Obligate Marine Actinomycete Research Articles

Microbe Profile: Salinispora tropica: natural products and the evolution of a unique marine bacterium.

Salinispora tropica was originally cultured from tropical marine sediments and described as the first obligate marine actinomycete genus. Soon after its discovery, it yielded the potent proteasome inhibitor salinosporamide A, a structurally novel natural product that is currently in phase III clinical trials for the treatment of cancer. If approved, it will be the first natural product derived from a cultured marine microbe to achieve clinical relevance. S. tropica produces many other biologically active natural products, including some linked to chemical defence, thus providing ecological context for their production. However, genomic analyses reveal that most natural product biosynthetic gene clusters remain orphan, suggesting that more compounds await discovery. The abundance of biosynthetic gene clusters in S. tropica supports the concept that the small molecules they encode serve important ecological functions, while their evolutionary histories suggest a potential role in promoting diversification. Better insights into the ecological functions of microbial natural products will help inform future discovery efforts.

Genome and metabolome mining of marine obligate Salinisporsatrains to discover new natural products.

Marine microorganisms are receiving more attention as a promising potential source of new natural products. In the present study, we performed genomic and metabolomic analyses to explore the metabolic potential of the obligate marine actinomycete genus Salinispora. The genomes of thirty Salinispora strains were prospected in search of biosynthetic gene clusters including polyketide synthase (PKS), nonribosomal peptide synthetase (NPRS), terpene, indole, lantibiotics, and siderophores. We determined considerable diversity of natural product biosynthetic gene clusters in their genome. There were a total of 1428 putative gene clusters involved in the biosynthesis of various bioactive natural products. Furthermore, 1509 ketosynthase (KS) and condensation (C) domains were detected by using NapDoS belonging to PKS and NRPS genes, respectively. Metabolic profiling was performed by a nontargeted LC-MS/MS approach combined with spectral networking using Global Natural Product Social Molecular Networking (GNPS). Dereplication and tentative identification of natural products were evaluated for common chemical properties and their associated pathways. Significant bioactive natural products such as lomaiviticin C, 7-OH-staurosporine, staurosporine, and cyanosporaside B were determined. More importantly, an unknown glycosylated compound associated with an NRPS/PKS-I hybrid gene cluster in Salinispora pacifica CNY703 was established through chemical and genomic analyses.

Corynebacterium alimapuense sp. nov., an obligate marine actinomycete isolated from sediment of Valparaíso bay, Chile.

A novel Gram-positive, non-motile, non-spore-forming and aerobic bacterium, designated strain VA37-3T, was isolated from a marine sediment sample collected at 19.2 m water depth from Valparaíso bay, Chile. Strain VA37-3T exhibits 97.6 % 16S rRNA gene sequence similarity to Corynebacterium marinum D7015T, 96.4 % to Corynebacterium humireducens MFC-5T and 96 % to Corynebacterium testudinoris M935/96/4T; and a rpoB gene sequence similarity of 85.1 % to Corynebacterium pollutisoli VMS11T, both analyses suggesting that strain VA37-3T represents a novel species of Corynebacterium. Physiological testing indicated that strain VA37-3T requires artificial sea water or sodium-supplemented media for growth, representing the first obligate marine actinomycete of the genus Corynebacterium. The genome of the proposed new species, along with the type strains of its most closely related species were sequenced and characterized. In silico genome-based similarity analyses revealed an ANIb of 72.8 % (C. marinum D7015T), ANIm of 85.0 % (Corynebacterium mustelae DSM 45274T), tetra of 0.90 (Corynebacterium callunae DSM 20147T) and ggdc of 24.7 % (Corynebacterium kutscheri DSM 20755T) when compared with the closest related strains. The genomic DNA G+C content of strain VA37-3T was 57.0 %. Chemotaxonomic assessment of strain VN6-2T showed the major fatty acids were C18 : 1ω9c and C16 : 0. Menaquinones predominantly consisted of MK-8(II-H2). Polar lipids consisted of diphosphatidylglycerol, glycolipids, phosphatidylglycerol, phosphoglycolipid and phosphatidylinositol. Mycolic acids also were present. Overall, the results from phylogenetic, phenotypic and genomic analyses confirmed that strain VA37-3T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium alimapuense sp. nov. is proposed, with VA37-3T as the type strain (=CCUG 69366T=NCIMB 15118T).

