Sponge Haliclona Simulans Research Articles

Purification and biochemical characterization of SM14est, a PET-hydrolyzing enzyme from the marine sponge-derived Streptomyces sp. SM14.

The successful enzymatic degradation of polyester substrates has fueled worldwide investigation into the treatment of plastic waste using bio-based processes. Within this realm, marine-associated microorganisms have emerged as a promising source of polyester-degrading enzymes. In this work, we describe the hydrolysis of the synthetic polymer PET by SM14est, a polyesterase which was previously identified from Streptomyces sp. SM14, an isolate of the marine sponge Haliclona simulans. The PET hydrolase activity of purified SM14est was assessed using a suspension-based assay and subsequent analysis of reaction products by UV-spectrophotometry and RP-HPLC. SM14est displayed a preference for high salt conditions, with activity significantly increasing at sodium chloride concentrations from 100 mM up to 1,000 mM. The initial rate of PET hydrolysis by SM14est was determined to be 0.004 s-1 at 45°C, which was increased by 5-fold to 0.02 s-1 upon addition of 500 mM sodium chloride. Sequence alignment and structural comparison with known PET hydrolases, including the marine halophile PET6, and the highly efficient, thermophilic PHL7, revealed conserved features of interest. Based on this work, SM14est emerges as a useful enzyme that is more similar to key players in the area of PET hydrolysis, like PHL7 and IsPETase, than it is to its marine counterparts. Salt-tolerant polyesterases such as SM14est are potentially valuable in the biological degradation of plastic particles that readily contaminate marine ecosystems and industrial wastewaters.

Large-scale mariculture of the sponge Haliclona simulans on floating rafts in Zhao'an Bay, Fujian Province, China

Marine sponges are an important source of bioactive products and biomaterials. However, there is a bottleneck of biomass supply for sponge utilization. To overcome this bottleneck, mariculture was considered to be the most viable solution. Mariculture of the marine sponge Haliclona simulans was conducted on floating rafts in Zhao'an Bay, Fujian Province, China. The explants were suspended 0.5–2 m below the water surface to grow naturally using the mesh method. Two independent trials with different scales were performed in this study. In a small-scale trial with 30 sponge explants, with daily maintenance, the survival rate of H. simulans was 93.3% over 224 days. To determine whether harvesting twice yearly yields greater output than annual harvesting, the 30 H. simulans were randomly divided into two equivalent groups. The annual harvest group (1 harvest per year, 1PY) was harvested in 224 days, and the twice harvest groups (2 harvest per year, 2PY) were harvested at 92 days and 224 days, with approximately 30% of the biomass was retained during the first harvest to allow regrowth. The results showed that the total biomass accumulation rate was 629.27 ± 256.74% for the 1PY group and 1014.73 ± 252.56% for the 2PY group. The biomass accumulation rate of 2PY was approximately 61.26% higher than that of 1PY. To obtain more accurate yield data under industry scale, a large-scale mariculture was independently conducted with about 7500 explants and covered a sea area about 500 m2, in which each area of 0.2 m2 was occupied by a horizontal rope, and each rope held 3 vertically suspended explants. Lost or dead explants were replanted during routine maintenance. A total of 330 kg wet weight biomass was obtained from the first harvest after 92 days of cultivation, and 272 kg wet weight biomass was obtained from the second harvest after 224 days. A total of 45.05 kg dry weight sponge was obtained after drying. This finding indicates that under our mariculture methods and harvesting strategy, the yield of H. simulans can reach approximately 900 kg dry weight biomass per hectare per year. This research provides valuable data for large-scale sponge production and new methods for the industrial development of sponge products.

