Bioactivity Screening and Gene-Trait Matching across Marine Sponge-Associated Bacteria

Marine sponges harbor diverse microbial communities that represent a significant source of natural products. In the present study, extracts of 21 sponge-associated bacteria were screened for their antimicrobial and anticancer activity, and their genomes were mined for secondary metabolite biosynthetic gene clusters (BGCs). Phylogenetic analysis assigned the strains to four major phyla in the sponge microbiome, namely Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Bioassays identified one extract with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and more than 70% of the total extracts had a moderate to high cytotoxicity. The most active extracts were derived from the Proteobacteria and Actinobacteria, prominent for producing bioactive substances. The strong bioactivity potential of the aforementioned strains was also evident in the abundance of BGCs, which encoded mainly beta-lactones, bacteriocins, non-ribosomal peptide synthetases (NRPS), terpenes, and siderophores. Gene-trait matching was performed for the most active strains, aiming at linking their biosynthetic potential with the experimental results. Genetic associations were established for the anti-MRSA and cytotoxic phenotypes based on the similarity of the detected BGCs with BGCs encoding natural products with known bioactivity. Overall, our study highlights the significance of combining in vitro and in silico approaches in the search of novel natural products of pharmaceutical interest.     

Advances in Marine Microbial Symbionts in the China Sea and Related Pharmaceutical Metabolites

Marine animals and plants such as sponges, sea squirts, corals, worms and algae host diverse and abundant symbiotic microorganisms. Marine microbial symbionts are possible the true producers or take part in the biosynthesis of some bioactive marine natural products isolated from the marine organism hosts. Investigation of the pharmaceutical metabolites may reveal the biosynthesis mechanisms of related natural products and solve the current problem of supply limitation in marine drug development. This paper reviews the advances in diversity revelation, biological activity and related pharmaceutical metabolites, and functional genes of marine microbial symbionts from the China Sea.     

Genome Mining for Antimicrobial Compounds in Wild Marine Animals-Associated Enterococci

New ecosystems are being actively mined for new bioactive compounds. Because of the large amount of unexplored biodiversity, bacteria from marine environments are especially promising. Further, host-associated microbes are of special interest because of their low toxicity and compatibility with host health. Here, we identified and characterized biosynthetic gene clusters encoding antimicrobial compounds in host-associated enterococci recovered from fecal samples of wild marine animals remote from human-affected ecosystems. Putative biosynthetic gene clusters in the genomes of 22 Enterococcus strains of marine origin were predicted using antiSMASH5 and Bagel4 bioinformatic software. At least one gene cluster encoding a putative bioactive compound precursor was identified in each genome. Collectively, 73 putative antimicrobial compounds were identified, including 61 bacteriocins (83.56%), 10 terpenes (13.70%), and 2 (2.74%) related to putative nonribosomal peptides (NRPs). Two of the species studied, Enterococcus avium and Enterococcus mundtti, are rare causes of human disease and were found to lack any known pathogenic determinants but yet possessed bacteriocin biosynthetic genes, suggesting possible additional utility as probiotics. Wild marine animal-associated enterococci from human-remote ecosystems provide a potentially rich source for new antimicrobial compounds of therapeutic and industrial value and potential probiotic application.     

A Multi-Omics Characterization of the Natural Product Potential of Tropical Filamentous Marine Cyanobacteria

Microbial natural products are important for the understanding of microbial interactions, chemical defense and communication, and have also served as an inspirational source for numerous pharmaceutical drugs. Tropical marine cyanobacteria have been highlighted as a great source of new natural products, however, few reports have appeared wherein a multi-omics approach has been used to study their natural products potential (i.e., reports are often focused on an individual natural product and its biosynthesis). This study focuses on describing the natural product genetic potential as well as the expressed natural product molecules in benthic tropical cyanobacteria. We collected from several sites around the world and sequenced the genomes of 24 tropical filamentous marine cyanobacteria. The informatics program antiSMASH was used to annotate the major classes of gene clusters. BiG-SCAPE phylum-wide analysis revealed the most promising strains for natural product discovery among these cyanobacteria. LCMS/MS-based metabolomics highlighted the most abundant molecules and molecular classes among 10 of these marine cyanobacterial samples. We observed that despite many genes encoding for peptidic natural products, peptides were not as abundant as lipids and lipopeptides in the chemical extracts. Our results highlight a number of highly interesting biosynthetic gene clusters for genome mining among these cyanobacterial samples.     

