Bioprospecting Sponge-Associated Marine Cyanobacteria to Produce Bioactive Compounds.
ABSTRACT: Marine cyanobacteria are considered a prolific source of bioactive natural products with a range of biotechnological and pharmacological applications. However, data on the production of natural compounds from sponge-associated cyanobacteria are scarce. This study aimed to assess the potential of sponge-associated cyanobacteria strains representing different taxonomic groups for the production of bioactive compounds and the biological activity of their extracts. Phylogenetic analysis of sponge-associated cyanobacteria and screening for the presence of genes encoding non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) were performed. Methanol extracts of the sponge-associated strains were analyzed for cyanotoxin production and tested for antioxidant activity and cytotoxic activity against several human cancer cell lines and pathogenic bacteria. PKS were detected in all sponge-associated strains examined, indicating the metabolic potential of the isolates. PKS genes were more ubiquitous than NRPS genes. Cyanotoxins (i.e., cylindrospermopsin, anatoxin-a, nodularin, and microcystins) were not detected in any of the sponge-associated cyanobacterial strains. Strains belonging to Leptothoe, Pseudanabaena, and Synechococcus were found to have activity mainly against Staphylococcus aureus. In addition, sponge-associated Leptothoe strains (TAU-MAC 0915, 1015, 1115, and 1215) were found to be highly cytotoxic and in most cases more effective against human cancer cell lines than against normal cells. Extracts with the most promising bioactivity deserve further investigation in order to isolate and identify the bioactive molecule(s).
Project description:Interest in the study of marine sponges and their associated microbiome has increased both for ecological reasons and for their great biotechnological potential. In this work, heterotrophic bacteria associated with three specimens of the marine sponge Erylus deficiens, were isolated in pure culture, phylogenetically identified and screened for antimicrobial activity. The isolation of bacteria after an enrichment treatment in heterotrophic medium revealed diversity in bacterial composition with only Pseudoalteromonas being shared by two specimens. Of the 83 selected isolates, 58% belong to Proteobacteria, 23% to Actinobacteria and 19% to Firmicutes. Diffusion agar assays for bioactivity screening against four bacterial strains and one yeast, revealed that a high number of the isolated bacteria (68.7%) were active, particularly against Candida albicans and Vibrio anguillarum. Pseudoalteromonas, Microbacterium, and Proteus were the most bioactive genera. After this preliminary screening, the bioactive strains were further evaluated in liquid assays against C. albicans, Bacillus subtilis and Escherichia coli. Filtered culture medium and acetone extracts from three and 5 days-old cultures were assayed. High antifungal activity against C. albicans in both aqueous and acetone extracts as well as absence of activity against B. subtilis were confirmed. Higher levels of activity were obtained with the aqueous extracts when compared to the acetone extracts and differences were also observed between the 3 and 5 day-old extracts. Furthermore, a low number of active strains was observed against E. coli. Potential presence of type-I polyketide synthases (PKS-I) and non-ribosomal peptide synthetases (NRPSs) genes were detected in 17 and 30 isolates, respectively. The high levels of bioactivity and the likely presence of associated genes suggest that Erylus deficiens bacteria are potential sources of novel marine bioactive compounds.
Project description:Heterotrophic bacteria associated with two specimens of the marine sponge Erylus discophorus were screened for their capacity to produce bioactive compounds against a panel of human pathogens (Staphylococcus aureus wild type and methicillin-resistant S. aureus (MRSA), Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumanii, Candida albicans and Aspergillus fumigatus), fish pathogen (Aliivibrio fischeri) and environmentally relevant bacteria (Vibrio harveyi). The sponges were collected in Berlengas Islands, Portugal. Of the 212 isolated heterotrophic bacteria belonging to Alpha- and Gammaproteobacteria, Actinobacteria and Firmicutes, 31% produced antimicrobial metabolites. Bioactivity was found against both Gram positive and Gram negative and clinically and environmentally relevant target microorganisms. Bioactivity was found mainly against B. subtilis and some bioactivity against S. aureus MRSA, V. harveyi and A. fisheri. No antifungal activity was detected. The three most bioactive genera were Pseudovibrio (47.0%), Vibrio (22.7%) and Bacillus (7.6%). Other less bioactive genera were Labrenzia, Acinetobacter, Microbulbifer, Pseudomonas, Gordonia, Microbacterium, Micrococcus and Mycobacterium, Paenibacillus and Staphylococcus. The search of polyketide I synthases (PKS-I) and nonribosomal peptide synthetases (NRPSs) genes in 59 of the bioactive bacteria suggested the presence of PKS-I in 12 strains, NRPS in 3 strains and both genes in 3 strains. Our results show the potential of the bacterial community associated with Erylus discophorus sponges as producers of bioactive compounds.
