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The problems of Prochloron.

Prokaryotic green algae (prochlorophytes), which contain chlorophylls a and b but no bilin pigments, may be phylogenetically related to ancestral chloroplasts if symbiogenesis occurred. They may be otherwise related to eukaryotic chlorophytes. They could have evolved from cyanophytes by loss of phycobilin and gain of chlorophyll b synthesis. These possibilities are briefly discussed. Relevant evidence from biochemical studies in many collaborative laboratories is now becoming available for the resolution of such questions.

Biological Evolution↗

The nomenclatural types of the orders Acholeplasmatales, Halanaerobiales, Halobacteriales, Methanobacteriales, Methanococcales, Methanomicrobiales, Planctomycetales, Prochlorales, Sulfolobales, Thermococcales, Thermoproteales and Verrucomicrobiales are the genera Acholeplasma, Halanaerobium, Halobacterium, Methanobacterium, Methanococcus, Methanomicrobium, Planctomyces, Prochloron, Sulfolobus, Thermococcus, Thermoproteus and Verrucomicrobium, respectively. Opinion 79.

The Judicial Commission of the International Committee on Systematics of Prokaryotes has corrected the nomenclatural types of 12 orders: Acholeplasmatales, Halanaerobiales, Halobacteriales, Methanobacteriales, Methanococcales, Methanomicrobiales, Planctomycetales, Prochlorales, Sulfolobales, Thermococcales, Thermoproteales and Verrucomicrobiales.

Archaea↗

Vertical transmission of photosymbionts in the colonial ascidian Didemnum molle: the larval tunic prevents symbionts from attaching to the anterior part of larvae.

Morphological processes in the vertical transmission of photosymbionts were investigated in the Prochloron-bearing ascidian Didemnum molle. Prochloron cells were found exclusively in the common cloacal cavity of the colony, attached mainly to the tunic lining of the cavity wall. Oocytes were found in the abdominal region of each zooid, but no Prochloron cells were associated with this stage. During embryogenesis, embryos moved into the tunic core of the colony and were always separated from Prochloron cells in the cloacal cavity by the tunic matrix, until they hatched out from the tunic core. In swimming larvae, Prochloron cells covered the surface of the posterior half of the larval trunk, whereas a thin larval tunic layer covered the anterior half, where no Prochloron cells were found. The tunic of the posterior half of the larval trunk had many folds that enfolded the Prochloron cells and may be adhesive in order to acquire Prochloron cells from the mother colony. The thin larval tunic layer is probably not adhesive and protects the anterior half of the trunk from interference by Prochloron cells with sensory receptors and adhesive organs.

Animals↗

Localization of symbiotic cyanobacteria in the colonial ascidian Trididemnum miniatum (Didemnidae, Ascidiacea).

Trididemnum miniatum is a colonial ascidian harboring the photosymbiotic prokaryote Prochloron sp. These bacterial cells are located in the tunic of the host animal. The present study revealed, by ultrastructural analysis, that the Prochloron cells were exclusively distributed and proliferated in the tunic. They were shown to be embedded in the tunic matrix and to have no direct contact with ascidian cells. Some tunic cells of the ascidians, however, did phagocytize and digest the symbiont. Round cell masses were sometimes found in the tunic and appeared to consist of disintegrating cyanobacterial cells. The thoracic epidermis of ascidian zooids was often digitated, and the epidermal cells extended microvilli into the tunic. Since there were no Prochloron cells in the alimentary tract of the ascidian zooids, the photosymbionts would not be considered part of the typical diet of the host ascidians. Thin layer chromatography showed that the symbionts possessed both chlorophyll a and b, while a 16S rRNA gene phylogeny supported the identification of the photosymbiont of T. miniatum as Prochloron sp.

Animals↗

Natural combinatorial peptide libraries in cyanobacterial symbionts of marine ascidians.

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.

Amino Acid Sequence↗

Identification of

[8-vinyl]-Protochlorophyllide a1 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.

Journal Article↗

Novel cytotoxic compounds from the ascidian Lissoclinum bistratum.

The isolation and structures of two new cyclic hexapeptides and two new macrocyclic ethers from the aplousobranch ascidian Lissoclinum bistratum are described. Their structures were determined by two-dimensional NMR techniques. The hexapeptides, named bistratamide A and bistratamide B, differ only by the presence or absence of one double bond. They were tested for cytotoxicity toward human fibroblast and tumor cell lines and displayed similar toxicities to the octapeptides called patellamides from Lissoclinum patella. The peptides are found within the obligate algal symbiont Prochloron but clearly differ from peptides isolated from the same Prochloron of L. patella. The macrocyclic ethers isolated from L. bistratum are exceedingly potent in cytotoxicity. They have been named bistratenes A and B, and structures for these compounds are proposed.