Comprehensive Genomic Analysis of Marine Strain Streptomyces sp. 891, an Excellent Producer of Chrysomycin A with Therapeutic Potential

Chrysomycin A is one of the most promising therapeutic candidates for treating infections caused by multidrug-resistant Gram-positive bacteria. By hybridizing next-step generation (Illumina) and third-generation (PacBio) sequencing technologies, a high-quality chromosome-level genome together with a plasmid was firstly assembled for chrysomycin A-producing marine strain 891. Phylogenetic analysis of the 16S rRNA gene and genome sequences revealed that this strain unambiguously belonged to the genus Streptomyces, and its genomic features and functional genes were comprehensively analyzed and annotated. AntiSMASH analysis of this strain unveiled one key biosynthetic gene cluster, T2PKS, responsible for the biosynthesis of chrysomycin, the biosynthesis pathway of which was putatively proposed. These findings definitely shed light on further investigation for construction of a robust industrial strain with high-yield chrysomycin A production using genetic engineering techniques and combinatorial biology approaches.     

Co-Cultivation—A Powerful Emerging Tool for Enhancing the Chemical Diversity of Microorganisms

Marine-derived bacteria and fungi are promising sources of novel bioactive compounds that are important for drug discovery programs. However, as encountered in terrestrial microorganisms there is a high rate of redundancy that results in the frequent re-discovery of known compounds. Apparently only a part of the biosynthetic genes that are harbored by fungi and bacteria are transcribed under routine laboratory conditions which involve cultivation of axenic microbial strains. Many biosynthetic genes remain silent and are not expressed in vitro thereby seriously limiting the chemical diversity of microbial compounds that can be obtained through fermentation. In contrast to this, co-cultivation (also called mixed fermentation) of two or more different microorganisms tries to mimic the ecological situation where microorganisms always co-exist within complex microbial communities. The competition or antagonism experienced during co-cultivation is shown to lead to a significantly enhanced production of constitutively present compounds and/or to an accumulation of cryptic compounds that are not detected in axenic cultures of the producing strain. This review highlights the power of co-cultivation for increasing the chemical diversity of bacteria and fungi drawing on published studies from the marine and from the terrestrial habitat alike.     

Insights into the Antimicrobial Activities and Metabolomes of Aquimarina (Flavobacteriaceae,Bacteroidetes) Species from the Rare Marine Biosphere

Two novel natural products, the polyketide cuniculene and the peptide antibiotic aquimarin, were recently discovered from the marine bacterial genus Aquimarina. However, the diversity of the secondary metabolite biosynthetic gene clusters (SM-BGCs) in Aquimarina genomes indicates a far greater biosynthetic potential. In this study, nine representative Aquimarina strains were tested for antimicrobial activity against diverse human-pathogenic and marine microorganisms and subjected to metabolomic and genomic profiling. We found an inhibitory activity of most Aquimarina strains against Candida glabrata and marine Vibrio and Alphaproteobacteria species. Aquimarina sp. Aq135 and Aquimarina muelleri crude extracts showed particularly promising antimicrobial activities, amongst others against methicillin-resistant Staphylococcus aureus. The metabolomic and functional genomic profiles of Aquimarina spp. followed similar patterns and were shaped by phylogeny. SM-BGC and metabolomics networks suggest the presence of novel polyketides and peptides, including cyclic depsipeptide-related compounds. Moreover, exploration of the ‘Sponge Microbiome Project’ dataset revealed that Aquimarina spp. possess low-abundance distributions worldwide across multiple marine biotopes. Our study emphasizes the relevance of this member of the microbial rare biosphere as a promising source of novel natural products. We predict that future metabologenomics studies of Aquimarina species will expand the spectrum of known secondary metabolites and bioactivities from marine ecosystems.     