Ligand Based-Pharmacophore Modeling and Extended Bi oactivity Prediction for Salinosporamide A, B and C from Marine Actino mycetes Salinispora tropica.

Actinomycetes produce structurally unique secondary metabolites with pharmaceutically essential bioactivities. Salinispora, an obligate marine actinomycete, produces structurally varied and unique secondary metabolites. There is plenty of scope for development of drugs from the novel compounds isolated from Salinispora. Anticancer, antibacterial and anti-protozoa activities have been shown for Salinosporamides A, B and C, the secondary metabolites identified from Salinispora, which make them interesting subjects for further extended biological activity prediction. An in silico ligand based-pharmacophore approach was used for the prediction of extended biological targets for salinosporamide A, B and C. Pharmacophore models of salinosporamide A, B and C were generated individually and screened against known drug databases. The drugs with best fitness score were shortlisted, and their respective targets pertaining to their bioactivity were retrieved. The predicted biological drug targets were docked with salinosporamide A, B and C for validation. The glucocorticoid receptor and methionine aminopeptidase 2 showed good docking score and binding energy with salinosporamide A, B and C. Molecular dynamics studies of the protein-ligand complexes showed stable interactions suggesting that the predicted new targets for salinosporamides might be promising. The glucocorticoid receptor and methionine aminopeptidase 2 could be possible new drug targets of bioactivity of salinosporamides. These proteins could be the druggable targets for antiinflammatory and anticancer activity of salinosporamides.

Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining

An esterase gene, encoding a 325-amino-acid protein (SAestA), was mined form obligate marine actinomycete strain Salinispora arenicola CNP193 genome sequence. Phylogenetic analysis of the deduced amino acid sequence showed that the enzyme belonged to the family IV of lipolytic enzymes. The gene was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The molecular weight of His-tagged SAestA is ∼38kDa. SAestA-His6 was active in a temperature (5–40°C) and pH range (7.0–11.0), and maximal activity was determined at pH 9.0 and 30°C. The activity was severely inhibited by Hg2+, Cu2+, and Zn2+. In particular, this enzyme showed remarkable stability in presence of organic solvents (25%, v/v) with log P>2.0 even after incubation for 7 days. All these characteristics suggested that SAestA may be a potential candidate for application in industrial processes in aqueous/organic media.

Substrate specificity and biochemical characterization of an adenylation domain from an obligate marine actinomycete

Salinispora arenicola CNS-205 was a first-isolated obligate marine actinomycete. A gene (sare0357), annotated as ''amino acid adenylation domain'' located on the genome of Salinispora arenicola CNS-205, was cloned and characterized. The recombinant target protein Sare0357 was expressed in E. coli. Sare0357 specifically recognized and activated tryptophan (Trp) and phenylalanine (Phe). The basic kinetic parameters of Sare0357 for Trp were K m = 0.04 mM, V max = 2.1 μM/min, k cat = 14.2 min(-1), and for Phe were K m = 0.03 mM, V max = 1.6 μM/min, kcat = 10.4 min(-1). Our data elucidated Sare0357 biological role and biochemical properties as a Trp and Phe-activating adenylation domain.