Acetamides↗

Glyceraldehyde-3-phosphate dehydrogenase gene diversity in eubacteria and eukaryotes: evidence for intra- and inter-kingdom gene transfer.

Cyanobacteria contain up to three highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes: gap1, gap2, and gap3. Genes gap1 and gap2 are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants and have recently been shown to play distinct key roles in catabolic and anabolic carbon flow, respectively, of the unicellular cyanobacterium Synechocystis sp. PCC6803. In the present study, sequences of 10 GAPDH genes distributed across the cyanobacteria Prochloron didemni, Gloeobacter violaceus PCC7421, and Synechococcus PCC7942 and the alpha-proteobacterium Paracoccus denitrificans and the beta-proteobacterium Ralstonia solanacearum were determined. Prochloron didemni possesses homologs to the gap2 and gap3 genes from Anabaena, Gloeobacter harbors gap1 and gap2 homologs, and Synechococcus possesses gap1, gap2, and gap3. Paracoccus harbors two highly divergent gap genes that are related to gap3, and Ralstonia possesses a homolog of the gap1 gene. Phylogenetic analyses of these sequences in the context of other eubacterial and eukaryotic GAPDH genes reveal that divergence across eubacterial gap1, and gap2, and gap3 genes is greater than that between eubacterial gap1 and eukaroytic glycolytic GapC or between eubacterial gap2 and eukaryotic Calvin cycle GapAB. These data strongly support previous analyses which suggested that eukaryotes acquired their nuclear genes for GapC and GapAB via endosymbiotic gene transfer from the antecedents of mitochondria and chloroplasts, and extend the known range of sequence diversity of the antecedent eubacterial genes. Analyses of available GAPDH sequences from other eubacterial sources indicate that the glycosomal gap gene from trypanosomes (cytosolic in Euglena) and the gap gene from the spirochete Treponema pallidum are each other's closest relatives. This specific relationship can therefore not reflect organismal evolution but must be the result of an interkingdom gene transfer, the direction of which cannot be determined with certainty at present. Contrary to this, the origin of the cytosolic Gap gene from trypanosomes can now be clearly defined as gamma-proteobacterial, since the newly established Ralstonia sequence (beta-proteobacteria) branches basally to the gamma-proteobacterial/trypanosomal assemblage.

Amino Acid Sequence↗

The prochlorophytes: are they more than just chlorophyll a/b-containing cyanobacteria?

The prochlorophytes are a diverse group of photosynthetic prokaryotes that fall within the cyanobacterial lineage, yet lack phycobilisomes as light harvesting structures. Instead, the prochlorophytes have a light-harvesting apparatus composed of the higher plant pigments chlorophylls a and b. This review discusses the evolutionary relationships among these bacteria, with focus on the structure and function of the photosynthetic apparatus. This analysis yields a consensus from studies both on Prochloron sp. and Prochlorothrix hollandica as to how the thylakoid membrane is organized. Overall, we propose that the structure of the light-harvesting complexes (LHC) from prochlorophytes is very different from those of chloroplast systems, and is evolutionarily very ancient. The functional association of the light-harvesting apparatus with photosystem I (PSI) in both Prochlorothrix and Prochloron, as well as a demonstrated capacity for PSI-dependent anoxygenic photosynthesis in Prochlorothrix, may indicate that there is an increased dependence on cyclic photophosphorylation in these organisms. Finally, the structure of the prochlorophyte thylakoid membrane is discussed with respect to the forces that drive thylakoid membrane stacking in prochlorophytes and chloroplasts. We suggest that the light-harvesting structures in prochlorophytes play little, if any, role in this process.

Chlorophyll↗

Chlorophyll b and phycobilins in the common ancestor of cyanobacteria and chloroplasts.

Photosynthetic organisms have a variety of accessory pigments, on which their classification has been based. Despite this variation, it is generally accepted that all chloroplasts are derived from a single cyanobacterial ancestor. How the pigment diversity has arisen is the key to revealing their evolutionary history. Prochlorophytes are prokaryotes which perform oxygenic photosynthesis using chlorophyll b, like land plants and green algae (Chlorophyta), and were proposed to be the ancestors of chlorophyte chloroplasts. However, three known prochlorophytes (Prochloron didemni, Prochlorothrix hollandica and Prochlorococcus marinus) have been shown to be not the specific ancestors of chloroplasts, but only diverged members of the cyanobacteria, which contain phycobilins but lack chlorophyll b. Consequently it has been proposed that the ability to synthesize chlorophyll b developed independently several times in prochlorophytes and in the ancestor of chlorophytes. Here we have isolated the chlorophyll b synthesis genes (chlorophyll a oxygenase) from two prochlorophytes and from major groups of chlorophytes. Phylogenetic analyses show that these genes share a common evolutionary origin. This indicates that the progenitors of oxygenic photosynthetic bacteria, including the ancestor of chloroplasts, had both chlorophyll b and phycobilins.

