Paenibacillus spongiae sp. nov. isolated from deep-water marine sponge Theonella swinhoei.

A novel bacterial strain, designated as PHS-Z3T, was isolated from a marine sponge belonging to the genus Theonella on the Puerto Galera Deep Monkey, Philippines. Cells of PHS-Z3T were Gram-stain-positive, motile, oxidase- and catalase-positive, white-pigmented, spore-forming, short rods that could grow at 10-40 °C (optimum, 20 °C), pH 6.0-9.5 (optimum, pH 7.5) and with 2-16 % (w/v) NaCl (optimum, 7 %). The 16S rRNA gene sequence of PHS-Z3T showed 97.9 %, 96.7 %, and 96.2 % identities to Paenibacillus mendelii C/2T, Paenibacillus oenotherae DT7-4T and Paenibacillus aurantiacus RC11T, respectively. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that PHS-Z3T formed an independent cluster with Paenibacillus mendelii C/2T. The total genome of PHS-Z3T was approximately 7 613 364 bp in size with a DNA G+C content of 51.6 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between PHS-Z3T and other type strains of species of the genus Paenibacillus were 68.0-81.4 % [ANI by blast (ANIb)], 83.0-88.0 % [ANI by MUMmer (ANIm)] and 12.7-32.1 % (dDDH). The dDDH and ANI values were below the standard cut-off criteria for delineation of bacterial species. The percentage of conserved proteins (POCP) values between the genome of PHS-Z3T and those of members of the genus Paenibacillus were 39.7-75.7 %, while the average amino acid identity (AAI) values were 55.9-83.7 %. The sole respiratory quinone in the strain was MK-7, and the predominant fatty acids were anteiso-C15 : 0 and C16 : 0. The major polar lipids of PHS-Z3T consisted of diphosphatidylglycerol, phospholipid and phosphatidylglycerol. The characteristic amino acid in the cell wall of PHS-Z3T was diamino heptanoic acid (meso-DAP). On the basis of the molecular, physiological, biochemical and chemotaxonomic features, strain PHS-Z3T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus spongiae sp. nov. is proposed, with the type strain PHS-Z3T (=MCCC 1K07848T=KCTC 43443T).

Cyclotheonellazoles D-I, Potent Elastase Inhibitory Thiazole-Containing Cyclic Peptides from Theonella sp. (2131).

Six new thiazole-containing cyclic peptides, the cyclotheonellazoles D-I (1-6), were isolated from the Australian marine sponge Theonella sp. (2131) with their structures assigned by comprehensive 1D and 2D NMR spectroscopic and MS spectrometric analyses, Marfey's derivatization studies, and comparison with time-dependent density functional theory (TDDFT) calculated ECD data. The Type 2 azole-homologated peptides herein comprise up to five nonproteinogenic amino acids, including the protease transition state mimic α-keto-ß-amino acid residue 3-amino-4-methyl-2-oxohexanoic acid (Amoha), while 1-3 also contain a terminal hydantoin residue not previously found in cyclotheonellazoles. The keramamides A (7) and L (8) were reisolated affording expanded exploration of their biological activities. The peptides were examined for protease inhibitory activities against two mammalian serine proteases (elastase and chymotrypsin) and SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), a validated antiviral therapeutic target for COVID-19. Peptides 1-6 and keramamide A (7) displayed potent nanomolar inhibition of elastase (IC50 16.0 to 61.8 nM), while 7 also contained modest inhibition of chymotrypsin and SARS-CoV-2 3CLpro (IC50 0.73 and 1.1 µM, respectively). The cyclotheonellazoles D-E (1-3) do not affect the viability of human breast, ovarian, and colon cancer cells (>100 µM), with the cytotoxicity previously reported for keramamide L (8) not replicated (inactive >20 µM).

Theonella: A Treasure Trove of Structurally Unique and Biologically Active Sterols.

