RESUMO
Patellamides are highly bioactive compounds found along with other cyanobactins in the symbiosis between didemnid ascidians and the enigmatic cyanobacterium Prochloron. The biosynthetic pathway of patellamide synthesis is well understood, the relevant operons have been identified in the Prochloron genome and genes involved in patellamide synthesis are among the most highly transcribed cyanobacterial genes in hospite. However, a more detailed study of the in vivo dynamics of patellamides and their function in the ascidian-Prochloron symbiosis is complicated by the fact that Prochloron remains uncultivated despite numerous attempts since its discovery in 1975. A major challenge is to account for the highly dynamic microenvironmental conditions experienced by Prochloron in hospite, where light-dark cycles drive rapid shifts between hyperoxia and anoxia as well as pH variations from pH ~6 to ~10. Recently, work on patellamide analogues has pointed out a range of different catalytic functions of patellamide that could prove essential for the ascidian-Prochloron symbiosis and could be modulated by the strong microenvironmental dynamics. Here, we review fundamental properties of patellamides and their occurrence and dynamics in vitro and in vivo. We discuss possible functions of patellamides in the ascidian-Prochloron symbiosis and identify important knowledge gaps and needs for further experimental studies.
Assuntos
Peptídeos Cíclicos/metabolismo , Prochloron/metabolismo , Urocordados/metabolismo , Animais , Humanos , Concentração de Íons de Hidrogênio , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/farmacologia , Prochloron/genética , Simbiose , Urocordados/genéticaRESUMO
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 â¼ 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.
Assuntos
Microambiente Celular/fisiologia , Poríferos/microbiologia , Prochloron/classificação , Simbiose/genética , Urocordados/microbiologia , Animais , Biofilmes , DNA Ribossômico/genética , Variação Genética , Luz , Fotossíntese/genética , Fotossíntese/fisiologia , Filogenia , Prochloron/genética , RNA Ribossômico 16S/genéticaRESUMO
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.
Assuntos
Recifes de Corais , Prochloron/genética , Rhodospirillaceae/genética , Urocordados/microbiologia , Sequência de Aminoácidos , Animais , Azóis/química , Azóis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Metagenoma , Modelos Biológicos , Dados de Sequência Molecular , Filogenia , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Prochloron/fisiologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Rhodospirillaceae/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Simbiose/genética , Simbiose/fisiologia , Urocordados/fisiologiaRESUMO
Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Prochloron/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos Cíclicos/genética , Prochloron/genética , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/metabolismo , Simbiose , Urocordados/microbiologiaRESUMO
We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O(2) levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp. characterized by strong depletion of visible light and a dynamic chemical microenvironment ranging from hyperoxia in light to anoxia in darkness and (iii) a biofilm covering the underside of the animal, where light is depleted of visible wavelengths and enriched in near-infrared radiation (NIR). Variable chlorophyll fluorescence imaging demonstrated photosynthetic activity, and hyperspectral imaging revealed a diversity of photopigments in all microhabitats. Amplicon sequencing revealed the dominance of cyanobacteria in all three layers. Sequences representing the chlorophyll d containing cyanobacterium Acaryochloris marina and anoxygenic phototrophs were abundant on the underside of the ascidian in shallow waters but declined in deeper waters. This depth dependency was supported by a negative correlation between A. marina abundance and collection depth, explained by the increased attenuation of NIR as a function of water depth. The combination of microenvironmental analysis and fine-scale sampling techniques used in this investigation gives valuable first insights into the distribution, abundance and diversity of bacterial communities associated with tropical ascidians. In particular, we show that microenvironments and microbial diversity can vary significantly over scales of a few millimeters in such habitats; which is information easily lost by bulk sampling.
Assuntos
Biofilmes , Cianobactérias/fisiologia , Prochloron/fisiologia , Urocordados/microbiologia , Animais , Sequência de Bases , Carbono/metabolismo , Clorofila/análise , Análise por Conglomerados , Cianobactérias/genética , Ecossistema , Luz , Imagem Óptica , Oxigênio/metabolismo , Fotossíntese , Análise de Componente Principal , Prochloron/genética , RNA Ribossômico 16S/genéticaRESUMO
Deep metagenome mining is a new method for engineering natural product pathways, focusing on examining symbiotic organisms. The method has been applied to a family of compounds known as cyanobactins, which are ribosomally synthesized peptides produced by cyanobacteria. Often, these cyanobacteria live symbiotically with marine animals, leading to production of natural products in whole animal samples. Here, we focus on methods to identify, clone, and study cyanobactin natural product genes from axenic organisms and metagenomic environments. The application to deep metagenome mining is described, along with other potential targets of this methodology.
Assuntos
Cianobactérias/genética , Cianobactérias/metabolismo , Genoma Bacteriano/fisiologia , Biossíntese Peptídica/genética , Peptídeos/metabolismo , Ribossomos/metabolismo , Genoma Bacteriano/genética , Peptídeos/genética , Prochloron/genética , Prochloron/metabolismo , Ribossomos/genéticaRESUMO
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.
Assuntos
Variação Genética , Prochloron/classificação , Prochloron/genética , Simbiose , Urocordados/microbiologia , Animais , Austrália , Ecossistema , Geografia , Japão , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , RNA Ribossômico 16S/classificação , Estados UnidosRESUMO
MOTIVATION: The Glimmer gene-finding software has been successfully used for finding genes in bacteria, archaea and viruses representing hundreds of species. We describe several major changes to the Glimmer system, including improved methods for identifying both coding regions and start codons. We also describe a new module of Glimmer that can distinguish host and endosymbiont DNA. This module was developed in response to the discovery that eukaryotic genome sequencing projects sometimes inadvertently capture the DNA of intracellular bacteria living in the host. RESULTS: The new methods dramatically reduce the rate of false-positive predictions, while maintaining Glimmer's 99% sensitivity rate at detecting genes in most species, and they find substantially more correct start sites, as measured by comparisons to known and well-curated genes. We show that our interpolated Markov model (IMM) DNA discriminator correctly separated 99% of the sequences in a recent genome project that produced a mixture of sequences from the bacterium Prochloron didemni and its sea squirt host, Lissoclinum patella. AVAILABILITY: Glimmer is OSI Certified Open Source and available at http://cbcb.umd.edu/software/glimmer.
