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.

A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster.

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.

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.

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.

Complete Genome Sequence of Luteitalea sp. Strain TBR-22.

We report a complete genome sequence of a novel bacterial isolate, strain TBR-22, belonging to the class Vicinamibacteria of the phylum Acidobacteria, which was isolated from duckweed fronds. The genome expands our knowledge of the lifestyle of this abundant but rarely characterized phylum.

Draft Genome Sequence of Bryobacteraceae Strain F-183.

Here, we report a draft genome sequence of a bacterial strain, F-183, isolated from a duckweed frond. Strain F-183 belongs to the family Bryobacteraceae of the phylum Acidobacteria, and its genomic information would contribute to understanding the ecophysiology of this abundant but rarely characterized phylum.

The 'Shellome' of the Crocus Clam Tridacna crocea Emphasizes Essential Components of Mollusk Shell Biomineralization.

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules - in particular proteins - that are essential for molluscan shell formation.

Novel Plant-Associated Acidobacteria Promotes Growth of Common Floating Aquatic Plants, Duckweeds.

Duckweeds are small, fast growing, and starch- and protein-rich aquatic plants expected to be a next generation energy crop and an excellent biomaterial for phytoremediation. Despite such an importance, very little is known about duckweed-microbe interactions that would be a key biological factor for efficient industrial utilization of duckweeds. Here we first report the duckweed growth promoting ability of bacterial strains belonging to the phylum Acidobacteria, the members of which are known to inhabit soils and terrestrial plants, but their ecological roles and plant-microbe interactions remain largely unclear. Two novel Acidobacteria strains, F-183 and TBR-22, were successfully isolated from wild duckweeds and phylogenetically affiliated with subdivision 3 and 6 of the phylum, respectively, based on 16S rRNA gene sequence analysis. In the co-culture experiments with aseptic host plants, the F-183 and TBR-22 strains visibly enhanced growth (frond number) of six duckweed species (subfamily Lemnoideae) up to 1.8-5.1 times and 1.6-3.9 times, respectively, compared with uninoculated controls. Intriguingly, both strains also increased the chlorophyll content of the duckweed (Lemna aequinoctialis) up to 2.4-2.5 times. Under SEM observation, the F-183 and TBR-22 strains were epiphytic and attached to the surface of duckweed. Taken together, our findings suggest that indigenous plant associated Acidobacteria contribute to a healthy growth of their host aquatic plants.

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.

Chromosomal Inversion Polymorphisms in Two Sympatric Ascidian Lineages.

Chromosomal rearrangements can reduce fitness of heterozygotes and can thereby prevent gene flow. Therefore, such rearrangements can play a role in local adaptation and speciation. In particular, inversions are considered to be a major potential cause for chromosomal speciation. There are two closely related, partially sympatric lineages of ascidians in the genus Ciona, which we call type-A and type-B animals in the present study. Although these invertebrate chordates are largely isolated reproductively, hybrids can be found in wild populations, suggesting incomplete prezygotic barriers. Although the genome of type-A animals has been decoded and widely used, the genome for type-B animals has not been decoded at the chromosomal level. In the present study, we sequenced the genomes of two type-B individuals from different sides of the English Channel (in the zone of sympatry with type-A individuals) and compared them at the chromosomal level with the type-A genome. Although the overall structures were well conserved between type A and type B, chromosomal alignments revealed many inversions differentiating these two types of Ciona; it is probable that the frequent inversions have contributed to separation between these two lineages. In addition, comparisons of the genomes between the two type-B individuals revealed that type B had high rates of inversion polymorphisms and nucleotide polymorphisms, and thus type B might be in the process of differentiation into multiple new types or species. Our results suggest an important role of inversions in chromosomal speciation of these broadcasting spawners.

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.

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.

Functional shell matrix proteins tentatively identified by asymmetric snail shell morphology.

Molluscan shell matrix proteins (SMPs) are essential in biomineralization. Here, we identify potentially important SMPs by exploiting the asymmetric shell growth in snail, Lymnaea stagnalis. Asymmetric shells require bilaterally asymmetric expression of SMP genes. We examined expression levels of 35,951 transcripts expressed in the left and right sides of mantle tissue of the pond snail, Lymnaea stagnalis. This transcriptome dataset was used to identify 207 SMPs by LC-MS/MS. 32 of the 207 SMP genes show asymmetric expression patterns, which were further verified for 4 of the 32 SMPs using quantitative PCR analysis. Among asymmetrically expressed SMPs in dextral snails, those that are more highly expressed on the left side than the right side are 3 times more abundant than those that are more highly expressed on the right than the left, suggesting potentially inhibitory roles of SMPs in shell formation. The 32 SMPs thus identified have distinctive features, such as conserved domains and low complexity regions, which may be essential in biomineralization.

Identification, cloning and heterologous expression of biosynthetic gene cluster for desertomycin.