Project description:The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.
Project description:Oceans hold a stunning number of unique microorganisms, which remain unstudied by culture-dependent methods due to failures in establishing the right conditions for these organisms to grow. In this work, an isolation effort inspired by the iChip was performed using marine sediments from Memoria beach, Portugal. The isolates obtained were identified by 16S rRNA gene analysis, fingerprinted using BOX-PCR and ERIC-PCR, searched for the putative presence of secondary metabolism genes associated with polyketide synthase I (PKS-I) and non-ribosomal peptide synthetases (NRPS), screened for antimicrobial activity against <i>Escherichia coli</i> ATCC 25922 and <i>Staphylococcus aureus</i> ATCC 29213, and had bioactive extracts dereplicated by LC/HRMS. Of the 158 isolated strains, 96 were affiliated with the phylum <i>Actinomycetota</i>, PKS-I and NRPS genes were detected in 53 actinomycetotal strains, and 11 proved to be bioactive (10 against <i>E. coli</i>, 1 against <i>S. aureus</i> and 1 against both pathogens). Further bioactivities were explored using an "one strain many compounds" approach, with six strains showing continued bioactivity and one showing a novel one. Extract dereplication showed the presence of several known bioactive molecules and potential novel ones in the bioactive extracts. These results indicate the use of the bacteria isolated here as sources of new bioactive natural products.
Project description:Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.
Project description:Coral reefs are experiencing increasing anthropogenic impacts that result in substantial declines of reef-building corals and a change of community structure towards other benthic invertebrates or macroalgae. Reefs around Zanzibar are exposed to untreated sewage and runoff from the main city Stonetown. At many of these sites, sponge cover has increased over the last years. Sponges are one of the top spatial competitors on reefs worldwide. Their success is, in part, dependent on their strong chemical defenses against predators, microbial attacks and other sessile benthic competitors. This is the first study that investigates the bioactive properties of sponge species in the Western Indian Ocean region. Crude extracts of the ten most dominant sponge species were assessed for their chemical defenses against 35 bacterial strains (nine known as marine pathogens) using disc diffusion assays and general cytotoxic activities were assessed with brine shrimp lethality assays. The three chemically most active sponge species were additionally tested for their allelopathic properties against the scleractinian coral competitor Porites sp.. The antimicrobial assays revealed that all tested sponge extracts had strong antimicrobial properties and that the majority (80%) of the tested sponges were equally defended against pathogenic and environmental bacterial strains. Additionally, seven out of ten sponge species exhibited cytotoxic activities in the brine shrimp assay. Moreover, we could also show that the three most bioactive sponge species were able to decrease the photosynthetic performance of the coral symbionts and thus were likely to impair the coral physiology.
Project description:Marine environments are a fruitful source of bioactive compounds some of which are the newest leading drugs in medicinal therapeutics. Of particular importance are organisms like sponges and macroalgae and their associated microbiome. Planctomycetes, abundant in macroalgae biofilms, are promising producers of bioactive compounds since they share characteristics, like large genomes and complex life cycles, with the most bioactive bacteria, the Actinobacteria. Furthermore, genome mining revealed the presence of secondary metabolite pathway genes or clusters in 13 analyzed Planctomycetes genomes. In order to assess the antimicrobial production of a large and diverse collection of Planctomycetes isolated from macroalgae from the Portuguese coast, molecular, and bioactivity assays were performed in 40 bacteria from several taxa. Two genes commonly associated with the production of bioactive compounds, nonribosomal peptide synthetases (NRPS), and polyketide synthases (PKS) genes were screened. Molecular analysis revealed that 95% of the planctomycetes potentially have one or both secondary bioactive genes; 85% amplified with PKS-I primers and 55% with NRPS primers. Some of the amplified genes were confirmed to be involved in secondary metabolite pathways. Using bioinformatic tools their biosynthetic pathways were predicted. The secondary metabolite genomic potential of strains LF1, UC8, and FC18 was assessed using in silico analysis of their genomes. Aqueous and organic extracts of the Planctomycetes were evaluated for their antimicrobial activity against an environmental Escherichia coli, E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and a clinical isolate of Candida albicans. The screening assays showed a high number of planctomycetes with bioactive extracts revealing antifungal (43%) and antibacterial (54%) activity against C. albicans and B. subtilis, respectively. Bioactivity was observed in strains from Rhodopirellula lusitana, R. rubra, R. baltica, Roseimaritima ulvae, and Planctomyces brasiliensis. This study confirms the bioactive capacity of Planctomycetes to produce antimicrobial compounds and encourages further studies envisaging molecule isolation and characterization for the possible discovery of new drugs.