A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge

To tackle the growing problem of antibiotic resistance, it is essential to identify new bioactive compounds that are effective against resistant microbes and safe to use. Natural products and their derivatives are, and will continue to be, an important source of these molecules. Sea sponges harbour a diverse microbiome that co-exists with the sponge, and these bacterial communities produce a rich array of bioactive metabolites for protection and resource competition. For these reasons, the sponge microbiota constitutes a potential source of clinically relevant natural products. To date, efforts in bioprospecting for these compounds have focused predominantly on sponge specimens isolated from shallow water, with much still to be learned about samples from the deep sea. Here we report the isolation of a new Micromonospora strain, designated 28ISP2-46^T, recovered from the microbiome of a mid-Atlantic deep-sea sponge. Whole-genome sequencing reveals the capacity of this bacterium to produce a diverse array of natural products, including kosinostatin and isoquinocycline B, which exhibit both antibiotic and antitumour properties. Both compounds were isolated from 28ISP2-46^T fermentation broths and were found to be effective against a plethora of multidrug-resistant clinical isolates. This study suggests that the marine production of isoquinocyclines may be more widespread than previously supposed and demonstrates the value of targeting the deep-sea sponge microbiome as a source of novel microbial life with exploitable biosynthetic potential.     

Diversity and Biosynthetic Potential of Culturable Microbes Associated with Toxic Marine Animals

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus.     

Bacillimidazoles A−F,Imidazolium-Containing Compounds Isolated from a Marine Bacillus

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A–F (1–6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1–6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1–6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1–6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.     

Harnessing the Potential of Halogenated Natural Product Biosynthesis by Mangrove-Derived Actinomycetes

Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH₂-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.     

Integrated Omics Strategy Reveals Cyclic Lipopeptides Empedopeptins from Massilia sp. YMA4 and Their Biosynthetic Pathway

Empedopeptins—eight amino acid cyclic lipopeptides—are calcium-dependent antibiotics that act against Gram-positive bacteria such as Staphylococcus aureus by inhibiting cell wall biosynthesis. However, to date, the biosynthetic mechanism of the empedopeptins has not been well identified. Through comparative genomics and metabolomics analysis, we identified empedopeptin and its new analogs from a marine bacterium, Massilia sp. YMA4. We then unveiled the empedopeptin biosynthetic gene cluster. The core nonribosomal peptide gene null-mutant strains (ΔempC, ΔempD, and ΔempE) could not produce empedopeptin, while dioxygenase gene null-mutant strains (ΔempA and ΔempB) produced several unique empedopeptin analogs. However, the antibiotic activity of ΔempA and ΔempB was significantly reduced compared with the wild-type, demonstrating that the hydroxylated amino acid residues of empedopeptin and its analogs are important to their antibiotic activity. Furthermore, we found seven bacterial strains that could produce empedopeptin-like cyclic lipopeptides using a genome mining approach. In summary, this study demonstrated that an integrated omics strategy can facilitate the discovery of potential bioactive metabolites from microbial sources without further isolation and purification.     

Polyketide Synthases in the Microbiome of the Marine Sponge Plakortis halichondrioides: A Metagenomic Update

Sponge-associated microorganisms are able to assemble the complex machinery for the production of secondary metabolites such as polyketides, the most important class of marine natural products from a drug discovery perspective. A comprehensive overview of polyketide biosynthetic genes of the sponge Plakortis halichondrioides and its symbionts was obtained in the present study by massively parallel 454 pyrosequencing of complex and heterogeneous PCR (Polymerase Chain Reaction) products amplified from the metagenomic DNA of a specimen of P. halichondrioides collected in the Caribbean Sea. This was accompanied by a survey of the bacterial diversity within the sponge. In line with previous studies, sequences belonging to supA and swfA, two widespread sponge-specific groups of polyketide synthase (PKS) genes were dominant. While they have been previously reported as belonging to Poribacteria (a novel bacterial phylum found exclusively in sponges), re-examination of current genomic sequencing data showed supA and swfA not to be present in the poribacterial genome. Several non-supA, non-swfA type-I PKS fragments were also identified. A significant portion of these fragments resembled type-I PKSs from protists, suggesting that bacteria may not be the only source of polyketides from P. halichondrioides, and that protistan PKSs should receive further investigation as a source of novel polyketides.     

