Anticancer Potential of Compounds from the Brazilian Blue Amazon.

"Blue Amazon" is used to designate the Brazilian Economic Exclusive Zone, which covers an area comparable in size to that of its green counterpart. Indeed, Brazil flaunts a coastline spanning 8000 km through tropical and temperate regions and hosting part of the organisms accredited for the country's megadiversity status. Still, biodiversity may be expressed at different scales of organization; besides species inventory, genetic characteristics of living beings and metabolic expression of their genes meet some of these other layers. These metabolites produced by terrestrial creatures traditionally and lately added to by those from marine organisms are recognized for their pharmaceutical value, since over 50% of small molecule-based medicines are related to natural products. Nonetheless, Brazil gives a modest contribution to the field of pharmacology and even less when considering marine pharmacology, which still lacks comprehensive in-depth assessments toward the bioactivity of marine compounds so far. Therefore, this review examined the last 40 years of Brazilian natural products research, focusing on molecules that evidenced anticancer potential-which represents ~ 15% of marine natural products isolated from Brazilian species. This review discusses the most promising compounds isolated from sponges, cnidarians, ascidians, and microbes in terms of their molecular targets and mechanisms of action. Wrapping up, the review delivers an outlook on the challenges that stand against developing groundbreaking natural products research in Brazil and on a means of surpassing these matters.

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills.

Shipworms are marine wood-boring bivalve mollusks (family Teredinidae) that harbor a community of closely related Gammaproteobacteria as intracellular endosymbionts in their gills. These symbionts have been proposed to assist the shipworm host in cellulose digestion and have been shown to play a role in nitrogen fixation. The genome of one strain of Teredinibacter turnerae, the first shipworm symbiont to be cultivated, was sequenced, revealing potential as a rich source of polyketides and nonribosomal peptides. Bioassay-guided fractionation led to the isolation and identification of two macrodioloide polyketides belonging to the tartrolon class. Both compounds were found to possess antibacterial properties, and the major compound was found to inhibit other shipworm symbiont strains and various pathogenic bacteria. The gene cluster responsible for the synthesis of these compounds was identified and characterized, and the ketosynthase domains were analyzed phylogenetically. Reverse-transcription PCR in addition to liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry revealed the transcription of these genes and the presence of the compounds in the shipworm, suggesting that the gene cluster is expressed in vivo and that the compounds may fulfill a specific function for the shipworm host. This study reports tartrolon polyketides from a shipworm symbiont and unveils the biosynthetic gene cluster of a member of this class of compounds, which might reveal the mechanism by which these bioactive metabolites are biosynthesized.

Genomic and phenotypic attributes of novel salinivibrios from stromatolites, sediment and water from a high altitude lake.

BACKGROUND: Salinivibrios are moderately halophilic bacteria found in salted meats, brines and hypersaline environments. We obtained three novel conspecific Salinivibrio strains closely related to S. costicola, from Socompa Lake, a high altitude hypersaline Andean lake (approx. 3,570 meters above the sea level). RESULTS: The three novel Salinivibrio spp. were extremely resistant to arsenic (up to 200 mM HAsO42-), NaCl (up to 15%), and UV-B radiation (19 KJ/m2, corresponding to 240 minutes of exposure) by means of phenotypic tests. Our subsequent draft genome ionsequencing and RAST-based genome annotation revealed the presence of genes related to arsenic, NaCl, and UV radiation resistance. The three novel Salinivibrio genomes also had the xanthorhodopsin gene cluster phylogenetically related to Marinobacter and Spiribacter. The genomic taxonomy analysis, including multilocus sequence analysis, average amino acid identity, and genome-to-genome distance revealed that the three novel strains belong to a new Salinivibrio species. CONCLUSIONS: Arsenic resistance genes, genes involved in DNA repair, resistance to extreme environmental conditions and the possible light-based energy production, may represent important attributes of the novel salinivibrios, allowing these microbes to thrive in the Socompa Lake.

Polyketide synthase gene diversity within the microbiome of the sponge Arenosclera brasiliensis, endemic to the Southern Atlantic Ocean.

