Nicole B. Lopanik

Potent cytotoxins produced by a microbial symbiont protect host larvae from predation

Larvae of the sessile marine invertebrate Bugula neritina (Bryozoa) are protected by an effective chemical defense. From the larvae, we isolated three bryostatin-class macrocyclic polyketides, including the novel bryostatin 20, that deterred feeding by a common planktivorous fish that co-occurs with B. neritina. A unique bacterial symbiont of B. neritina, Endobugula sertula, was hypothesized as the putative source of the bryostatins. We show that: (1) bryostatins are concentrated in B. neritina larvae and protect them against predation by fish; (2) the adults are not defended by bryostatins; and (3) E. sertula produces bryostatins. This study represents the first example from the marine environment of a microbial symbiont producing an anti-predator defense for its host and, in this case, specifically for the host’s larval stage, which is exceptionally vulnerable to predators.

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.

Chemical defensive symbioses in the marine environment

Summary Marine organisms are a prolific source of natural products, many of which have become the target of pharmacological investigations. As well as being bioactive, these compounds can play an important role in the survival of the organisms and potentially affect community structure. Sessile invertebrates such as sponges, cnidarians, bryozoans and tunicates, in particular, are an especially rich source of ecologically relevant compounds. These organisms are typically soft-bodied and unable to escape predators, and as such, they rely on chemical defence for their persistence. These invertebrates are also frequently hosts for microbial symbionts. Marine microbes are a prolific source of bioactive natural products, many of which can be allelopathic and prevent the growth of pathogens. For sessile marine invertebrates, which often have both bioactive natural products and microbial symbionts, it is logical to hypothesize that microbial symbionts may produce the secondary metabolites. While symbionts are often thought to be responsible for producing bioactive natural products that play a role in host defence, relatively few studies have experimentally demonstrated that symbiont-produced compounds defend the hosts. One reason for this is the difficulty of manipulating the host–symbiont relationship, which is often obligate for one or both partners. Given the importance of natural products to marine invertebrates for defence, the prevalence of symbiosis in the marine environment and the diverse metabolic capabilities of micro-organisms, symbiont-mediated host chemical defence may be more prevalent than currently understood. In this review, I document evidence supporting chemical defensive symbioses in marine organisms, discuss commonalities and differences among the diverse relationships, and provide future research directions. Epibiont-produced defensive compounds would seem to result in the most efficient manner of protection, as the compounds are most accessible to the predators. By contrast, endosymbiont-produced compounds may need to be transported to exposed tissues. Elucidating mechanisms to transport symbiont-produced defensive compounds within the host will provide greater insight into the breadth and complexity of host–symbiont interactions. Another important unexplored issue is how the hosts are able to tolerate symbiont-produced metabolites that are often eucaryotic cell effectors. A greater understanding of defensive symbiosis will result from detailed studies of the co-evolution of predator, host and symbiont, and if or how predators can influence the production of defensive metabolites.

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 [β-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.

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.

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

ABSTRACT “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.

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 9α-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.

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.

Antibiotic activity and microbial community of the temperate sponge, Haliclona sp.

Sessile marine invertebrates engage in a diverse array of beneficial interactions with bacterial symbionts. One feature of some of these relationships is the presence of bioactive natural products that can defend the holobiont from predation, competition or disease. In this study, we investigated the antimicrobial activity and microbial community of a common temperate sponge from coastal North Carolina.

Inferring metabolic pathway activity levels from RNA-Seq data

BackgroundAssessing 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.ResultsXPathway 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).ConclusionsOur 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.