Secondary Metabolites and Biosynthetic Gene Clusters Analysis of Deep-Sea Hydrothermal Vent-Derived Streptomyces sp. SCSIO ZS0520

Streptomyces sp. SCSIO ZS0520 is a deep-sea hydrothermal vent-derived actinomycete. Our previous metabolism investigation showed that Streptomyces sp. SCSIO ZS0520 is a producer of cytotoxic actinopyrones. Here, another four types of secondary metabolites were identified, including six salinomycin isomers (2–7), the macrolide elaiophylin (8), the triterpene N-acetyl-aminobacteriohopanetriol (9), and the pyrone minipyrone (10). Among them, compounds 2–6 and 10 are new compounds. To understand the biosynthetic pathway of these compounds, a bioinformatic analysis of the whole genome was carried out, which identified 34 secondary metabolite biosynthetic gene clusters. Next, the biosynthetic pathways responsive to four types of products were deduced on the basis of gene function predictions and structure information. Taken together, these findings prove the metabolite potential of ZS0520 and lay the foundations to solve the remaining biosynthetic issues in four types of marine natural products.     

Actinomycetes from Red Sea Sponges: Sources for Chemical and Phylogenetic Diversity

The diversity of actinomycetes associated with marine sponges collected off Fsar Reef (Saudi Arabia) was investigated in the present study. Forty-seven actinomycetes were cultivated and phylogenetically identified based on 16S rRNA gene sequencing and were assigned to 10 different actinomycete genera. Eight putatively novel species belonging to genera Kocuria, Mycobacterium, Nocardia, and Rhodococcus were identified based on sequence similarity values below 98.2% to other 16S rRNA gene sequences available in the NCBI database. PCR-based screening for biosynthetic genes including type I and type II polyketide synthases (PKS-I, PKS-II) as well as nonribosomal peptide synthetases (NRPS) showed that 20 actinomycete isolates encoded each at least one type of biosynthetic gene. The organic extracts of nine isolates displayed bioactivity against at least one of the test pathogens, which were Gram-positive and Gram-negative bacteria, fungi, human parasites, as well as in a West Nile Virus protease enzymatic assay. These results emphasize that marine sponges are a prolific resource for novel bioactive actinomycetes with potential for drug discovery.     

Biosynthesis of Polyunsaturated Fatty Acids in Marine Invertebrates: Recent Advances in Molecular Mechanisms

Virtually all polyunsaturated fatty acids (PUFA) originate from primary producers but can be modified by bioconversions as they pass up the food chain in a process termed trophic upgrading. Therefore, although the main primary producers of PUFA in the marine environment are microalgae, higher trophic levels have metabolic pathways that can produce novel and unique PUFA. However, little is known about the pathways of PUFA biosynthesis and metabolism in the levels between primary producers and fish that are largely filled by invertebrates. It has become increasingly apparent that, in addition to trophic upgrading, de novo synthesis of PUFA is possible in some lower animals. The unequivocal identification of PUFA biosynthetic pathways in many invertebrates is complicated by the presence of other organisms within them. These organisms include bacteria and algae with PUFA biosynthesis pathways, and range from intestinal flora to symbiotic relationships that can involve PUFA translocation to host organisms. This emphasizes the importance of studying biosynthetic pathways at a molecular level, and the continual expansion of genomic resources and advances in molecular analysis is facilitating this. The present paper highlights recent research into the molecular and biochemical mechanisms of PUFA biosynthesis in marine invertebrates, particularly focusing on cephalopod molluscs.     

Comparative Analysis of Glycoside Hydrolases Activities from Phylogenetically Diverse Marine Bacteria of the Genus Arenibacter

A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active β-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH) families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.     