Microbes associated with marine sponges are considered important producers of bioactive, structurally unique polyketides. The synthesis of such secondary metabolites involves type I polyketide synthases (PKSs), which are enzymes that reach a maximum complexity degree in bacteria. The Haplosclerida sponge Arenosclera brasiliensis hosts a complex microbiota and is the source of arenosclerins, alkaloids with cytotoxic and antibacterial activity. In the present investigation, we performed high-throughput sequencing of the ketosynthase (KS) amplicon to investigate the diversity of PKS genes present in the metagenome of A. brasiliensis. Almost 4,000 ketosynthase reads were recovered, with about 90% annotated automatically as bacterial. A total of 235 bacterial KS contigs was rigorously assembled from this sequence pool and submitted to phylogenetic analysis. A great diversity of six type I PKS groups has been consistently detected in our phylogenetic reconstructions, including a novel and A. brasiliensis-exclusive group. Our study is the first to reveal the diversity of type I PKS genes in A. brasiliensis as well as the potential of its microbiome to serve as a source of new polyketides.

The chemistry and biology of organic guanidine derivatives.

The chemistry and biology of organic natural guanidines are reviewed, including the isolation, structure determination, synthesis, biosynthesis and biological activities of alkaloids, non-ribosomal peptides, guanidine-bearing terpenes, polyketides and shikimic acid derivatives from natural sources.

Secondary Metabolism in the Gill Microbiota of Shipworms (Teredinidae) as Revealed by Comparison of Metagenomes and Nearly Complete Symbiont Genomes.

Shipworms play critical roles in recycling wood in the sea. Symbiotic bacteria supply enzymes that the organisms need for nutrition and wood degradation. Some of these bacteria have been grown in pure culture and have the capacity to make many secondary metabolites. However, little is known about whether such secondary metabolite pathways are represented in the symbiont communities within their hosts. In addition, little has been reported about the patterns of host-symbiont co-occurrence. Here, we collected shipworms from the United States, the Philippines, and Brazil and cultivated symbiotic bacteria from their gills. We analyzed sequences from 22 shipworm gill metagenomes from seven shipworm species and from 23 cultivated symbiont isolates. Using (meta)genome sequencing, we demonstrate that the cultivated isolates represent all the major bacterial symbiont species and strains in shipworm gills. We show that the bacterial symbionts are distributed among shipworm hosts in consistent, predictable patterns. The symbiotic bacteria harbor many gene cluster families (GCFs) for biosynthesis of bioactive secondary metabolites, only <5% of which match previously described biosynthetic pathways. Because we were able to cultivate the symbionts and to sequence their genomes, we can definitively enumerate the biosynthetic pathways in these symbiont communities, showing that ∼150 of ∼200 total biosynthetic gene clusters (BGCs) present in the animal gill metagenomes are represented in our culture collection. Shipworm symbionts occur in suites that differ predictably across a wide taxonomic and geographic range of host species and collectively constitute an immense resource for the discovery of new biosynthetic pathways corresponding to bioactive secondary metabolites.IMPORTANCE We define a system in which the major symbionts that are important to host biology and to the production of secondary metabolites can be cultivated. We show that symbiotic bacteria that are critical to host nutrition and lifestyle also have an immense capacity to produce a multitude of diverse and likely novel bioactive secondary metabolites that could lead to the discovery of drugs and that these pathways are found within shipworm gills. We propose that, by shaping associated microbial communities within the host, the compounds support the ability of shipworms to degrade wood in marine environments. Because these symbionts can be cultivated and genetically manipulated, they provide a powerful model for understanding how secondary metabolism impacts microbial symbiosis.

Integrating Computational Methods to Investigate the Macroecology of Microbiomes.

Studies in microbiology have long been mostly restricted to small spatial scales. However, recent technological advances, such as new sequencing methodologies, have ushered an era of large-scale sequencing of environmental DNA data from multiple biomes worldwide. These global datasets can now be used to explore long standing questions of microbial ecology. New methodological approaches and concepts are being developed to study such large-scale patterns in microbial communities, resulting in new perspectives that represent a significant advances for both microbiology and macroecology. Here, we identify and review important conceptual, computational, and methodological challenges and opportunities in microbial macroecology. Specifically, we discuss the challenges of handling and analyzing large amounts of microbiome data to understand taxa distribution and co-occurrence patterns. We also discuss approaches for modeling microbial communities based on environmental data, including information on biological interactions to make full use of available Big Data. Finally, we summarize the methods presented in a general approach aimed to aid microbiologists in addressing fundamental questions in microbial macroecology, including classical propositions (such as "everything is everywhere, but the environment selects") as well as applied ecological problems, such as those posed by human induced global environmental changes.

