Metabolomic Profiling and Molecular Networking of Nudibranch-Associated Streptomyces sp. SCSIO 001680.

Antibiotic-resistant bacteria are the primary source of one of the growing public health problems that requires global attention, indicating an urgent need for new antibiotics. Marine ecosystems are characterized by high biodiversity and are considered one of the essential sources of bioactive chemical compounds. Bacterial associates of marine invertebrates are commonly a source of active medicinal and natural products and are important sources for drug discovery. Hence, marine invertebrate-associated microbiomes are a fruitful resource for excavating novel genes and bioactive compounds. In a previous study, we isolated Streptomyces sp. SCSIO 001680, coded as strain 63, from the Red Sea nudibranch Chromodoris quadricolor, which exhibited antimicrobial and antitumor activity. In addition, this isolate harbors several natural product biosynthetic gene clusters, suggesting it has the potential to produce bioactive natural products. The present study aimed to investigate the metabolic profile of the isolated Streptomyces sp. SCSIO 001680 (strain 63) and to predict their potential role in the host's survival. The crude metabolic extracts of strain 63 cultivated in two different media were characterized by ultra-high-performance liquid chromatography and high-resolution mass spectrometry. The metabolomics approach provided us with characteristic chemical fingerprints of the cellular processes and the relative abundance of specific compounds. The Global Products Social Molecular Networking database was used to identify the metabolites. While 434 metabolites were detected in the extracts, only a few compounds were identified based on the standards and the public spectral libraries, including desferrioxamines, marineosin A, and bisucaberin, halichoblelide, alternarin A, pachastrelloside A, streptodepsipeptide P1 1B, didemnaketal F, and alexandrolide. This finding suggests that this strain harbors several novel compounds. In addition, the metabolism of the microbiome of marine invertebrates remains poorly represented. Thus, our data constitute a valuable complement to the study of metabolism in the host microbiome.

The natural product biosynthetic potential of Red Sea nudibranch microbiomes.

BACKGROUND: Antibiotic resistance is a growing problem that can be ameliorated by the discovery of novel drug candidates. Bacterial associates are often the source of pharmaceutically active natural products isolated from marine invertebrates, and thus, important targets for drug discovery. While the microbiomes of many marine organisms have been extensively studied, microbial communities from chemically-rich nudibranchs, marine invertebrates that often possess chemical defences, are relatively unknown. METHODS: We applied both culture-dependent and independent approaches to better understand the biochemical potential of microbial communities associated with nudibranchs. Gram-positive microorganisms isolated from nudibranchs collected in the Red Sea were screened for antibacterial and antitumor activity. To assess their biochemical potential, the isolates were screened for the presence of natural product biosynthetic gene clusters, including polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, using PCR. The microbiomes of the nudibranchs were investigated by high-throughput sequencing of 16S rRNA amplicons. RESULTS: In screens against five model microorganisms, 51% of extracts displayed antimicrobial activity against more than one organism, and 19% exhibited antitumor activity against Ehrlich's ascites carcinoma. Sixty-four percent of isolates contained PKS and NRPS genes, suggesting their genomes contain gene clusters for natural product biosynthesis. Thirty-five percent were positive for more than one class of biosynthetic gene. These strains were identified as belonging to the Firmicutes and Actinobacteria phyla via 16S rRNA gene sequencing. In addition, 16S rRNA community amplicon sequencing revealed all bacterial isolates were present in the uncultured host-associated microbiome, although they were a very small percentage of the total community. Taken together, these results indicate that bacteria associated with marine nudibranchs are potentially a rich source of bioactive compounds and natural product biosynthetic genes.

The Phylum Bryozoa: From Biology to Biomedical Potential.

Less than one percent of marine natural products characterized since 1963 have been obtained from the phylum Bryozoa which, therefore, still represents a huge reservoir for the discovery of bioactive metabolites with its ~6000 described species. The current review is designed to highlight how bryozoans use sophisticated chemical defenses against their numerous predators and competitors, and which can be harbored for medicinal uses. This review collates all currently available chemoecological data about bryozoans and lists potential applications/benefits for human health. The core of the current review relates to the potential of bryozoan metabolites in human diseases with particular attention to viral, brain, and parasitic diseases. It additionally weighs the pros and cons of total syntheses of some bryozoan metabolites versus the synthesis of non-natural analogues, and explores the hopes put into the development of biotechnological approaches to provide sustainable amounts of bryozoan metabolites without harming the natural environment.

Inferring metabolic pathway activity levels from RNA-Seq data.