Assuntos
Mapeamento Cromossômico/métodos , DNA Bacteriano/genética , Decapodiformes/genética , Genoma Bacteriano/genética , Prochloron/genética , Software , Simbiose/genética , Algoritmos , Animais , Artefatos , Decapodiformes/microbiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodosRESUMO
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.
Assuntos
Prochloron/isolamento & purificação , Simbiose , Urocordados/microbiologia , Animais , Sequência de Bases , Clorofila/metabolismo , Clorofila A , Cromatografia em Camada Fina/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Dados de Sequência Molecular , Fotobiologia , Filogenia , Prochloron/classificação , Prochloron/genética , Prochloron/ultraestrutura , RNA Ribossômico 16S/genética , Urocordados/ultraestruturaRESUMO
Post-translationally modified ribosomal peptides are unusual natural products and many have potent biological activity. The biosynthetic processes involved in their formation have been delineated for some, but the patellamides represent a unique group of these metabolites with a combination of a macrocycle, small heterocycles and d-stereocentres. The genes encoding for the patellamides show very low homology to known biosynthetic genes and there appear to be no explicit genes for the macrocyclisation and epimerisation steps. Using a combination of literature data and large-scale molecular dynamics calculations with explicit solvent, we propose that the macrocyclisation and epimerisation steps are spontaneous and interdependent and a feature of the structure of the linear peptide. Our study suggests the steps in the biosynthetic route are heterocyclisation, macrocyclisation, followed by epimerisation and finally dehydrogenation. This study is presented as testable hypothesis based on literature and theoretical data to be verified by future detailed experimental investigations.
Assuntos
Peptídeos Cíclicos/biossíntese , Ciclização , Isomerismo , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos Cíclicos/química , Prochloron/química , Prochloron/genética , Prochloron/metabolismo , Tiazóis/químicaRESUMO
Prochloron spp. 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 by Prochloron spp., 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 the Prochloron didemni sequencing project, we identified patellamide biosynthetic genes and confirmed their function by heterologous expression of the whole pathway in Escherichia 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 in Trichodesmium erythraeum IMS101, an important bloom-forming cyanobacterium.
Assuntos
Bacteriocinas/metabolismo , Peptídeos Cíclicos/biossíntese , Prochloron/metabolismo , Simbiose , Urocordados/microbiologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cianobactérias/genética , Primers do DNA , Escherichia coli , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Palau , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Prochloron/genética , Análise de Sequência de DNARESUMO
Didemnid family ascidians commonly harbor obligate cyanobacterial symbionts, Prochloron spp., which have been proposed to biosynthesize cyclic peptides. Here, it is shown that Prochloron spp. do indeed contain genes for nonribosomal peptide biosynthesis, although genes for cyclic peptide biosynthesis have not yet been characterized. A peptide synthetase-containing open reading frame of unknown function was cloned from the Prochloron symbionts of some didemnid ascidians, but not from others. These data indicate that Prochloron spp. have variable secondary metabolic potential.
Assuntos
Peptídeos Cíclicos/biossíntese , Prochloron/genética , Prochloron/metabolismo , Urocordados/química , Animais , Sequência de Bases , Cianobactérias , Dados de Sequência Molecular , Estrutura Molecular , Papua Nova Guiné , Peptídeos Cíclicos/químicaRESUMO
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.
Assuntos
Cianobactérias/genética , Genes de RNAr , Poríferos/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Animais , Cianobactérias/classificação , Cianobactérias/crescimento & desenvolvimento , DNA Ribossômico/análise , DNA Ribossômico/genética , Dados de Sequência Molecular , Filogenia , Prochloron/classificação , Prochloron/genética , Prochloron/crescimento & desenvolvimento , Mapeamento por RestriçãoRESUMO
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.
Assuntos
Clorofila/análise , Cloroplastos/química , Cianobactérias/química , Evolução Molecular , Oxigenases/genética , Ficocianina/análise , Pirróis/análise , Sequência de Aminoácidos , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Cloroplastos/classificação , Cianobactérias/classificação , Cianobactérias/enzimologia , Cianobactérias/genética , Eucariotos/química , Eucariotos/enzimologia , Eucariotos/genética , Dados de Sequência Molecular , Ficobilinas , Filogenia , Prochloron/química , Prochloron/enzimologia , Prochloron/genética , Prochlorothrix/química , Prochlorothrix/enzimologia , Prochlorothrix/genética , Homologia de Sequência de Aminoácidos , TetrapirróisRESUMO
Prochloron is a genus of prokaryotic algae with photosynthetic pigments like those of chlorophytes. Prochlorophytes are almost invariably found associated as symbionts with marine protochordates (didemnid ascidians), and so far none has been successfully grown in sustained culture away from in host. Based on materials collected from nature, information of various sorts (biochemical, physiological, cytological and fine-structural) has been obtained, indicating many resemblances (and probably close phylogenetic affinities) between prochlorophytes and cyanophytes. Nevertheless they are distinguished by certain unique combinations of characters. Some of the data support the symbiogenesis theory for the origin of green-plant chloroplasts. Other possibilities are briefly discussed.