The gill-associated microbiome is the main source of wood plant polysaccharide hydrolases and secondary metabolite gene clusters in the mangrove shipworm Neoteredo reynei.

Teredinidae are a family of highly adapted wood-feeding and wood-boring bivalves, commonly known as shipworms, whose evolution is linked to the acquisition of cellulolytic gammaproteobacterial symbionts harbored in bacteriocytes within the gills. In the present work we applied metagenomics to characterize microbiomes of the gills and digestive tract of Neoteredo reynei, a mangrove-adapted shipworm species found over a large range of the Brazilian coast. Comparative metagenomics grouped the gill symbiont community of different N. reynei specimens, indicating closely related bacterial types are shared. Similarly, the intestine and digestive gland communities were related, yet were more diverse than and showed no overlap with the gill community. Annotation of assembled metagenomic contigs revealed that the gill symbiotic community of N. reynei encodes a plethora of plant cell wall polysaccharides degrading glycoside hydrolase encoding genes, and Biosynthetic Gene Clusters (BGCs). In contrast, the digestive tract microbiomes seem to play little role in wood digestion and secondary metabolites biosynthesis. Metagenome binning recovered the nearly complete genome sequences of two symbiotic Teredinibacter strains from the gills, a representative of Teredinibacter turnerae "clade I" strain, and a yet to be cultivated Teredinibacter sp. type. These Teredinibacter genomes, as well as un-binned gill-derived gammaproteobacteria contigs, also include an endo-ß-1,4-xylanase/acetylxylan esterase multi-catalytic carbohydrate-active enzyme, and a trans-acyltransferase polyketide synthase (trans-AT PKS) gene cluster with the gene cassette for generating ß-branching on complex polyketides. Finally, we use multivariate analyses to show that the secondary metabolome from the genomes of Teredinibacter representatives, including genomes binned from N. reynei gills' metagenomes presented herein, stands out within the Cellvibrionaceae family by size, and enrichments for polyketide, nonribosomal peptide and hybrid BGCs. Results presented here add to the growing characterization of shipworm symbiotic microbiomes and indicate that the N. reynei gill gammaproteobacterial community is a prolific source of biotechnologically relevant enzymes for wood-digestion and bioactive compounds production.

Endophytic fungus Pseudofusicoccum stromaticum produces cyclopeptides and plant-related bioactive rotenoids.

In the present study, we integrated liquid chromatography high-resolution mass spectrometry (LC-HRMS) and high-throughput DNA sequencing for prospecting cytotoxic specialized metabolites from Pseudofusicoccum stromaticum, an endophytic fungus associated to the medicinal plant Myracrodruon urundeuva. LC-HRMS profiling allowed identifying putatively eleven compounds in the ethyl acetate extract from P. stromaticum broth. Additionally, a chemical fractionation guided by cytotoxicity combined with spectrometric analysis resulted in the isolation of three compounds: the cyclopeptide cyclo-l-Phe-d-Leu-l-Leu-l-Leu-l-lle along with the known rotenoids rotenolone and tephrosin. MTT assay showed that tephrosin (IC50 0.51 µg mL-1) has strong cytotoxic effect and may be pointed out as the compound responsible for the antiproliferative activity of P. stromaticum. Next Generation Sequencing (NGS) and genome mining of P. stromaticum draft genome revealed 56 contigs codifying specialized metabolites biosynthesis-related enzymes. Nearly half of such genes (44.6%) could be mapped to orphan Biosynthetic Gene Clusters (BGCs) of related plant pathogens belonging to family Botryosphaeriaceae. Also, screening for rotenoids biosynthetic enzymes led to characterization of a putative chalcone isomerase-like (CHI-like) protein. This is the first report of rotenoids biosynthesized by a fungus, unveiling a unique ability of P. stromaticum.

Diversity and antimicrobial potential of culturable heterotrophic bacteria associated with the endemic marine sponge Arenosclera brasiliensis.

Marine sponges are the oldest Metazoa, very often presenting a complex microbial consortium. Such is the case of the marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro State, Brazil. In this investigation we characterized the diversity of some of the culturable heterotrophic bacteria living in association with A. brasiliensis and determined their antimicrobial activity. The genera Endozoicomonas (N = 32), Bacillus (N = 26), Shewanella (N = 17), Pseudovibrio (N = 12), and Ruegeria (N = 8) were dominant among the recovered isolates, corresponding to 97% of all isolates. Approximately one third of the isolates living in association with A. brasiliensis produced antibiotics that inhibited the growth of Bacillus subtilis, suggesting that bacteria associated with this sponge play a role in its health.