Cultivation of sponge Haliclona simulans juveniles in a floating sea raft

Large-scale mariculture of a marine sponge requires a sustainable seed stock that can be raised artificially. To find the optimized breeding methods for Haliclona simulans juveniles, a study with three stages, starting from free swimming larvae, was performed in a field laboratory placed on a floating sea raft. In the first step, six different substrates were compared, and polystyrene plastic petri dishes were found to be the optimal substrates for settlement and metamorphosis of H. simulans swimming larvae. Then, in the second stage, 30-days growth experiments with 20 conditions were performed for the larvae settled on plastic petri dishes. The optimized devices and conditions suited for the early-stage cultivation of sponge larvae were obtained. In the final stage, a 4-months growth experiment was performed with the guidance of the optimized conditions acquired from the former stages. At the end of the experiment, the group with the highest growth rate achieved an average length of 18.44 ± 10.73 mm, and the largest juvenile length reached 33.4 mm, equivalent to a 45-folds increase in length or over 20,000-folds increase in volume when compared to the larvae that just settled. Thus, we established a feasible and low-cost sponge-larval mariculture pattern, upon which the culturing procedures can be improved and the sustainable seed stock needed for sponge large-scale culture becomes possible.

Comparative Genomics of Marine Sponge-Derived Streptomyces spp. Isolates SM17 and SM18 With Their Closest Terrestrial Relatives Provides Novel Insights Into Environmental Niche Adaptations and Secondary Metabolite Biosynthesis Potential.

The emergence of antibiotic resistant microorganisms has led to an increased need for the discovery and development of novel antimicrobial compounds. Frequent rediscovery of the same natural products (NPs) continues to decrease the likelihood of the discovery of new compounds from soil bacteria. Thus, efforts have shifted toward investigating microorganisms and their secondary metabolite biosynthesis potential, from diverse niche environments, such as those isolated from marine sponges. Here we investigated at the genomic level two Streptomyces spp. strains, namely SM17 and SM18, isolated from the marine sponge Haliclona simulans, with previously reported antimicrobial activity against clinically relevant pathogens; using single molecule real-time (SMRT) sequencing. We performed a series of comparative genomic analyses on SM17 and SM18 with their closest terrestrial relatives, namely S. albus J1074 and S. pratensis ATCC 33331 respectively; in an effort to provide further insights into potential environmental niche adaptations (ENAs) of marine sponge-associated Streptomyces, and on how these adaptations might be linked to their secondary metabolite biosynthesis potential. Prediction of secondary metabolite biosynthetic gene clusters (smBGCs) indicated that, even though the marine isolates are closely related to their terrestrial counterparts at a genomic level; they potentially produce different compounds. SM17 and SM18 displayed a better ability to grow in high salinity medium when compared to their terrestrial counterparts, and further analysis of their genomes indicated that they possess a pool of 29 potential ENA genes that are absent in S. albus J1074 and S. pratensis ATCC 33331. This ENA gene pool included functional categories of genes that are likely to be related to niche adaptations and which could be grouped based on potential biological functions such as osmotic stress, defense; transcriptional regulation; symbiotic interactions; antimicrobial compound production and resistance; ABC transporters; together with horizontal gene transfer and defense-related features.

A Novel Inexpensive Media for the Growth of Escherichia coli Expressing a Recombinant Lipase

There is an ever increasing demand for industrial enzymes for different applications to satisfy the needs of the world's ever‐growing population. Nigeria currently is at a precipice and urgent measures are needed for self‐sustainability in a distressed economy for most of her industrial processes. The progression of synthetic biology techniques has been a continuous driving force for sustainable development on industrial enzymes. Lipases (EC 3.1.1.3) are important biocatalysts due to their utility in various industrial applications. For industrial processes, it is necessary to define the best set of conditions for cell growth, protein induction and recovery. This work therefore investigates the conditions necessary for optimum expression of novel recombinant lipase (sourced originally from marine sponge Haliclona simulans metagenome) in Escherichia coli.A codon‐optimized gene that codes for lipase Lpc53E1, a member of the group VIII family of lipases from Haliclona simulans was heterologously expressed in E. coli by induction of graded concentrations (0.1 mM, 0.25 mM, 0.5 mM and 1 mM) of Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) when the cell's density (OD600) reached 0.4, 0.5, 0.6 and 0.7 accordingly. Analysis of the SDS‐PAGE revealed the presence of lipase with an approximate molecular weight (MW) of 44 kDa. The recombinant lipase exhibited a higher halo‐tolerance and thermostable properties up to 60°C when the cell density reached 0.7 OD600 after induction with 0.5 mM IPTG. A novel and newly formulated inexpensive media for cell growth of the E. coli culture showed distinct colonies comparable to the LB control. This suggests an alternative and cheaper media for this microbial culture. Thus this inexpensive media from locally sourced materials would reduce the cost of operations in industries requiring lipase in developing countries.Support or Funding InformationThe grant for this research was awarded by the Central Research Committee, University of Lagos, Nigeria (CRC, 2017/01)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The hydroid of the medusa Lizzia blondina Forbes, 1848