Project description:Phycobiliprotein-containing water and carotenoid-containing methanolic extracts of three different cyanobacteria, Pseudanabaena sp., Spirulina sp. and Lyngbya sp., were studied for their DPPH scavenging, iso-bolographic studies, and anti-nephrolithe activities. The best EC50 values for DPPH scavenging were in Lyngbya water (LW, 18.78 ± 1.57 mg·mg(-1) DPPH) and Lyngbya methanol (LM, 59.56 ± 37.38 mg·mg(-1) DPPH) extracts. Iso-bolographic analysis revealed most of the combinations of extracts were antagonistic to each other, although LM-Spirulina methanol (SM) 1:1 had the highest synergistic rate of 86.65%. In vitro digestion studies showed that DPPH scavenging activity was considerably decreased in all extracts except for Pseudanabaena methanol (PM) and LM after the simulated digestion. All of the extracts were effective in reducing the calcium oxalate crystal size by nearly 60%-65% compared to negative control, while PM and Spirulina water (SW) extracts could inhibit both nucleation and aggregation of calcium oxalate by nearly 60%-80%.
Project description:This study aimed to investigate the PCR-based screening strategy for the prediction of the antimicrobial biosynthetic potential of the selected Streptomyces strains originated from an extreme environment (Cholistan Desert, Pakistan). The biosynthetic potential was determined by using both molecular and culture-dependent screening approaches. The four biosynthetic genes clusters, including the pks-1, nrps, cyp P450 hydroxylase (cyps), and glycopeptide oxy b genes, were investigated in the selected strains by PCR amplification, sequencing, and by subsequent bioinformatics approaches. Among the 40 selected Streptomyces strains, 33 strains possessed the nrps gene, 17 strains carried the pks-1 gene, four strains were found to have the cyps gene, and none of the strain carried oxy b gene. The Streptomyces strains including NR-1, NR-10, NR-14, and NR-15 were investigated for in vitro antifungal activity against Fusarium oxysporum, Rhizoctonia solani, and Aspergillus sp. The extracts were analyzed for chemical profiling (TLC and HPLC-UV), and a unique pattern of secondary metabolites was observed. The selected strains exhibited pronounced antifungal activity against the fungal test strains with the zone of inhibition up to 17, 18, and 19 mm, respectively. The study depicts that gene-based screening can be successfully applied to identify potentially bioactive strains by usin a single screening process. This PCR-based approach is rapid and can be used for sorting out and selecting the potential candidate among actinobacterial culture collections. Such a preselection or strain prioritization consequently decreases the time and efforts required for selecting the potential bioactive strain, which then can be subjected to the detailed chemical analysis.This study aimed to investigate the PCR-based screening strategy for the prediction of the antimicrobial biosynthetic potential of the selected Streptomyces strains originated from an extreme environment (Cholistan Desert, Pakistan). The biosynthetic potential was determined by using both molecular and culture-dependent screening approaches. The four biosynthetic genes clusters, including the pks-1, nrps, cyp P450 hydroxylase (cyps), and glycopeptide oxy b genes, were investigated in the selected strains by PCR amplification, sequencing, and by subsequent bioinformatics approaches. Among the 40 selected Streptomyces strains, 33 strains possessed the nrps gene, 17 strains carried the pks-1 gene, four strains were found to have the cyps gene, and none of the strain carried oxy b gene. The Streptomyces strains including NR-1, NR-10, NR-14, and NR-15 were investigated for in vitro antifungal activity against Fusarium oxysporum, Rhizoctonia solani, and Aspergillus sp. The extracts were analyzed for chemical profiling (TLC and HPLC-UV), and a unique pattern of secondary metabolites was observed. The selected strains exhibited pronounced antifungal activity against the fungal test strains with the zone of inhibition up to 17, 18, and 19 mm, respectively. The study depicts that gene-based screening can be successfully applied to identify potentially bioactive strains by usin a single screening process. This PCR-based approach is rapid and can be used for sorting out and selecting the potential candidate among actinobacterial culture collections. Such a preselection or strain prioritization consequently decreases the time and efforts required for selecting the potential bioactive strain, which then can be subjected to the detailed chemical analysis.
Project description:Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs.