Dopamine-Derived Guanidine Alkaloids from a Didemnidae Tunicate: Isolation, Synthesis, and Biological Activities.

Mellpaladines A-C (1-3) and dopargimine (4) are dopamine-derived guanidine alkaloids isolated from a specimen of Palauan Didemnidae tunicate as possible modulators of neuronal receptors. In this study, we isolated the dopargimine derivative 1-carboxydopargimine (5), three additional mellpaladines D-F (6-8), and serotodopalgimine (9), along with a dimer of serotonin, 5,5'-dihydroxy-4,4'-bistryptamine (10). The structures of these compounds were determined based on spectrometric and spectroscopic analyses. Compound 4 and its congeners dopargine (11), nordopargimine (15), and 2-(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)ethan-1-amine (16) were synthetically prepared for biological evaluations. The biological activities of all isolated compounds were evaluated in comparison with those of 1-4 using a mouse behavioral assay upon intracerebroventricular injection, revealing key functional groups in the dopargimines and mellpaladines for in vivo behavioral toxicity. Interestingly, these alkaloids also emerged during a screen of our marine natural product library aimed at identifying antiviral activities against dengue virus, SARS-CoV-2, and vesicular stomatitis Indiana virus (VSV) pseudotyped with Ebola virus glycoprotein (VSV-ZGP).

Function of the algicidal bacterium Pseudomonas sp. Go58 isolated from the biofilm on a water plant, and its active compounds, pyoluteorins.

In the development of applications to mitigate nuisance cyanobacterial blooms, environmentally friendly approaches have gained much attention. Recently, we found a bacterial strain Go58, which was isolated from the biofilm of a water plant, that showed potent algicidal activity against the bloom-forming cyanobacterium Microcystis aeruginosa. Whole genome sequencing of strain Go58 suggested that it is potentially a novel species closely related to Pseudomonas protegens. Pyoluteorins were obtained from the culture broth of this strain, and they demonstrated high toxicity against cultured cyanobacterial species, including M. aeruginosa and Anabaena cylindrica, but less toxicity against eukaryotic microalgae and other aquatic organisms. The production of pyoluteorin was enhanced by the presence of the target cyanobacterium. When a wild-caught microalgal consortium was treated with either strain Go58 or pyoluteorin, both efficiently suppressed the growth of harmful wild cyanobacteria, but promoted the growth of some specific eukaryotic microalgae. Since P. protegens is globally ubiquitous and highly anticipated to be a biopesticide for infectious diseases in the field of agriculture, the similar bacterial group identified in this study may also have potential as a safe on-site collectable biological countermeasure for controlling cyanobacterial blooms.

Diverse Aromatic Metabolites in the Solitary Tunicate Cnemidocarpa irene.

Fourteen aromatic metabolites (6-19) were isolated from an aqueous extract of the solitary tunicate Cnemidocarpa irene collected in Hokkaido, Japan. The structures of the metabolites were determined based on the spectroscopic interpretations, including one- and two-dimensional NMR, mass spectra, UV, and circular dichroism data. The biopterin analogue 10 modulated the behavior of mice after intracerebroventricular injection and showed a weak affinity to ionotropic glutamate receptor subtypes. Analyses of fluorescent coelomic fluid of the tunicate revealed that pterin 12 was responsible for the fluorescence of the blood cells, while ß-carbolines 1 and 3 were fluorescent compounds in the serum. The metabolic profiles in adults, juveniles, larvae, and eggs of the animal differed substantially, suggesting that the metabolism of the animal, especially biosynthesis of aromatic secondary metabolites, changes over different life stages.

Questiomycins, Algicidal Compounds Produced by the Marine Bacterium Alteromonas sp. D and Their Production Cue.

Questiomycin A (1) along with three new compounds, questiomycins C-E (2-4), were isolated from culture of Alteromonas sp. D, an algicidal marine bacterium, guided by algal lethality assay using the raphidophyte, Chattonella antiqua, one of the causative organisms of harmful algal bloom. The structures of 1-4 were assigned on the basis of their spectrometric and spectroscopic data. Compounds 1 to 4 exhibited algicidal activity against C. antiqua with LC50 values ranging from 0.18 to 6.37 M. Co-cultivation experiment revealed that 1 was produced only when the microalgae and the bacterium are in close contact, suggesting that some interactions between them trigger the biosynthesis of questiomycins. These results suggested that the algicidal bacteria such as Alteromonas sp. D can control microalgae chemically in marine ecosystem.

Cloning of the Bisucaberin B Biosynthetic Gene Cluster from the Marine Bacterium Tenacibaculum mesophilum, and Heterologous Production of Bisucaberin B.

The biosynthetic gene cluster for bisucaberin B (1, bsb gene cluster), an N-hydroxy-N-succinyl diamine (HSD)-based siderophore, was cloned from the marine bacterium Tenacibaculum mesophilum, originated from a marine sponge. The bsb gene cluster consists of six open reading frames (ORFs), in contrast to the four ORFs typically seen in biosynthetic gene clusters of the related molecules. Heterologous expression of the key enzyme, BsbD2, which is responsible for the final biosynthetic step of 1 resulted in production of bisucaberin B (1), but not bisucaberin (2) a macrocyclic counterpart of 1. To date, numbers of related enzymes producing macrocyclic analogues have been reported, but this work represents the first example of the HSD-based siderophore biosynthetic enzyme which exclusively produces a linear molecule rather than macrocyclic counterparts.

Elemental composition and ultrafine structure of the skeleton in shell-bearing protists-A case study of phaeodarians and radiolarians.

Cross-sections were prepared by ultramicrotome (UM) and focused ion beam (FIB) system in order to examine the skeletal structure of ecologically and geologically important shell-bearing protists: phaeodarians and radiolarians. The elemental composition of the skeleton was clarified by the energy dispersive X-ray spectroscopy, suggesting that the skeletons of both groups are mainly made of amorphous silica (SiO2·nH2O) with other minor elements (Na, Mg, Al, Cl, K, Ca and Fe) and that these two groups have similar elemental composition, compared with other siliceous organisms (diatoms and sponges). However, the structural difference among the two groups was confirmed: phaeodarian skeletons are porous, unlike radiolarians with solid skeletons. It was also revealed that the phaeodarian skeleton contains concentric layered structure with spaces, presumably related to the ontogenetic skeleton formation. The distinction in the skeletal ultrafine structure (porous/solid and non-dense/dense) would reflect the ecological difference among the two groups and could be an effective criterion to determine whether microfossils belong to Radiolaria or Phaeodaria. The UM and FIB combined method presented in this study could be a useful approach to examine the chemical and structural characteristics of unculturable and/or rare microorganisms.

KB343, a Cyclic Tris-guanidine Alkaloid from Palauan Zoantharian Epizoanthus illoricatus.

A new tris-guanidine alkaloid, KB343 (1), was isolated from the aqueous extract of a Palauan zoantharian, Epizoanthus illoricatus. The structure of 1 was determined on the basis of spectral analyses of 1 and its derivatives. The absolute configuration for 1 was determined upon comparison of the CD spectrum of 1 to those obtained from density functional theory calculations. The structure of 1 is highly unusual, as three guanidine groups are present in one ring system.

N-Methyl-ß-carbolinium Salts and an N-Methylated 8-Oxoisoguanine as Acetylcholinesterase Inhibitors from a Solitary Ascidian, Cnemidocarpa irene.

New brominated ß-carbolines irenecarbolines A (1) and B (4) along with known ß-carbolines 2 and 3 and a new 8-oxoisoguanine derivative, 5, were isolated from a solitary ascidian, Cnemidocarpa irene. The structures of these compounds were determined on the basis of their spectral data. All, except for 3, inhibited the action of acetylcholinesterase (AchE). The activities of 1 and 5 were comparable to those of galantamine, a clinically used AchE inhibitor. Compounds 1 and 2 were found to be present in high concentrations in blood, and fluorescence was observed in certain types of cells found in the blood of the tunicate.

Production of avaroferrin and putrebactin by heterologous expression of a deep-sea metagenomic DNA.

The siderophore avaroferrin (1), an inhibitor of Vibrio swarming that was recently identified in Shewanella algae B516, was produced by heterologous expression of the biosynthetic gene cluster cloned from a deep-sea sediment metagenomic DNA, together with two analogues, bisucaberin (2) and putrebactin (3). Avaroferrin (1) is a macrocyclic heterodimer of N-hydroxy-N-succinyl cadaverine (4) and N-hydroxy-N-succinyl-putrescine (5), whereas analogues 2 and 3 are homodimers of 4 and 5, respectively. Heterologous expression of two other related genes from culturable marine bacteria resulted in production of compounds 1-3, but in quite different proportions compared with production through expression of the metagenomic DNA.

Protoaculeine B, a putative N-terminal residue for the novel peptide toxin aculeines.

A new polyamine-modified indole derivative protoaculeine B (1) was isolated from Okinawan marine sponge Axinyssa aculeata. The structure of 1 was assigned on the basis of spectral data along with chemical transformations. Because the structure of 1 greatly inferred the N-terminal amino acid for highly modified peptide toxin aculeines, the probable structure for aculeine B was proposed on the basis of high-resolution mass spectral analysis.

Heterologous production of desferrioxamines with a fusion biosynthetic gene cluster.

Desferrioxamines E (1), D2 (2), X1 (3), and X2 (4), four macrocyclic N-hydroxy-N-succinyl diamine-based siderophores, were produced efficiently by heterologous expression of a fusion biosynthetic gene cluster. This expression system consisted of three genes (mbsA-C) from marine metagenomic DNA and one gene (dfoC(C)) from the terrestrial bacterium Erwinia amylovora. The first three genes are functional in the production of the common monomers N-hydroxy-N-succinyl cadaverine (5, HSC) and N-hydroxy-N-succinyl putrescine (6, HSP), whereas dfoC(C) catalyzes the oligomerization and the macrocyclization reactions of compounds 5 and 6 to form compounds 1-4. This fusion gene cluster system provides a convenient expression platform for various biosynthetic genes of HSC-HSP based siderophores by simply switching the fourth gene by the cassette process.

Bisucaberin B, a linear hydroxamate class siderophore from the marine bacterium Tenacibaculum mesophilum.

A siderophore, named bisucaberin B, was isolated from Tenacibaculum mesophilum bacteria separated from a marine sponge collected in the Republic of Palau. Using spectroscopic and chemical methods, the structure of bisucaberin B (1) was clearly determined to be a linear dimeric hydroxamate class siderophore. Although compound 1 is an open form of the known macrocyclic dimer bisucaberin (2), and was previously described as a bacterial degradation product of desferrioxamine B (4), the present report is the first description of the de novo biosynthesis of 1. To the best of our knowledge, compound 1 is the first chemically characterized siderophore isolated from a bacterium belonging to the phylum Bacteroidetes.

Phylogenetically novel LuxI/LuxR-type quorum sensing systems isolated using a metagenomic approach.

A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-L-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-L-homoserine lactone (C(12)-HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria.

Heterologous production of bisucaberin using a biosynthetic gene cluster cloned from a deep sea metagenome.

A siderophore biosynthetic gene cluster was cloned from a metagenomic library generated from deep sea sediment. The gene cluster was successfully expressed in Escherichia coli to produce bisucaberin, a siderophore originally reported from the marine bacterium Alteromonas haloplanktis. The cloned bisucaberin biosynthetic gene cluster was moderately similar to that of the known bisucaberin producer Vibrio salmonicida. However, the cloned gene cluster consists of four genes rather than three genes found in the V. salmonicida cluster. The low overall homology of the amino acid and nucleotide sequences with those of other species suggests that the cloned genes were derived from one of the unsequenced bacteria including uncultured species.

Cloning and heterologous expression of the vibrioferrin biosynthetic gene cluster from a marine metagenomic library.

A biosynthetic gene cluster of siderophore consisting of five open reading frames (ORFs) was cloned by functional screening of a metagenomic library constructed from tidal-flat sediment. Expression of the cloned biosynthetic genes in Escherichia coli led to the production of vibrioferrin, a siderophore originally reported for the marine bacterium Vibrio parahaemolyticus. To the best of our knowledge, this is the first example of heterologous production of a siderophore by biosynthetic genes cloned from a metagenomic library. The cloned cluster was one of the largest of the clusters obtained by functional screening. In this study, we demonstrated and extended the possibility of function-based metagenomic research.