BACKGROUND: Assessing pathway activity levels is a plausible way to quantify metabolic differences between various conditions. This is usually inferred from microarray expression data. Wide availability of NGS technology has triggered a demand for bioinformatics tools capable of analyzing pathway activity directly from RNA-Seq data. In this paper we introduce XPathway, a set of tools that compares pathway activity analyzing mapping of contigs assembled from RNA-Seq reads to KEGG pathways. The XPathway analysis of pathway activity is based on expectation maximization and topological properties of pathway graphs. RESULTS: XPathway tools have been applied to RNA-Seq data from the marine bryozoan Bugula neritina with and without its symbiotic bacterium "Candidatus Endobugula sertula". We successfully identified several metabolic pathways with differential activity levels. The expression of enzymes from the identified pathways has been further validated through quantitative PCR (qPCR). CONCLUSIONS: Our results show that XPathway is able to detect and quantify the metabolic difference in two samples. The software is implemented in C, Python and shell scripting and is capable of running on Linux/Unix platforms. The source code and installation instructions are available at http://alan.cs.gsu.edu/NGS/?q=content/xpathway .

Latitudinal variation of a defensive symbiosis in the Bugula neritina (Bryozoa) sibling species complex.

Mutualistic relationships are beneficial for both partners and are often studied within a single environment. However, when the range of the partners is large, geographical differences in selective pressure may shift the relationship outcome from positive to negative. The marine bryozoan Bugula neritina is a colonial invertebrate common in temperate waters worldwide. It is the source of bioactive polyketide metabolites, the bryostatins. Evidence suggests that an uncultured vertically transmitted symbiont, "Candidatus Endobugula sertula", hosted by B. neritina produces the bryostatins, which protect the vulnerable larvae from predation. Studies of B. neritina along the North American Atlantic coast revealed a complex of two morphologically similar sibling species separated by an apparent biogeographic barrier: the Type S sibling species was found below Cape Hatteras, North Carolina, while Type N was found above. Interestingly, the Type N colonies lack "Ca. Endobugula sertula" and, subsequently, defensive bryostatins; their documented northern distribution was consistent with traditional biogeographical paradigms of latitudinal variation in predation pressure. Upon further sampling of B. neritina populations, we found that both host types occur in wider distribution, with Type N colonies living south of Cape Hatteras, and Type S to the north. Distribution of the symbiont, however, was not restricted to Type S hosts. Genetic and microscopic evidence demonstrates the presence of the symbiont in some Type N colonies, and larvae from these colonies are endowed with defensive bryostatins and contain "Ca. Endobugula sertula". Molecular analysis of the symbiont from Type N colonies suggests an evolutionarily recent acquisition, which is remarkable for a symbiont thought to be transmitted only vertically. Furthermore, most Type S colonies found at higher latitudes lack the symbiont, suggesting that this host-symbiont relationship is more flexible than previously thought. Our data suggest that the symbiont, but not the host, is restricted by biogeographical boundaries.

Host differentially expressed genes during association with its defensive endosymbiont.

Mutualism, a beneficial relationship between two species, often requires intimate interaction between the host and symbiont to establish and maintain the partnership. The colonial marine bryozoan Bugula neritina harbors an as yet uncultured endosymbiont, "Candidatus Endobugula sertula," throughout its life stages. The bacterial symbiont is the putative source of bioactive complex polyketide metabolites, the bryostatins, which chemically defend B. neritina larvae from predation. Despite the presence of "Ca. Endobugula sertula" in all life stages of the host, deterrent bryostatins appear to be concentrated in reproductive portions of the host colony, suggesting an interaction between the two partners to coordinate production and distribution of the metabolites within the colony. In this study, we identified host genes that were differentially expressed in control colonies and in colonies cured of the symbiont. Genes that code for products similar to glycosyl hydrolase family 9 and family 20 proteins, actin, and a Rho-GDP dissociation inhibitor were significantly downregulated (more than twice) in antibiotic-cured non-reproductive zooids compared to control symbiotic ones. Differential expression of these genes leads us to hypothesize that the host B. neritina may regulate the distribution of the symbiont within the colony via mechanisms of biofilm degradation and actin rearrangement, and consequently, influences bryostatin localization to bestow symbiont-associated protection to larvae developing in the reproductive zooids.

Polyketide synthase pathways identified from a metagenomic library are derived from soil Acidobacteria.

Polyketides are structurally diverse secondary metabolites, many of which have antibiotic or anticancer activity. Type I modular polyketide synthase (PKS) genes are typically large and encode repeating enzymatic domains that elongate and modify the nascent polyketide chain. A fosmid metagenomic library constructed from an agricultural soil was arrayed and the macroarray was screened for the presence of conserved ketosynthase [ß-ketoacyl synthase (KS)] domains, enzymatic domains present in PKSs. Thirty-four clones containing KS domains were identified by Southern hybridization. Many of the KS domains contained within metagenomic clones shared significant similarity to PKS or nonribosomal peptide synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. However, analysis of complete clone insert sequences indicated that the blast analysis for KS domains did not reflect the true phylogenetic origin of many of these metagenomic clones that had a %G+C content and significant sequence similarity to genes from members of the phylum Acidobacteria. This conclusion of an Acidobacteria origin for several clones was further supported by evidence that cultured soil Acidobacteria from different subdivisions have genetic loci closely related to PKS domains contained within metagenomic clones, suggesting that Acidobacteria may be a source of novel polyketides. This study also demonstrates the utility of combining data from culture-dependent and -independent investigations in expanding our collective knowledge of microbial genomic diversity.