The marine environment is considered a vast source in the discovery of structurally unique bioactive secondary metabolites. Among marine invertebrates, the sponge Theonella spp. represents an arsenal of novel compounds ranging from peptides, alkaloids, terpenes, macrolides, and sterols. In this review, we summarize the recent reports on sterols isolated from this amazing sponge, describing their structural features and peculiar biological activities. We also discuss the total syntheses of solomonsterols A and B and the medicinal chemistry modifications on theonellasterol and conicasterol, focusing on the effect of chemical transformations on the biological activity of this class of metabolites. The promising compounds identified from Theonella spp. possess pronounced biological activity on nuclear receptors or cytotoxicity and result in promising candidates for extended preclinical evaluations. The identification of naturally occurring and semisynthetic marine bioactive sterols reaffirms the utility of examining natural product libraries for the discovery of new therapeutical approach to human diseases.

Metagenomic and metatranscriptomic exploration of the Egyptian Red Sea sponge Theonella sp. associated microbial community.

Marine sponges associated microorganisms are considered to be prolific source of bioactive natural products. Omics-based techniques such as metagenomics and metatranscriptomics have been used as effective tools to discover natural products. In this study, we used integrated metagenomic and metatranscriptomic analysis of three samples of the Egyptian Red Sea sponge Theonella sp. microbiome to obtain a complete picture of its biosynthetic activity to produce bioactive compounds. Our data revealed high biodiversity of the Egyptian sponge microbiota represented by 38 bacterial phyla with Candidate Phylum Poribacteria as the most abundant phyla with an average of 27.5% of the microbial community. The analysis also revealed high biosynthetic activity of the sponge microbiome through detecting different types of biosynthetic gene clusters (BGCs) with predicted antibacterial, cytotoxic and inhibitory bioactivity and the majority of these clusters were found to be actively transcribed. The complete BGCs of the cytotoxic theonellamide and misakinolide were detected and found to be actively transcribed. The majority of the detected BGCs were predicted to be novel as they did not show any similarity with any known cluster in the MIBiG database.

Two Onnamide Analogs from the Marine Sponge Theonella conica: Evaluation of Geometric Effects in the Polyene Systems on Biological Activity.

Two previously unreported onnamide analogs, 2Z- and 6Z-onnamides A (1 and 2), were isolated from the marine sponge Theonella conica collected at Amami-Oshima Is., Kagoshima Prefecture, Japan. Structures of compounds 1 and 2 were elucidated by spectral analysis. Structure-activity relationships (SARs) for effects on histone modifications and cytotoxicity against HeLa and P388 cells were characterized. The geometry in the polyene systems of onnamides affected the histone modification levels and cytotoxicity.

New Theonellapeptolides from Indonesian Marine Sponge Theonella swinhoei as Anti-Austerity Agents.

We reported three new members of the theonellapeptolide family from theonellapeptolide II series, namely theonellapeptolides IIb (1), IIa (2), IIc (3), and three known members-IId (4), IIe (5), and Id (6)-from Kodingarengan marine sponge Theonella swinhoei collected in Makassar, Indonesia. The structures of tridecadepsipeptides 1-3, including the absolute configurations of their amino acids, were determined by the integrated NMR and tandem MS analyses followed by Marfey's analysis. To the best of our knowledge, 1 and 2 are the first theonellapeptolide-type compounds to have a valine residue with D configuration at residue position 6. The isolated theonellapeptolide-type compounds 1-6 showed selective cytotoxic activity against human pancreatic MIA PaCa-2 cancer cells in a nutrient-deprived medium. Among them, the most potent preferential cytotoxicity was observed in new theonellapeptolide IIc (3) and known IId (4), IIe (5), and Id (6).

Arsenate reducing bacteria isolated from the marine sponge Theonella swinhoei: Bioremediation potential.