Streptomyces oceani sp. nov., a new obligate marine actinomycete isolated from a deep-sea sample of seep authigenic carbonate nodule in South China Sea

A novel aerobic actinomycete strain, designated as SCSIO 02100(T), was isolated from a deep sea sediment sample collected from Northern South China Sea at a depth of 578 m. This isolate requires sea water or a sodium-supplemented medium for growth. BLAST searches based on the almost full length of the 16S rRNA gene sequence, showed that strain SCSIO 02100(T) had the highest similarities with Streptomyces armeniacus (JCM 3070(T)) (97.1 %). Phylogenetic trees reconstructed on the basis of 16S rRNA gene sequences revealed that strain SCSIO 02100(T) formed a distinct lineage with S. nanshensis SCSIO 01066(T) with 96.9 % similarity. Further analysis of the polyphasic taxonomic data, including morphological, phenotypic and chemotaxonomic properties, showed that strain SCSIO 02100(T) could be readily distinguished from the most closely related members of the genus Streptomyces. Thus, based on the polyphasic taxonomic data, a novel species, Streptomyces oceani sp. nov., is proposed, with the type strain SCSIO 02100(T) (=DSM 42043(T) = CGMCC 4.7007(T)).

Identification of Sare0718 As an Alanine-Activating Adenylation Domain in Marine Actinomycete Salinispora arenicola CNS-205

BackgroundAmino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters.Methodology/Principal FindingsIn order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated “amino acid adenylation domain”. Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are Km = 0.1164±0.0159 (mM), Vmax = 3.1484±0.1278 (µM/min), kcat = 12.5936±0.5112 (min−1).Conclusions/SignificanceBy revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on.

Comparative genomics reveals evidence of marine adaptation in Salinispora species

BackgroundActinobacteria represent a consistent component of most marine bacterial communities yet little is known about the mechanisms by which these Gram-positive bacteria adapt to life in the marine environment. Here we employed a phylogenomic approach to identify marine adaptation genes in marine Actinobacteria. The focus was on the obligate marine actinomycete genus Salinispora and the identification of marine adaptation genes that have been acquired from other marine bacteria.ResultsFunctional annotation, comparative genomics, and evidence of a shared evolutionary history with bacteria from hyperosmotic environments were used to identify a pool of more than 50 marine adaptation genes. An Actinobacterial species tree was used to infer the likelihood of gene gain or loss in accounting for the distribution of each gene. Acquired marine adaptation genes were associated with electron transport, sodium and ABC transporters, and channels and pores. In addition, the loss of a mechanosensitive channel gene appears to have played a major role in the inability of Salinispora strains to grow following transfer to low osmotic strength media.ConclusionsThe marine Actinobacteria for which genome sequences are available are broadly distributed throughout the Actinobacterial phylogenetic tree and closely related to non-marine forms suggesting they have been independently introduced relatively recently into the marine environment. It appears that the acquisition of transporters in Salinispora spp. represents a major marine adaptation while gene loss is proposed to play a role in the inability of this genus to survive outside of the marine environment. This study reveals fundamental differences between marine adaptations in Gram-positive and Gram-negative bacteria and no common genetic basis for marine adaptation among the Actinobacteria analyzed.

Salinipyrones and Pacificanones, Mixed-Precursor Polyketides from the Marine Actinomycete Salinispora pacifica

Chemical examination of a phylogenetically unique strain of the obligate marine actinomycete Salinispora pacifica led to the discovery of four new polyketides, salinipyrones A and B ( 1, 2) and pacificanones A and B ( 3, 4). These compounds appear to be derived from a mixed-precursor polyketide biosynthesis involving acetate, propionate, and butyrate building blocks. Spectral analysis, employing NMR, IR, UV, and CD methods and chemical derivatization, was used to assign the structures and absolute configurations of these new metabolites. Salinipyrones A and B displayed exactly opposite CD spectra, indicating their pseudoenantiomeric relationship. This relationship was shown to be a consequence of the geometric isomerization of one double bond. The phenomenon of polyketide module skipping is proposed to explain the unusual biosynthesis of the salinipyrones and the pacificanones.

