Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis.

The smallest reported bacterial genome belongs to Tremblaya princeps, a symbiont of Planococcus citri mealybugs (PCIT). Tremblaya PCIT not only has a 139 kb genome, but possesses its own bacterial endosymbiont, Moranella endobia. Genome and transcriptome sequencing, including genome sequencing from a Tremblaya lineage lacking intracellular bacteria, reveals that the extreme genomic degeneracy of Tremblaya PCIT likely resulted from acquiring Moranella as an endosymbiont. In addition, at least 22 expressed horizontally transferred genes from multiple diverse bacteria to the mealybug genome likely complement missing symbiont genes. However, none of these horizontally transferred genes are from Tremblaya, showing that genome reduction in this symbiont has not been enabled by gene transfer to the host nucleus. Our results thus indicate that the functioning of this three-way symbiosis is dependent on genes from at least six lineages of organisms and reveal a path to intimate endosymbiosis distinct from that followed by organelles.

The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest.

The crown-of-thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-building corals throughout the Indo-Pacific region. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of reef ecosystems. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products that underlie species-specific communication and could potentially be used in biocontrol strategies. We focused on water-borne chemical plumes released from aggregating COTS, which make the normally sedentary starfish become highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS consists largely of signalling factors and hydrolytic enzymes, and includes an expanded and rapidly evolving set of starfish-specific ependymin-related proteins. These secreted proteins may be detected by members of a large family of olfactory-receptor-like G-protein-coupled receptors that are expressed externally, sometimes in a sex-specific manner. This study provides insights into COTS-specific communication that may guide the generation of peptide mimetics for use on reefs with COTS outbreaks.

New azodyrecins identified by a genome mining-directed reactivity-based screening.

Only a few azoxy natural products have been identified despite their intriguing biological activities. Azodyrecins D-G, four new analogs of aliphatic azoxides, were identified from two Streptomyces species by a reactivity-based screening that targets azoxy bonds. A biological activity evaluation demonstrated that the double bond in the alkyl side chain is important for the cytotoxicity of azodyrecins. An in vitro assay elucidated the tailoring step of azodyrecin biosynthesis, which is mediated by the S-adenosylmethionine (SAM)-dependent methyltransferase Ady1. This study paves the way for the targeted isolation of aliphatic azoxy natural products through a genome-mining approach and further investigations of their biosynthetic mechanisms.

Hemichordate genomes and deuterostome origins.

Acorn worms, also known as enteropneust (literally, 'gut-breathing') hemichordates, are marine invertebrates that share features with echinoderms and chordates. Together, these three phyla comprise the deuterostomes. Here we report the draft genome sequences of two acorn worms, Saccoglossus kowalevskii and Ptychodera flava. By comparing them with diverse bilaterian genomes, we identify shared traits that were probably inherited from the last common deuterostome ancestor, and then explore evolutionary trajectories leading from this ancestor to hemichordates, echinoderms and chordates. The hemichordate genomes exhibit extensive conserved synteny with amphioxus and other bilaterians, and deeply conserved non-coding sequences that are candidates for conserved gene-regulatory elements. Notably, hemichordates possess a deuterostome-specific genomic cluster of four ordered transcription factor genes, the expression of which is associated with the development of pharyngeal 'gill' slits, the foremost morphological innovation of early deuterostomes, and is probably central to their filter-feeding lifestyle. Comparative analysis reveals numerous deuterostome-specific gene novelties, including genes found in deuterostomes and marine microbes, but not other animals. The putative functions of these genes can be linked to physiological, metabolic and developmental specializations of the filter-feeding ancestor.

Identification of functional cytochrome P450 and ferredoxin from Streptomyces sp. EAS-AB2608 by transcriptional analysis and their heterologous expression.

Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.

Comparative genomics of four strains of the edible brown alga, Cladosiphon okamuranus.