Arsenic (As) contamination of freshwater resources constitutes a major environmental issue affecting over 200 million people worldwide. Although the use of microorganisms for the bioremediation of As has been well studied, only very few candidates have been identified to date. Here, we investigated bacteria associated with the Red Sea sponge Theonella swinhoei and their potential to reduce As in a low-salinity liquid medium. This Indo-Pacific common sponge has been shown to hyper-accumulate As, at an average concentration of 8600 mg/g-1 in an environment uncontaminated by arsenic or barium. Four isolated strains of bacteria exhibited arsenic reduction potential by transforming inorganic As in the form of arsenate (iAsV) to arsenite (iAsIII). Two of these isolates were identified as Alteromonas macleodii and Pseudovibrio ascidisceicola, and the other two isolates, both belonging to the same species, were identified as Pseudovibrio denitrificans. The four isolates were then cultured in a low-salinity iAsV-rich medium (5 mM) and As concentration was measured over time using a specifically designed high-performance liquid chromatograph coupled to a mass spectrometer (HPLC-MS). Out of the four isolates, A. macleodii and P. ascidisceicola grew successfully in a low-salinity liquid medium and reduced AsV to AsIII at an average rate of 0.094 and 0.083 mM/h, respectively, thereby demonstrating great potential for the bioremediation of As-contaminated groundwater.

Theonellamides J and K and 5-cis-Apoa-theopalauamide, Bicyclic Glycopeptides of the Red Sea Sponge Theonella swinhoei.

Theonella swinhoei is a fairly common inhabitant of reefs throughout the Indian and Pacific Oceans. Metabolomic analyses of samples of T. swinhoei collected in different depths in the Gulf of Aqaba revealed two chemotypes differing in the profiles of the theonellamides they produce, some of which seem to be unknown. Driven by this finding, we examined a sample of T. swinhoei collected more than 40 years ago in the southern part of the Gulf of Aqaba. Large-scale extract of this sample yielded four theonellamides, the known theopalauamide (4), as the major component, and three new metabolites, theonellamide J (1), 5-cis-Apoa-theopalauamide (2), and theonellamide K (3), as the minor components. The planar structure of these complex cyclic glycopeptides was elucidated by combination of 1D and 2D NMR techniques and HRESIMS. The absolute configuration of the amino acids was established by Marfey's and advanced Marfey's methods, and the absolute configuration of its galactose unit using "Tanaka's method" for monosaccharides. The biological activity of the pure compounds was tested for antibacterial activity and for cytotoxicity to HTC-116 cell line. The compounds presented significant cytotoxicity against the HTC-116 cell line, illuminating the importance of the Apoa subunit for the activity.

Oxygenated Theonellastrols: Interpretation of Unusual Chemical Behaviors Using Quantum Mechanical Calculations and Stereochemical Reassignment of 7α-Hydroxytheonellasterol.

A total of eight new oxygenated 4-exo-methylene sterols, 1-8, together with one artifact 9 and six known sterols 11-16, were isolated from the marine sponge Theonella swinhoei collected from the Bohol province in Philippines. Structures of sterols 1-8 were determined from 1D and 2D NMR data. Among the sterols, 8α-hydroxytheonellasterol (4) spontaneously underwent an allylic 1,3-hydroxyl shift to produce 15α-hydroxytheonellasterol (9) as an artifact; this was rationalized by quantum mechanical calculations of the transition state. In addition, the 1,2-epoxy alcohol subunit of 8α-hydroxy-14,15-ß-epoxytheonellasterol (5) was assigned using the Gauge-Independent Atomic Orbital (GIAO) NMR chemical shift calculations and subsequent DP4+ analysis. Finally, comparison of the 13C chemical shifts of isolated 7α-hydroxytheonellasterol (6) with the reported values revealed significant discrepancies at C-6, C-7, C-8, and C-14, leading to reassignment of the C-7 stereochemistry in the known structure.

Swinhopeptolides A and B: Cyclic Depsipeptides from the Sponge Theonella swinhoei That Inhibit Ras/Raf Interaction.

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.

Characterization of an Orphan Type†III Polyketide Synthase Conserved in Uncultivated "Entotheonella" Sponge Symbionts.