Polyketide ß-branching in bryostatin biosynthesis: identification of surrogate acetyl-ACP donors for BryR, an HMG-ACP synthase.

In vitro analysis of natural product biosynthetic gene products isolated from unculturable symbiotic bacteria is necessary to probe the functionalities of these enzymes. Herein, we report the biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) homolog implicated in ß-branching at C13 and C21 of the core ring system from the bryostatin metabolic pathway (Bry). We confirmed the activity of BryR using two complementary methods, radio-SDS PAGE, and Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS). The activity of BryR depended on pairing of the native acetoacetyl-BryM3 acceptor acyl carrier protein (ACP) with an appropriate donor acetyl-ACP from a heterologous HMGS cassette. Additionally, the ability of BryR to discriminate between various ACPs was assessed using a surface plasmon resonance (SPR)-based protein-protein binding assay. Our data suggest that specificity for a protein-bound acyl group is a distinguishing feature between HMGS homologs found in PKS or PKS/NRPS biosynthetic pathways and those of primary metabolism. These findings reveal an important example of molecular recognition between protein components that are essential for biosynthetic fidelity in natural product assembly and modification.

In vivo and in vitro trans-acylation by BryP, the putative bryostatin pathway acyltransferase derived from an uncultured marine symbiont.

The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete open reading frame (ORF) (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.

Analysis of a parallel branch in the mitomycin biosynthetic pathway involving the mitN-encoded aziridine N-methyltransferase.

Mitomycin C is a natural product with potent alkylating activity, and it is an important anticancer drug and antibiotic. mitN, one of three genes with high similarity to methyltransferases, is located within the mitomycin biosynthetic gene cluster. An inframe deletion in mitN of the mitomycin biosynthetic pathway was generated in Streptomyces lavendulae to produce the DHS5373 mutant strain. Investigation of DHS5373 revealed continued production of mitomycin A and mitomycin C in addition to the accumulation of a new mitomycin analog, 9-epi-mitomycin C. The mitN gene was overexpressed in Escherichia coli, and the histidine-tagged protein (MitN) was purified to homogeneity. Reaction of 9-epi-mitomycin C with MitN in the presence of S-adenosylmethionine yielded mitomycin E showing that the enzyme functions as an aziridine N-methyltransferase. Likewise, MitN was also shown to convert mitomycin A to mitomycin F under the same reaction conditions. We conclude that MitN plays an important role in a parallel biosynthetic pathway leading to the subclass of mitomycins with 9alpha-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.

Identification of the putative bryostatin polyketide synthase gene cluster from "Candidatus Endobugula sertula", the uncultivated microbial symbiont of the marine bryozoan Bugula neritina.

The bryostatins are protein kinase C modulators with unique structural features and potential anticancer and neurological activities. These complex polyketides were isolated from the marine bryozoan Bugula neritina, but recent studies indicate that they are produced by the uncultured symbiotic bacterium "Candidatus Endobugula sertula" ("E. sertula"). Here we present the putative biosynthetic genes: five modular polyketide synthase (PKS) genes, a discrete acyltransferase, a beta-ketosynthase, a hydroxy-methyl-glutaryl CoA synthase (HMG-CS), and a methyltransferase. The cluster was sequenced in two closely related "E. sertula" strains from different host species. In one strain the gene cluster is contiguous, while in the other strain it is split into two loci, with one locus containing the PKS genes and the other containing the accessory genes. Here, we propose a hypothesis for the biosynthesis of the bryostatins. Thirteen PKS modules form the core macrolactone ring, and the pendent methyl ester groups are added by the HMG-CS gene cassette. The resulting hypothetical compound bryostatin 0 is the common basis for the 20 known bryostatins. As "E. sertula" is to date uncultured, heterologous expression of this biosynthetic gene cluster has the potential of producing the bioactive bryostatins in large enough quantities for development into a pharmaceutical.

Isolation of two polyketide synthase gene fragments from the uncultured microbial symbiont of the marine bryozoan Bugula neritina.

"Candidatus Endobugula sertula," the uncultured microbial symbiont of the bryozoan Bugula neritina, produces ecologically and biomedically important polyketide metabolites called bryostatins. We isolated two gene fragments from B. neritina larvae that have high levels of similarity to polyketide synthase genes. These gene fragments are clearly associated with the symbiont and not with the host.