BACKGROUND: The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is one of the most important edible seaweeds, and it is cultivated for market primarily in Okinawa, Japan. Four strains, denominated S, K, O, and C, with distinctively different morphologies, have been cultivated commercially since the early 2000s. We previously reported a draft genome of the S-strain. To facilitate studies of seaweed biology for future aquaculture, we here decoded and analyzed genomes of the other three strains (K, O, and C). RESULTS: Here we improved the genome of the S-strain (ver. 2, 130 Mbp, 12,999 genes), and decoded the K-strain (135 Mbp, 12,511 genes), the O-strain (140 Mbp, 12,548 genes), and the C-strain (143 Mbp, 12,182 genes). Molecular phylogenies, using mitochondrial and nuclear genes, showed that the S-strain diverged first, followed by the K-strain, and most recently the C- and O-strains. Comparisons of genome architecture among the four strains document the frequent occurrence of inversions. In addition to gene acquisitions and losses, the S-, K-, O-, and C-strains possess 457, 344, 367, and 262 gene families unique to each strain, respectively. Comprehensive Blast searches showed that most genes have no sequence similarity to any entries in the non-redundant protein sequence database, although GO annotation suggested that they likely function in relation to molecular and biological processes and cellular components. CONCLUSIONS: Our study compares the genomes of four strains of C. okamuranus and examines their phylogenetic relationships. Due to global environmental changes, including temperature increases, acidification, and pollution, brown algal aquaculture is facing critical challenges. Genomic and phylogenetic information reported by the present research provides useful tools for isolation of novel strains.

Heterologous Expression of the Biosynthetic Gene Cluster for Verticilactam and Identification of Analogues.

Verticilactam and the new geometric isomers, verticilactams B and C, were produced by heterologous expression of the biosynthetic gene cluster for verticilactam using the Streptomyces avermitilis SUKA17 strain. Only verticilactam, a compound with a characteristic ß-ketoamide unit within a 16-membered polyketide macrolactam conjugated with an octalin skeleton, had been previously reported having been isolated from Streptomyces spiroverticillatus JC-8444. In this report, minor verticilactam derivatives were isolated from the transformed strain, and their structures elucidated by spectral analysis. Verticilactam B was a geometric isomer at Δ17 and Δ19, and verticilactam C was the Δ19 and Δ21 isomer. In addition, the absolute configuration of verticilactam was confirmed by ECD analysis and NMR chemical shifts. The stereochemistry assignments of the hydroxy groups at C-10 and C-12 were supported by the domain organization of the polyketide synthase identified in the verticilactam gene cluster. Verticilactam showed moderate activity against the malaria parasite Plasmodium falciparum 3D7 strain with no significant cytotoxicity or antimicrobial effects.

Small genome symbiont underlies cuticle hardness in beetles.

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle's cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella's sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host's cuticle formation and hardening. Notably, Nardonella's tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host's aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host's final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host-symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

Two divergent Symbiodinium genomes reveal conservation of a gene cluster for sunscreen biosynthesis and recently lost genes.

BACKGROUND: The marine dinoflagellate, Symbiodinium, is a well-known photosynthetic partner for coral and other diverse, non-photosynthetic hosts in subtropical and tropical shallows, where it comprises an essential component of marine ecosystems. Using molecular phylogenetics, the genus Symbiodinium has been classified into nine major clades, A-I, and one of the reported differences among phenotypes is their capacity to synthesize mycosporine-like amino acids (MAAs), which absorb UV radiation. However, the genetic basis for this difference in synthetic capacity is unknown. To understand genetics underlying Symbiodinium diversity, we report two draft genomes, one from clade A, presumed to have been the earliest branching clade, and the other from clade C, in the terminal branch. RESULTS: The nuclear genome of Symbiodinium clade A (SymA) has more gene families than that of clade C, with larger numbers of organelle-related genes, including mitochondrial transcription terminal factor (mTERF) and Rubisco. While clade C (SymC) has fewer gene families, it displays specific expansions of repeat domain-containing genes, such as leucine-rich repeats (LRRs) and retrovirus-related dUTPases. Interestingly, the SymA genome encodes a gene cluster for MAA biosynthesis, potentially transferred from an endosymbiotic red alga (probably of bacterial origin), while SymC has completely lost these genes. CONCLUSIONS: Our analysis demonstrates that SymC appears to have evolved by losing gene families, such as the MAA biosynthesis gene cluster. In contrast to the conservation of genes related to photosynthetic ability, the terminal clade has suffered more gene family losses than other clades, suggesting a possible adaptation to symbiosis. Overall, this study implies that Symbiodinium ecology drives acquisition and loss of gene families.