Uncultivated bacterial symbionts from the candidate genus "Entotheonella" have been shown to produce diverse natural products previously attributed to their sponge hosts. In addition to these known compounds, "Entotheonella" genomes contain rich sets of biosynthetic gene clusters that lack identified natural products. Among these is a small type III polyketide synthase (PKS) cluster, one of only three clusters present in all known "Entotheonella" genomes. This conserved "Entotheonella" PKS (cep) cluster encodes the type III PKS CepA and the putative methyltransferase CepB. Herein, the characterization of CepA as an enzyme involved in phenolic lipid biosynthesis is reported. In vitro analysis showed a specificity for alkyl starter substrates and the production of tri- and tetraketide pyrones and tetraketide resorcinols. The conserved distribution of the cep cluster suggests an important role for the phenolic lipid polyketides produced in "Entotheonella" variants.

Investigations into PoyH, a promiscuous protease from polytheonamide biosynthesis.

Polytheonamides are the most extensively modified ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) currently known. In RiPP biosynthesis, the processed peptide is usually released from a larger precursor by proteolytic cleavage to generate the bioactive terminal product of the pathway. For polytheonamides, which are members of a new RiPP family termed proteusins, we have recently shown that such cleavage is catalyzed by the cysteine protease PoyH acting on the precursor PoyA, both encoded in the polytheonamide biosynthetic gene cluster. We now report activity for PoyH under a variety of reaction conditions for different maturation states of PoyA and demonstrate a potential use of PoyH as a promiscuous protease to liberate and characterize RiPPs from other pathways. As a proof of concept, the identified recognition motif was introduced into precursors of the thiopeptide thiocillin and the lanthipeptide lichenicidin VK1, allowing for their site-specific cleavage with PoyH. Additionally, we show that PoyH cleavage is inhibited by PoyG, a previously uncharacterized chagasin-like protease inhibitor encoded in the polytheonamide gene cluster.

Colony-wise Analysis of a Theonella swinhoei Marine Sponge with a Yellow Interior Permitted the Isolation of Theonellamide I.

There are several examples of marine organisms whose metabolic profiles differ among conspecifics inhabiting the same region. We have analyzed the metabolic profile of each colony of a Theonella swinhoei marine sponge with a yellow interior and noticed the patchy distribution of one metabolite. This compound was isolated and its structure was studied by a combination of spectrometric analyses and chemical degradation, showing it to be a congener in the theonellamide class of bicyclic peptides. Theonellamides had previously been isolated by us only from T. swinhoei with a white interior and not from those with a yellow interior.

Swinhoeisterols from the South China Sea Sponge Theonella swinhoei.

Swinhoeisterols C-F (1-4), four new steroids having a rearranged 6/6/5/7 ring system, were isolated from the Xisha sponge Theonella swinhoei, together with the known analogue swinhoeisterol A (5). Their structures were determined based on spectroscopic analysis, TDDFT-ECD and optical rotation calculations, and biogenetic correlations. In an in vitro assay, compound 1 showed an inhibitory effect on (h)p300 with an IC50 value of 8.8 µM, whereas compounds 2-4 were not active.

New 4-methylidene sterols from the marine sponge Theonella swinhoei.

A series of 4-methylidene sterols including three new compounds 1-3, were isolated from the marine sponge Theonella swinhoei. The structures of new compounds were determined on the basis of spectroscopic analyses. Compounds 3, 5, and 6 showed cytotoxicities against U937, MCF-7, and PC-9 cancer cells with IC50 in the range of 1.6-8.8 µM. The new compound 3 exhibited remarkable proapoptotic activity in breast cancer cells. Mechanically, 3 significantly triggered reactive oxygen species (ROS) accumulation resulting in apoptosis and DNA damage in breast cancer cells.

Single-bacterial genomics validates rich and varied specialized metabolism of uncultivated Entotheonella sponge symbionts.