Turnerbactin, a novel triscatecholate siderophore from the shipworm endosymbiont Teredinibacter turnerae T7901.

Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.

Taxonomic and functional microbial signatures of the endemic marine sponge Arenosclera brasiliensis.

The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (∼150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome.

Genetic modification of Teredinibacter turnerae, an endosymbiont with biotechnological potential.

Teredinibacter turnerae belongs to a group of biotechnologically relevant bacteria. Gene transfer into T. turnerae was achieved by using pPROBE'-gfp[ASV] derived plasmids through conjugative mating with Escherichia coli DH5alpha pRK2073. Transferred plasmids were stably maintained and T. turnerae could also act as a donor to transfer these mobilizable plasmids. Constructs for both constitutive and IPTG-inducible gene expression were obtained, representing new tools for gene overexpression in T. turnerae.

Bryostatins: biological context and biotechnological prospects.

Bryostatins are a family of protein kinase C modulators that have potential applications in biomedicine. Found in miniscule quantities in a small marine invertebrate, lack of supply has hampered their development. In recent years, bryostatins have been shown to have potent bioactivity in the central nervous system, an uncultivated marine bacterial symbiont has been shown to be the likely natural source of the bryostatins, the bryostatin biosynthetic genes have been identified and characterized, and bryostatin analogues with promising biological activity have been developed and tested. Challenges in the development of bryostatins for biomedical and biotechnological application include the cultivation of the bacterial symbiont and heterologous expression of bryostatin biosynthesis genes. Continued exploration of the biology as well as the symbiotic origin of the bryostatins presents promising opportunities for discovery of additional bryostatins, and new functions for bryostatins.

Physiological traits of the symbiotic bacterium Teredinibacter turnerae isolated from the mangrove shipworm Neoteredo reynei.

Nutrition in the Teredinidae family of wood-boring mollusks is sustained by cellulolytic/nitrogen fixing symbiotic bacteria of the Teredinibacter clade. The mangrove Teredinidae Neoteredo reynei is popularly used in the treatment of infectious diseases in the north of Brazil. In the present work, the symbionts of N. reynei, which are strictly confined to the host's gills, were conclusively identified as Teredinibacter turnerae. Symbiont variants obtained in vitro were able to grow using casein as the sole carbon/nitrogen source and under reduced concentrations of NaCl. Furthermore, cellulose consumption in T. turnerae was clearly reduced under low salt concentrations. As a point of interest, we hereby report first hand that T. turnerae in fact exerts antibiotic activity. Furthermore, this activity was also affected by NaCl concentration. Finally, T. turnerae was able to inhibit the growth of Gram-negative and Gram-positive bacteria, this including strains of Sphingomonas sp., Stenotrophomonas maltophilia, Bacillus cereus and Staphylococcus sciuri. Our findings introduce new points of view on the ecology of T. turnerae, and suggest new biotechnological applications for this marine bacterium.

The complete genome of Teredinibacter turnerae T7901: an intracellular endosymbiont of marine wood-boring bivalves (shipworms).

Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2-40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels.

Physiological traits of the symbiotic bacterium Teredinibacter turnerae isolated from the mangrove shipworm Neoteredo reynei

Nutrition in the Teredinidae family of wood-boring mollusks is sustained by cellulolytic/nitrogen fixing symbiotic bacteria of the Teredinibacter clade. The mangrove Teredinidae Neoteredo reynei is popularly used in the treatment of infectious diseases in the north of Brazil. In the present work, the symbionts of N. reynei, which are strictly confined to the host's gills, were conclusively identified as Teredinibacter turnerae. Symbiont variants obtained in vitro were able to grow using casein as the sole carbon/nitrogen source and under reduced concentrations of NaCl. Furthermore, cellulose consumption in T. turnerae was clearly reduced under low salt concentrations. As a point of interest, we hereby report first hand that T. turnerae in fact exerts antibiotic activity. Furthermore, this activity was also affected by NaCl concentration. Finally, T. turnerae was able to inhibit the growth of Gram-negative and Gram-positive bacteria, this including strains of Sphingomonas sp., Stenotrophomonas maltophilia, Bacillus cereus and Staphylococcus sciuri. Our findings introduce new points of view on the ecology of T. turnerae, and suggest new biotechnological applications for this marine bacterium.