Arenicolides A—C, 26‐Membered Ring Macrolides from the Marine Actinomycete Salinispora arenicola.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Arenicolides A−C, 26-Membered Ring Macrolides from the Marine Actinomycete Salinispora arenicola

Chemical evaluation of the saline fermentation broth of several strains of the obligate marine actinomycete Salinispora arenicola has led to the identification of three new macrolide polyketides designated arenicolides A-C (1-3). The planar structures, elucidated via spectroscopic and chemical methods, consist of 26-membered polyunsaturated macrolactones containing repeating vicinal hydroxyl methoxyl moieties. The relative and absolute stereochemistries of 1-3 were assigned by a combination of J-based configurational analyses and chemical derivatization.

Chemical Biology

Chemical Biology

Cinnabaramides A−G: Analogues of Lactacystin and Salinosporamide from a Terrestrial Streptomycete

The cinnabaramides A-G (1-7) were isolated from a terrestrial strain of Streptomyces as potent and selective inhibitors of the human 20S proteasome. Their chemical and biological properties resemble those of salinosporamide A, a recently identified lead compound from an obligate marine actinomycete, which is currently under development as an anticancer agent. Cinnabaramides F and G (6, 7) combine essential structural features of salinosporamide A and lactacystin and show about equal potency in vitro, with IC50 values in the 1 nM range. The properties and phylogenetic position of the producer organism, the production and isolation of compounds 1-7, their structure elucidation by MS and NMR, and their biological activities are reported. Additionally, an X-ray crystal structure was obtained from cinnabaramide A (1).

Effects of Halogens on the Production of Salinosporamides by the Obligate Marine Actinomycete Salinispora tropica

We examined the effects of halogens on the production of salinosporamide A (NPI-0052) by the obligate marine actinomycete Salinispora tropica NPS465, specifically the production of analogs containing halogens other than chlorine. Adding NaF, NaBr and NaI directly to the production medium prepared in seawater containing -3% NaCl did not induce the production of the corresponding analogs. Replacing seawater with 2-3% NaI in the production medium enhanced the production of NPI-0052 by 2.1 fold. Replacing seawater with 2-3% NaBr in the production medium suppressed the production of NPI-0052 but induced the production of a brominated analog at very low yield. Using a stepwise enrichment of bromide in the seed cultures in order to reduce the chloride ion carried over to the production medium, the production of the brominated analog was enhanced by 4 fold. We also demonstrated that the growth of this obligate marine actinomycete is dependent upon sodium concentration, not chloride concentration.

Cyanosporasides A and B, Chloro- and Cyano-cyclopenta[a]indene Glycosides from the Marine Actinomycete “Salinispora pacifica”

[structure: see text] Two structurally novel cyclopenta[a]indene glycosides, cyanosporasides A and B (1 and 2) have been isolated from the culture broth of a new species of the obligate marine actinomycete genus Salinispora. The structures and absolute stereochemistries of these compounds were determined by spectral and chemical methods. The cyanosporasides possess a new 3-keto-pyranohexose sugar as well as a cyano- and chloro-substituted cyclopenta[a]indene ring system. The cyanosporasides are proposed to be cyclization products of an enediyne precursor.

New Cytotoxic Salinosporamides from the Marine Actinomycete Salinispora tropica.

AbstractFor Abstract see ChemInform Abstract in Full Text.

New Cytotoxic Salinosporamides from the Marine Actinomycete Salinispora tropica

An extensive study of the secondary metabolites produced by the obligate marine actinomycete Salinispora tropica (strain CNB-392), the producing microbe of the potent proteasome inhibitor salinosporamide A (1), has led to the isolation of seven related gamma-lactams. The most important of these compounds were salinosporamide B (3), which is the deschloro-analogue of 1, and salinosporamide C (4), which is a decarboxylated pyrrole analogue. New SAR data for all eight compounds, derived from extensive testing against the human colon carcinoma HCT-116 and the 60-cell-line panel at the NCI, indicate that the chloroethyl moiety plays a major role in the enhanced activity of 1.