A tiny, filiferan hydroid living within the sponge Haliclona simulans (Johnston, 1842) could be identified as the so far unknown polyp stage of the hydromedusa Lizzia blondina Forbes, 1848. This finding is based on two lines of evidence: (i) the direct observation that sponge pieces with the hydroid release young Lizzia blondina medusae, and (ii) 16S rRNA gene sequences obtained from DNA samples extracted from mixed sponge–hydroid samples were identifiable as either Lizzia blondina or sponge-related. Histological examination of the hydroid showed that it is colonial, and the individual polyps are connected through stolons which penetrate deeply into the sponge tissue. The polyps only protrude temporarily and partially for the purpose of feeding. The hydroid can retract its tentacles and the hypostome in an introvert-like pouch, becoming thus almost invisible on the sponge surface. The association of the Lizzia blondina hydroid with the sponges of the genus Haliclona Grant, 1841 is likely a rather specific relationship.

Draft Genome Sequence of the Antimycin-Producing Bacterium Streptomyces sp. Strain SM8, Isolated from the Marine Sponge Haliclona simulans.

ABSTRACTStreptomyces sp. strain SM8, isolated from Haliclona simulans, possesses antifungal and antibacterial activities and inhibits the calcineurin pathway in yeast. The draft genome sequence is 7,145,211 bp, containing 5,929 predicted coding sequences. Several secondary metabolite biosynthetic gene clusters are present, encoding known and novel metabolites, including antimycin.

A novel erythromycin resistance plasmid from Bacillus sp. strain HS24, isolated from the marine sponge Haliclona simulans.

A better understanding of the origin and natural reservoirs of resistance determinants is fundamental to efficiently tackle antibiotic resistance. This paper reports the identification of a novel 5.8 kb erythromycin resistance plasmid, from Bacillus sp. HS24 isolated from the marine sponge Haliclona simulans. pBHS24B has a mosaic structure and carries the erythromycin resistance gene erm(T). This is the first report of an erythromycin resistance plasmid from a sponge associated bacteria and of the Erm(T) determinant in the genus Bacillus.

Metabolomic profiling and genomic study of a marine sponge-associated Streptomyces sp.

Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.

Isolation and identification of antitrypanosomal and antimycobacterial active steroids from the sponge Haliclona simulans.

The marine sponge Haliclona simulans collected from the Irish Sea yielded two new steroids: 24-vinyl-cholest-9-ene-3β,24-diol and 20-methyl-pregn-6-en-3β-ol,5α,8α-epidioxy, along with the widely distributed 24-methylenecholesterol. One of the steroids possesses an unusually short hydrocarbon side chain. The structures were elucidated using nuclear magnetic resonance spectroscopy and confirmed using electron impact- and high resolution electrospray-mass spectrometry. All three steroids possess antitrypanosomal and anti-mycobacterial activity. All the steroids were found to possess low cytotoxicity against Hs27 which was above their detected antitrypanosomal potent concentrations.

Subtilomycin: A New Lantibiotic from Bacillus subtilis Strain MMA7 Isolated from the Marine Sponge Haliclona simulans

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.

Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans

BackgroundLipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome.ResultsA metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C β-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3–12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 μmol/min, respectively.ConclusionWe have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.