Unprecedented Cyclization Catalyzed by a Cytochrome P450 in Benzastatin Biosynthesis.

Benzastatins have unique structures probably derived from geranylated p-aminobenzoic acids. The indoline and tetrahydroquinoline scaffolds are presumably formed by cyclization of the geranyl moiety, but the cyclization mechanism was unknown. We studied the benzastatin biosynthetic gene cluster of Streptomyces sp. RI18; functions of the six enzymes encoded by it were analyzed by gene disruption in a heterologous host and in vitro enzyme assays. We propose the biosynthetic pathway for benzastatins in which a cytochrome P450 (BezE) is responsible for the cyclization of geranylated p-acetoxyaminobenzoic acids; BezE catalyzes elimination of acetic acid to form an iron nitrenoid, nitrene transfer to form an aziridine ring, and nucleophilic addition of hydroxide ion to C-10 and chloride ion to C-9 to generate the indoline and tetrahydroquinoline scaffolds, respectively. Discovery of this enzyme, which should be termed cytochrome P450 nitrene transferase, provides an important insight into the functional diversity of cytochrome P450.

Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster.

During genome mining for thioviridamide-like biosynthetic gene clusters that could produce polythioamide RiPP (ribosomally synthesized and post-translationally modified peptides), we discovered a novel cryptic biosynthetic gene cluster. During efforts to express this biosynthetic gene using heterologous expression of this biosynthetic gene cluster, a novel compound designated as neothioviridamide was produced. We report herein the cloning and heterologous expression of the neothioviridamide biosynthetic gene cluster and the isolation, structure determination, and cytotoxic activity of neothioviridamide.

Using the Acropora digitifera genome to understand coral responses to environmental change.

Despite the enormous ecological and economic importance of coral reefs, the keystone organisms in their establishment, the scleractinian corals, increasingly face a range of anthropogenic challenges including ocean acidification and seawater temperature rise. To understand better the molecular mechanisms underlying coral biology, here we decoded the approximately 420-megabase genome of Acropora digitifera using next-generation sequencing technology. This genome contains approximately 23,700 gene models. Molecular phylogenetics indicate that the coral and the sea anemone Nematostella vectensis diverged approximately 500 million years ago, considerably earlier than the time over which modern corals are represented in the fossil record (∼240 million years ago). Despite the long evolutionary history of the endosymbiosis, no evidence was found for horizontal transfer of genes from symbiont to host. However, unlike several other corals, Acropora seems to lack an enzyme essential for cysteine biosynthesis, implying dependency of this coral on its symbionts for this amino acid. Corals inhabit environments where they are frequently exposed to high levels of solar radiation, and analysis of the Acropora genome data indicates that the coral host can independently carry out de novo synthesis of mycosporine-like amino acids, which are potent ultraviolet-protective compounds. In addition, the coral innate immunity repertoire is notably more complex than that of the sea anemone, indicating that some of these genes may have roles in symbiosis or coloniality. A number of genes with putative roles in calcification were identified, and several of these are restricted to corals. The coral genome provides a platform for understanding the molecular basis of symbiosis and responses to environmental changes.

Sustained heterozygosity across a self-incompatibility locus in an inbred ascidian.