Marine sponges are prolific sources of unique bioactive natural products. The sponge Theonella swinhoei is represented by several distinct variants with largely nonoverlapping chemistry. For the Japanese chemotype Y harboring diverse complex polyketides and peptides, we previously provided genomic and functional evidence that a single symbiont, the filamentous, multicellular organism "Candidatus Entotheonella factor," produces almost all of these compounds. To obtain further insights into the chemistry of "Entotheonella," we investigated another phylotype, "Candidatus Entotheonella serta," present in the T. swinhoei WA sponge chemotype, a source of theonellamide- and misakinolide-type compounds. Unexpectedly, considering the lower chemical diversity, sequencing of individual bacterial filaments revealed an even larger number of biosynthetic gene regions than for Ca E. factor, with virtually no overlap. These included genes for misakinolide and theonellamide biosynthesis, the latter assigned by comparative genomic and metabolic analysis of a T. swinhoei chemotype from Israel, and by biochemical studies. The data suggest that both compound families, which were among the earliest model substances to study bacterial producers in sponges, originate from the same bacterium in T. swinhoei WA. They also add evidence that metabolic richness and variability could be a more general feature of Entotheonella symbionts.

Synthesis and evaluation of the mutagenicity of 3-alkylpyridine marine alkaloid analogues with anticancer potential.

Theonella sp is an important source of biologically-active 3-alkylpyridine alkaloids (3-APAs) that has shown a wide variety of promising biological effects. In the present work, two new 3-APAs analogues were synthesized based on molecular modeling studies to act as potential antimalarial agents. These theoneladin C analogues, containing the thiocyanate group in their chemical structures, were synthesized and evaluated against Plasmodium falciparum (IC50 values ranging from 2.3 to 5.5µM). The structural and energetic analysis demonstrated a high chemical affinity of the two analogues for their target, the heme group. However, despite the good antimalarial activity, the compounds exhibited high cytotoxicity and a lack of selectivity for human cell lines. These findings prompted us to evaluate the cytotoxicity of these compounds against human cancer cell lines. In order to better understand the mechanisms responsible for the toxicity, a variety of genotoxicity assays were performed in vitro. One of the compounds assayed presented an interesting selectivity and high toxicity to the human colon cancer cell line RKO-AS45-1. In addition, the results of the micronucleus assay, comet assay, Ames assay and annexin-V/propidium iodide staining showed that the synthetic alkaloids were able to induce chromosomal mis-segregation and trigger cell death by apoptosis. These results demonstrate that the compounds assessed herein may be promising prototypes of anticancer chemotherapeutic agents.

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA.

Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin-dependent halogenase with a broad substrate specificity, with potential as a useful new biocatalytic tool, is described.

Cyclotheonellazoles A-C, Potent Protease Inhibitors from the Marine Sponge Theonella aff. swinhoei.

The extract of a sample of the sponge Theonella aff. swinhoei collected in Madagascar exhibited promising in vitro antiplasmodial activity. The antiplasmodial activity was ascribed in part to the known metabolite swinholide A. Further investigation of the extract afforded three unusual cyclic peptides, cyclotheonellazoles A-C (1-3), which contain six nonproteinogenic amino acids out of the eight acid units that compose these natural products. Among these acids the most novel were 4-propenoyl-2-tyrosylthiazole and 3-amino-4-methyl-2-oxohexanoic acid. The structure of the compounds was elucidated by interpretation of the 1D and 2D NMR data, HRESIMS, and advanced Merfay's techniques. The new compounds were found to be nanomolar inhibitors of chymotrypsin and sub-nanomolar inhibitors of elastase, but did not present antiplasmodial activity.

Sponge-associated bacteria mineralize arsenic and barium on intracellular vesicles.

Arsenic and barium are ubiquitous environmental toxins that accumulate in higher trophic-level organisms. Whereas metazoans have detoxifying organs to cope with toxic metals, sponges lack organs but harbour a symbiotic microbiome performing various functions. Here we examine the potential roles of microorganisms in arsenic and barium cycles in the sponge Theonella swinhoei, known to accumulate high levels of these metals. We show that a single sponge symbiotic bacterium, Entotheonella sp., constitutes the arsenic- and barium-accumulating entity within the host. These bacteria mineralize both arsenic and barium on intracellular vesicles. Our results indicate that Entotheonella sp. may act as a detoxifying organ for its host.