-pheoporphyrin a5-monomethyl ester (old Fischer nomenclature). We prefer the name divinyl-protochlorophyllide (DV-Pchlide) which is commonly used in chlorophyll biosynthesis.1 was isolated from a Prochloron sp. associated with the host ascidian, Lissoclinum patella. To obtain sufficient amounts for identification of the purified pigment, suitable extraction procedures and HPLC systems were developed. The structure was finally elucidated by UV-VIS and fluorescence spectroscopy, mass spectrometry and NMR (rotating-frame Overhauser enhancement spectroscopy). [8-vinyl]-Protochlorophyllide a was originally detected only as an intermediate in chlorophyll biosynthesis. Although its presence as a light-harvesting pigment was previously suggested in some prochlorophytes and eukaryotic algae, this is the first unequivocal demonstration of [8-vinyl]-protochlorophyllide a in an oxygenic phototroph. We also show that [8-vinyl]-protochlorophyllide a occurs in Prochloron species of four other ascidians as well as in Micromonas pusilla and Prochlorococcus marinus. The possible role of this pigment in photosynthesis is discussed.

The clear waters surrounding tropical coral reefs typically are oligotrophic, yet these reefs are highly productive and support dense populations of marine organisms. This paradox is resolved in part because many coral reef invertebrates accommodate unicellular autotrophs (‘‘zooxanthellae,’’ Symbiodinium spp.; Prochlorales, Prochloron sp.; cyanobacteria) within their tissues. These photoautotrophic symbioses entail an exchange of nutrients between the endosymbionts and the animal hosts. Organic compounds produced by the microalgal partners are released to the hosts for their nutrition while inorganic metabolic wastes are recycled to fertilize the algae (Muscatine 1990). The requirement for photosynthetically active radiation (PAR, 400–700 nm) also exposes such algalinvertebrate symbioses and other primary producers to high levels of environmental ultraviolet radiation (UVR 5 295–400 nm) in tropical waters (Smith and Baker 1979, Fleischmann 1989). These waters are often low in UV-absorbing particulate and dissolved organic matter, and sea level fluences of solar UV are high because of the short atmospheric path length and thinness of the stratospheric ozone layer above the tropics (Baker et al. 1980). Photoautotrophic symbiosis thus presents an evolutionary challenge by precluding the morphological development of an optically opaque barrier (such as hair, scales, and feathers in higher vertebrates) and potentially allows damaging UV radiation to reach vulnerable biomolecules in both partners. The problem is exacerbated because the tissues are hyperoxic (.250% air saturation: Kuhl et al.

Phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris marina, containing chlorophyll d as major pigment. In the electron microscope the biliprotein aggregates appear as rod-shaped structures of 26.0×11.3 nm, composed of four ring-shaped subunits 5.8 nm thick and 11.7 nm in diameter. Spectral data indicate that the aggregates contain two types of biliproteins: phycocyanin and an allophycocyanin-type pigment, with very efficient energy transfer from the phycocyanin- to allophycocyanin-type constituent. The chromophore-binding polypeptides of the pigments have apparent molecular masses of 16.2 and 17.4 kDa. They crossreact with antibodies against phycocyanin and allophycocyanin from a red alga.

Photosynthesis by cells of Prochloron sp. freshly isolated from the ascidian host, Lissoclinum patella, collected from shallow waters in Palau, was severely inhibited by ultraviolet (UV) irradiation. No photoinhibition, however, was observed in Prochloron cells isolated from intact colonies after UV irradiation, suggesting some protection by the ascidian host. It was shown that UV protection was brought about by the thick gelatinous tunic covering the whole ascidian colony. Analysis revealed that the surface tunic of L. patella, although transparent to visible light, contains several UV-absorbing substances, identified by high-performance liquid chromatography as mycosporine-like amino acids (MAAs). The predominant MAA identified was shinorine (λmax = 334 nm), followed by mycosporine-glycine (λmax = 310 nm) and a small amount of palythine (λmax = 320 nm). Although isolated Prochloron cells also contained shinorine, on a protein-weight basis it was less than half of that observed in the host tunic. These results suggest that the surface tunic of a L. patella colony, which is transparent to visible light for photosynthesis, also contains UV-absorbing compounds that protect its photoautotrophic symbiont, Prochloron sp., from damage by the intense UV-irradiation that they receive daily in shallow, tropical marine waters.