Amino Acid Sequence↗

Unique origin and lateral transfer of prokaryotic chlorophyll-b and chlorophyll-d light-harvesting systems.

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.

Biological Evolution↗

Sequence analysis of 16S rRNA gene of cyanobacteria associated with the marine sponge Mycale (Carmia) hentscheli.

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.

Animals↗

UV protection in the photosymbiotic ascidian Didemnum molle inhabiting different depths.

Didemnum molle is a colonial ascidian that harbors the prokaryotic photosymbiont Prochloron in its cloacal cavity. Colonies occur over a relatively wide bathymetric range (approximately 0-30 m), and colony color is widely variable, partly depending on depth. Colonies in shallow sites are bright white, with densely distributed spicules, and often with brown or dark gray pigmentation, while colonies in deeper sites are less pigmented, with sparsely distributed spicules. Didemnum molle colonies contain mycosporine-like amino acids (MAAs) as UV-absorbing substances. These include mycosporine-glycine, shinorine, and porphyra-334. Among colonies from 5-, 10-, 15-, and 20-m depths, the concentration of total MAAs was significantly high at 10 m and low at 20 m. Colonies at 10 m need to maintain low spicule densities to have enough photosynthetically active radiation (PAR) to maintain the photosymbionts, and they probably concentrate MAAs to block UV radiation without attenuating PAR. Because high levels of PAR cause photoinhibition of photosynthesis, spicules and pigment cells would be more effective for photoprotection in shallow water. Colonies of D. molle may adjust the light conditions for photosymbionts by combining MAAs, spicules, and pigment cells in varying amounts.

Amino Acids↗

Sequence analysis and phylogenetic reconstruction of the genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the chlorophyll b-containing prokaryote Prochlorothrix hollandica.

Prochlorophytes similar to Prochloron sp. and Prochlorothrix hollandica have been suggested as possible progenitors of the plastids of green algae and land plants because they are prokaryotic organisms that possess chlorophyll b (chl b). We have sequenced the Prochlorothrix genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase(rubisco), rbcL and rbcS, for comparison with those of other taxa to assess the phylogenetic relationship of this species. Length differences in the large subunit polypeptide among all sequences compared occur primarily at the amino terminus, where numerous short gaps are present, and at the carboxy terminus, where sequences of Alcaligenes eutrophus and non-chlorophyll b algae are several amino acids longer. Some domains in the small subunit polypeptide are conserved among all sequences analyzed, yet in other domains the sequences of different phylogenetic groups exhibit specific structural characteristics. Phylogenetic analyses of rbcL and rbcS using Wagner parsimony analysis of deduced amino acid sequences indicate that Prochlorothrix is more closely related to cyanobacteria than to the green plastid lineage. The molecular phylogenies suggest that plastids originated by at least three separate primary endosymbiotic events, i.e., once each leading to green algae and land plants, to red algae, and to Cyanophora paradoxa. The Prochlorothrix rubisco genes show a strong GC bias, with 68% of the third codon positions being G or C. Factors that may affect the GC content of different genomes are discussed.

Amino Acid Sequence↗

Catalytically active hybrids formed in vitro between large and small subunits of different procaryotic ribulose bisphosphate carboxylases.

Ribulose bisphosphate carboxylase from the procaryotic green alga, Prochloron (the symbiont of Lissoclinum patellum), has eight large and eight small subunits, and a low affinity for CO2, similar to that of cyanobacterial carboxylases. The small subunits were progressively removed from this carboxylase and from that from the cyanobacterium, Synechococcus ACMM 323, by twice-repeated, mild-acid precipitation. This procedure produced large-subunit octamers, greatly depleted in small subunits, as well as isolated small subunits. Catalytic activity of the large-subunit preparations reflected their residual small-subunit content. The two large-subunit preparations were reconstituted with both homologous and heterologous small subunits. The reassembled enzymes were catalytically competent in all cases. When fully saturated with small subunits, the hybrid enzymes were only about 20% less active than the homologously reconstituted enzymes. Heterologous reconstitution underscores the essential function of the small subunits in catalysis.

Catalysis↗

New cyclic peptides with cytotoxic activity from the ascidian Lissoclinum patella.

The isolation and structures of a new patellamide (patellamide D) and two new lissoclinamides (lissoclinamides 4 and 5) from the aplousobranch ascidian Lissoclinum patella are described. Structures were determined largely by using two-dimensional NMR techniques and mass spectrometry. These peptides and other members of the patellamide and lissoclinamide families that have been reported previously are found within the obligate algal symbiont of the genus Prochloron. The cytotoxicities of the compounds toward fibroblast and tumor cell lines are reported. One of these compounds, lissoclinamide 4, is markedly more toxic than other members of the family. Structure-activity relationships are discussed.

Animals↗