From our in-house microbial genome database of secondary metabolite producers, we identified a candidate biosynthetic gene cluster for desertomycin from Streptomyces nobilis JCM4274. We report herein the cloning of the 127-kb entire gene cluster for desertomycin biosynthesis using bacterial artificial chromosome vector. The entire biosynthetic gene cluster for desertomycin was introduced in the heterologous host, Streptomyces lividans TK23, with an average yield of more than 130 mg l-1.

Divergent northern and southern populations and demographic history of the pearl oyster in the western Pacific revealed with genomic SNPs.

In the open ocean without terrain boundaries, marine invertebrates with pelagic larvae can migrate long distances using ocean currents, suggesting reduced genetic diversification. Contrary to this assumption, however, genetic differentiation is often observed in marine invertebrates. In the present study, we sought to explain how population structure is established in the western Pacific Ocean, where the strong Kuroshio Current maintains high levels of gene flow from south to north, presumably promoting genetic homogeneity. We determined the population structure of the pearl oyster, Pinctada fucata, in the Indo-Pacific Ocean using genome-wide genotyping data from multiple sampling localities. Cluster analysis showed that the western Pacific population is distinct from that of the Indian Ocean, and that it is divided into northern (Japanese mainland) and southern (Nansei Islands, China, and Cambodia) populations. Genetic differentiation of P. fucata can be explained by geographic barriers in the Indian Ocean and a local lagoon, and by environmental gradients of sea surface temperature (SST) and oxygen concentration in the western Pacific. A genome scan showed evidence of adaptive evolution in genomic loci, possibly associated with changes in environmental factors, including SST and oxygen concentration. Furthermore, Bayesian simulation demonstrated that the past population expansion and division are congruent with ocean warming after the last glacial period. It is highly likely that the environmental gradient forms a genetic barrier that diversifies P. fucata populations in the western Pacific. This hypothesis helps to explain genetic differentiation and possible speciation of marine invertebrates.

Novel macrolactam compound produced by the heterologous expression of a large cryptic biosynthetic gene cluster of Streptomyces rochei IFO12908.

In the course of our studies on the heterologous expression of giant biosynthetic genes, we discovered a novel cryptic biosynthetic gene cluster in Streptomyces rochei IFO12908. During our efforts to express biosynthetic genes using the host SUKA strain derived from Streptomyces avermitilis, a novel polyene macrolactam compound designated as JBIR-156 was produced. We report herein the cloning and heterologous expression of the JBIR-156 biosynthetic gene cluster, and the isolation, structure determination, and cytotoxic activity of this novel compound.

A Nearly Complete Genome of Ciona intestinalis Type A (C. robusta) Reveals the Contribution of Inversion to Chromosomal Evolution in the Genus Ciona.

Since its initial publication in 2002, the genome of Ciona intestinalis type A (Ciona robusta), the first genome sequence of an invertebrate chordate, has provided a valuable resource for a wide range of biological studies, including developmental biology, evolutionary biology, and neuroscience. The genome assembly was updated in 2008, and it included 68% of the sequence information in 14 pairs of chromosomes. However, a more contiguous genome is required for analyses of higher order genomic structure and of chromosomal evolution. Here, we provide a new genome assembly for an inbred line of this animal, constructed with short and long sequencing reads and Hi-C data. In this latest assembly, over 95% of the 123 Mb of sequence data was included in the chromosomes. Short sequencing reads predicted a genome size of 114-120 Mb; therefore, it is likely that the current assembly contains almost the entire genome, although this estimate of genome size was smaller than previous estimates. Remapping of the Hi-C data onto the new assembly revealed a large inversion in the genome of the inbred line. Moreover, a comparison of this genome assembly with that of Ciona savignyi, a different species in the same genus, revealed many chromosomal inversions between these two Ciona species, suggesting that such inversions have occurred frequently and have contributed to chromosomal evolution of Ciona species. Thus, the present assembly greatly improves an essential resource for genome-wide studies of ascidians.

Publisher Correction: Medusozoan genomes inform the evolution of the jellyfish body plan.

The version of this article originally published was not open access, but should have been open access. The error has been corrected, and the paper is now open access with a CC-BY license.

Medusozoan genomes inform the evolution of the jellyfish body plan.

Cnidarians are astonishingly diverse in body form and lifestyle, including the presence of a jellyfish stage in medusozoans and its absence in anthozoans. Here, we sequence the genomes of Aurelia aurita (a scyphozoan) and Morbakka virulenta (a cubozoan) to understand the molecular mechanisms responsible for the origin of the jellyfish body plan. We show that the magnitude of genetic differences between the two jellyfish types is equivalent, on average, to the level of genetic differences between humans and sea urchins in the bilaterian lineage. About one-third of Aurelia genes with jellyfish-specific expression have no matches in the genomes of the coral and sea anemone, indicating that the polyp-to-jellyfish transition requires a combination of conserved and novel, medusozoa-specific genes. While no genomic region is specifically associated with the ability to produce a jellyfish stage, the arrangement of genes involved in the development of a nematocyte-a phylum-specific cell type-is highly structured and conserved in cnidarian genomes; thus, it represents a phylotypic gene cluster.