Genome Mining and Metabolic Profiling Uncover Polycyclic Tetramate Macrolactams from Streptomyces koyangensis SCSIO 5802

We have previously shown deep-sea-derived Streptomyces koyangensis SCSIO 5802 to produce two types of active secondary metabolites, abyssomicins and candicidins. Here, we report the complete genome sequence of S. koyangensis SCSIO 5802 employing bioinformatics to highlight its potential to produce at least 21 categories of natural products. In order to mine novel natural products, the production of two polycyclic tetramate macrolactams (PTMs), the known 10-epi-HSAF (1) and a new compound, koyanamide A (2), was stimulated via inactivation of the abyssomicin and candicidin biosynthetic machineries. Detailed bioinformatics analyses revealed a PKS/NRPS gene cluster, containing 6 open reading frames (ORFs) and spanning ~16 kb of contiguous genomic DNA, as the putative PTM biosynthetic gene cluster (BGC) (termed herein sko). We furthermore demonstrate, via gene disruption experiments, that the sko cluster encodes the biosynthesis of 10-epi-HSAF and koyanamide A. Finally, we propose a plausible biosynthetic pathway to 10-epi-HSAF and koyanamide A. In total, this study demonstrates an effective approach to cryptic BGC activation enabling the discovery of new bioactive metabolites; genome mining and metabolic profiling methods play key roles in this strategy.     

Brominated Skeletal Components of the Marine Demosponges,Aplysina cavernicola and Ianthella basta: Analytical and Biochemical Investigations

Demosponges possess a skeleton made of a composite material with various organic constituents and/or siliceous spicules. Chitin is an integral part of the skeleton of different sponges of the order Verongida. Moreover, sponges of the order Verongida, such as Aplysina cavernicola or Ianthella basta, are well-known for the biosynthesis of brominated tyrosine derivates, characteristic bioactive natural products. It has been unknown so far whether these compounds are exclusively present in the cellular matrix or whether they may also be incorporated into the chitin-based skeletons. In the present study, we therefore examined the skeletons of A. cavernicola and I. basta with respect to the presence of bromotyrosine metabolites. The chitin-based-skeletons isolated from these sponges indeed contain significant amounts of brominated compounds, which are not easily extractable from the skeletons by common solvents, such as MeOH, as shown by HPLC analyses in combination with NMR and IR spectroscopic measurements. Quantitative potentiometric analyses confirm that the skeleton-associated bromine mainly withstands the MeOH-based extraction. This observation suggests that the respective, but yet unidentified, brominated compounds are strongly bound to the sponge skeletons, possibly by covalent bonding. Moreover, gene fragments of halogenases suggested to be responsible for the incorporation of bromine into organic molecules could be amplified from DNA isolated from sponge samples enriched for sponge-associated bacteria.     

Isolation,Phylogenetic and Gephyromycin Metabolites Characterization of New Exopolysaccharides-Bearing Antarctic Actinobacterium from Feces of Emperor Penguin

A new versatile actinobacterium designated as strain NJES-13 was isolated from the feces of the Antarctic emperor penguin. This new isolate was found to produce two active gephyromycin analogues and bioflocculanting exopolysaccharides (EPS) metabolites. Phylogenetic analysis based on pairwise comparison of 16S rRNA gene sequences showed that strain NJES-13 was closely related to Mobilicoccus pelagius Aji5-31^T with a gene similarity of 95.9%, which was lower than the threshold value (98.65%) for novel species delineation. Additional phylogenomic calculations of the average nucleotide identity (ANI, 75.9–79.1%), average amino acid identity (AAI, 52.4–66.9%) and digital DNA–DNA hybridization (dDDH, 18.6–21.9%), along with the constructed phylogenomic tree based on the up-to-date bacterial core gene (UBCG) set from the bacterial genomes, unequivocally separated strain NJES-13 from its close relatives within the family Dermatophilaceae. Hence, it clearly indicated that strain NJES-13 represented a putative new actinobacterial species isolated from the gut microbiota of mammals inhabiting the Antarctic. The obtained complete genome of strain NJES-13 consisted of a circular 3.45 Mb chromosome with a DNA G+C content of 67.0 mol%. Furthering genome mining of strain NJES-13 showed the presence of five biosynthetic gene clusters (BGCs) including one type III PKS responsible for the biosynthesis of the core of gephyromycins, and a series of genes encoding for bacterial EPS biosynthesis. Thus, based on the combined phylogenetic and active metabolites characterization presented in this study, we confidently conclude that strain NJES-13 is a novel, fresh actinobacterial candidate to produce active gephyromycins and microbial bioflocculanting EPS, with potential pharmaceutical, environmental and biotechnological implications.