Diversity and bioactive potential of endospore-forming bacteria cultured from the marine sponge Haliclona simulans

Despite the frequent isolation of endospore-formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore-forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds. A bank of presumptive aerobic spore-forming bacteria was isolated from the marine sponge H. simulans. These represented c. 1% of the total culturable bacterial population. A subgroup of thirty isolates was characterized using morphological, phenotypical and phylogenetic analysis. A large diversity of endospore-forming bacteria was present, with the thirty isolates being distributed through a variety of Bacillus and Paenibacillus species. These included ubiquitous species, such as B. subtilis, B. pumilus, B. licheniformis and B. cereus group, as well as species that are typically associated with marine habitats, such as B. aquimaris, B. algicola and B. hwajinpoensis. Two strains carried the aiiA gene that encodes a lactonase known to be able to disrupt quorum-sensing mechanisms, and various isolates demonstrated protease activity and antimicrobial activity against different pathogenic indicator strains, including Clostridium perfringens, Bacillus cereus and Listeria monocytogenes. The marine sponge H. simulans harbours a diverse collection of endospore-forming bacteria, which produce proteases and antibiotics. This diversity appears to be overlooked by culture-dependent and culture-independent methods that do not specifically target sporeformers. Marine sponges are an as yet largely untapped and poorly understood source of endospore-forming bacterial diversity with potential biotechnological, biopharmaceutical and probiotic applications. These results also indicate the importance of combining different methodologies for the comprehensive characterization of complex microbial populations such as those found in marine sponges.

Endoglucanase activities and growth of marine-derived fungi isolated from the spongeHaliclona simulans

The conversion of cheap cellulosic biomass to more easily fermentable sugars requires the use of costly cellulases. We have isolated a series of marine sponge-derived fungi and screened these for cellulolytic activity to determine the potential of this unique environmental niche as a source of novel cellulase activities. Fungi were isolated from the marine sponge Haliclona simulans. Phylogenetic analysis of these and other fungi previously isolated from H. simulans showed fungi from three phyla with very few duplicate species. Cellulase activities were determined using plate-based assays using different media and sea water concentrations while extracellular cellulase activities were determined using 3,5-dinitrosalicylic acid (DNSA)-based assays. Total and specific cellulase activities were determined using a range of incubation temperatures and compared to those for the cellulase overproducing mutant Hypocrea jecorina QM9414. Several of the strains assayed produced total or relative endoglucanase activities that were higher than H. jecorina, particularly at lower reaction temperatures. Marine sponges harbour diverse fungal species and these fungi are a good source of endoglucanase activities. Analysis of the extracellular endoglucanase activities revealed that some of the marine-derived fungi produced high endoglucanase activities that were especially active at lower temperatures. Marine-derived fungi associated with coastal marine sponges are a novel source of highly active endoglucanases with significant activity at low temperatures and could be a source of novel cellulase activities.

Isolation and Analysis of Bacteria with Antimicrobial Activities from the Marine Sponge Haliclona simulans Collected from Irish Waters

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.

Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from Irish waters and identification of polyketide synthase genes from the sponge metagenome

Samples of the sponge Haliclona simulans were collected from Irish waters and subjected to a culture-independent analysis to determine the microbial, polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) diversity. 16S rRNA gene libraries were prepared from total sponge, bacterial enriched sponge and seawater samples. Eight phyla from the Bacteria were detected in the sponge by phylogenetic analyses of the 16S rRNA gene libraries. The most abundant phylum in the total sponge library was the Proteobacteria (86%), with the majority of these clones being from the gamma-Proteobacteria (77%); two groups of clones were dominant and together made up 69% of the total. Both of these groups were related to other sponge-derived microbes and comprised novel genera. Within the other bacterial phyla groups of clones representing novel candidate genera within the phyla Verrucomicrobia and Lentisphaerae were also found. Selective enrichment of the bacterial component of the sponge prior to 16S rRNA gene analysis resulted in a 16S rRNA gene library dominated by a novel genus of delta-Proteobacteria, most closely related to the Bdellovibrio. The potential for the sponge microbiota to produce secondary metabolites was also analysed by polymerase chain reaction amplification of PKS and NRPS genes. While no NRPS sequences were isolated seven ketosynthase (KS) sequences were obtained from the sponge metagenome. Analyses of these clones revealed a diverse collection of PKS sequences which were most closely affiliated with PKS from members of the Cyanobacteria, Myxobacteria and Dinoflagellata.

The marine sponge Haliclona simulans harbours a novel and diverse microbial community with extensive genetic diversity in polyketide synthase genes, with potential biopharmaceutical applications

The marine sponge Haliclona simulans harbours a novel and diverse microbial community with extensive genetic diversity in polyketide synthase genes, with potential biopharmaceutical applications