Because self-incompatibility loci are maintained heterozygous and recombination within self-incompatibility loci would be disadvantageous, self-incompatibility loci are thought to contribute to structural and functional differentiation of chromosomes. Although the hermaphrodite chordate, Ciona intestinalis, has two self-incompatibility genes, this incompatibility system is incomplete and self-fertilization occurs under laboratory conditions. Here, we established an inbred strain of C. intestinalis by repeated self-fertilization. Decoding genome sequences of sibling animals of this strain identified a 2.4-Mbheterozygous region on chromosome 7. A self-incompatibility gene, Themis-B, was encoded within this region. This observation implied that this self-incompatibility locus and the linkage disequilibrium of its flanking region contribute to the formation of the 2.4-Mb heterozygous region, probably through recombination suppression. We showed that different individuals in natural populations had different numbers and different combinations of Themis-B variants, and that the rate of self-fertilization varied among these animals. Our result explains why self-fertilization occurs under laboratory conditions. It also supports the concept that the Themis-B locus is preferentially retained heterozygous in the inbred line and contributes to the formation of the 2.4-Mb heterozygous region. High structural variations might suppress recombination, and this long heterozygous region might represent a preliminary stage of structural differentiation of chromosomes.

Production of a Novel Amide-Containing Polyene by Activating a Cryptic Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08.

Streptomyces sp. MSC090213JE08 seems to have more than 20 cryptic biosynthetic gene clusters for secondary metabolites. We aimed to activate some of them by forced production of Streptomyces antibiotic regulatory protein (SARP) family transcriptional activators. We constructed seven recombinant strains, each of which contained a SARP gene under the control of a constitutive promoter, and subjected them to comparative metabolic profiling analysis. Four of the seven strains produced nine metabolites that were hardly detected in the control strains. We isolated a new metabolite (named ishigamide) from the SARP-7-expressing strain and determined its structure as 3-((2E,4E,6E,8E)-13-hydroxytetradeca-2,4,6,8-tetraenamido)propanoic acid. Genome scanning and gene disruption studies identified the ishigamide biosynthetic gene cluster adjacent to the SARP-7 gene. We think that a new subfamily of type II polyketide synthase is involved in the biosynthesis of the polyene structure of ishigamide.

Involvement of the Baeyer-Villiger Monooxygenase IfnQ in the Biosynthesis of Isofuranonaphthoquinone Scaffold of JBIR-76 and -77.

JBIR-76 and -77 are isofuranonaphthoquinones (IFNQs) isolated from Streptomyces sp. RI-77. Draft genome sequencing and gene disruption analysis of Streptomyces sp. RI-77 showed that a type II polyketide synthase (PKS) gene cluster (ifn cluster) was responsible for the biosynthesis of JBIR-76 and -77. It was envisaged that an octaketide intermediate (C16 ) could be synthesized by the minimal PKS (IfnANO) and that formation of the IFNQ scaffold (C13 ) would therefore require a C-C bond cleavage reaction. An ifnQ disruptant accumulated some shunt products (C15 ), which were presumably produced by spontaneous cyclization of the decarboxylated octaketide intermediate. Recombinant IfnQ catalyzed the Baeyer-Villiger oxidation of 1-(2-naphthyl)acetone, an analogue of the bicyclic octaketide intermediate. Based on these results, we propose a pathway for the biosynthesis of JBIR-76 and -77, involving IfnQ-catalyzed C-C bond cleavage as a key step in the formation of the IFNQ scaffold.

Identification and Characterization of the Streptazone†E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08.

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2-like superfamily are responsible for C-C bond formation coupled with epoxide ring opening to give the five-membered ring of streptazone E.

Capability of a large bacterial artificial chromosome clone harboring multiple biosynthetic gene clusters for the production of diverse compounds.

The biosynthetic gene clusters (BGCs) for the macrocyclic lactone-based polyketide compounds are extremely large-sized because the polyketide synthases that generate the polyketide chains of the basic backbone are of very high molecular weight. In developing a heterologous expression system for the large BGCs amenable to the production of such natural products, we selected concanamycin as an appropriate target. We obtained a bacterial artificial chromosome (BAC) clone with a 211-kb insert harboring the entire BGC responsible for the biosynthesis of concanamycin. Heterologous expression of this clone in a host strain, Streptomyces avermitilis SUKA32, permitted the production of concanamycin, as well as that of two additional aromatic polyketides. Structural elucidation identified these additional products as ent-gephyromycin and a novel compound that was designated JBIR-157. We describe herein sequencing and expression studies performed on these BGCs, demonstrating the utility of large BAC clones for the heterologous expression of cryptic or near-silent loci.

Genomic and transcriptomic analyses illuminate the molecular basis of the unique lifestyle of a tubeworm, Lamellibrachia satsuma.

Vestimentiferan tubeworms are representative members of deep-sea chemosynthetic ecosystems. In this study, we developed a draft genome and gene models and performed genomic and transcriptomic analyses of Lamellibrachia satsuma, the only vestimentiferan reported from the euphotic zone. The quality of the genome assembly and gene models is comparable to or higher than those of previously reported vestimentiferan tubeworms. Tissue-specific transcriptome sequencing revealed that Toll-like receptor genes and lineage-specific expanded bacteriolytic enzyme genes are highly expressed in the obturacular and vestimental regions, respectively, suggesting the importance of these tissues in defense against pathogens. On the other hand, globin subunit genes are expressed almost exclusively in the trunk region, supporting the hypothesis that the trophosome is the site of haemoglobin biosynthesis. Vestimentiferan-specific expanded gene families included chitinases, ion channels, and C-type lectins, suggesting the importance of these functions for vestimentiferans. C-type lectins in the trunk region, in particular, may be involved in recognition of pathogens, or in interactions between tubeworms and symbiotic bacteria. Our genomic and transcriptomic analyses enhance understanding of molecular mechanisms underlying the unique lifestyle of vestimentiferan tubeworms, particularly their obligate mutualism with chemosynthetic bacteria.

Prostacyclin receptor in tumor endothelial cells promotes angiogenesis in an autocrine manner.

Molecules highly expressed in tumor endothelial cells (TEC) are important for specific targeting of these cells. Previously, using DNA microarray analysis, we found that the prostacyclin receptor (IP receptor) gene was upregulated in TEC compared with normal endothelial cells (NEC). Although prostacyclin is implicated in re-endothelialization and angiogenesis, its role remains largely unknown in TEC. Moreover, the effect of the IP receptor on TEC has not been reported. In the present study we investigated the function of the IP receptor in TEC. The TEC were isolated from two types of human tumor xenografts in nude mice, while NEC were isolated from normal counterparts. Prostacyclin secretion levels in TEC were significantly higher than those in NEC, as shown using ELISA. Real-time RT-PCR showed that the IP receptor was upregulated in TEC compared with NEC. Furthermore, migration and tube formation of TEC were suppressed by the IP receptor antagonist RO1138452. Immunohistostaining showed that the IP receptor was specifically expressed in blood vessels of renal cell carcinoma specimens, but not in glomerular vessels of normal renal tissue. These findings suggest that the IP receptor is a TEC-specific marker and might be a useful therapeutic target.

The acidic amino acid-rich C-terminal domain of VanabinX enhances reductase activity, attaining 1.3- to 1.7-fold vanadium reduction.

Ascidians accumulate extremely high levels of vanadium (V) in their blood cells. Several V-related proteins, including V-binding proteins (vanabins), have been isolated from V-accumulating ascidians. In this study, to obtain a deeper understanding of vanabins, we performed de novo transcriptome analysis of blood cells from a V-rich ascidian, Ascidia sydneiensis samea, and constructed a database containing 8532 predicted proteins. We found a novel vanabin with a unique acidic amino acid-rich C-terminal domain, designated VanabinX, in the database and studied it in detail. Reverse-transcription polymerase chain reaction analysis revealed that VanabinX was detected in all adult tissues examined, and was most prominent in blood cells and muscle tissue. We prepared recombinant proteins and performed immobilized metal ion affinity chromatography and a NADPH-coupled V(V)-reductase assay. VanabinX bound to metal ions, with increasing affinity for Cu(II) > Zn(II) > Co(II), but not to V(IV). VanabinX reduced V(V) to V(IV) at a rate of 0.170 µM per micoromolar protein within 30 min. The C-terminal acidic domain enhanced the reduction of V(V) by Vanabin2 to 1.3-fold and of VanabinX itself to 1.7-fold in trans mode. In summary, we constructed a protein database containing 8532 predicted proteins expressed in blood cells; among them, we discovered a novel vanabin, VanabinX, which enhances V reduction by vanabins.