Mechanism of action of pseudopteroxazole and pseudopterosin G: Diterpenes from marine origin.

Pseudopteroxazole (Ptx) and the pseudopterosins are marine natural products with promising antibacterial potential. While Ptx has attracted interest for its antimycobacterial activity, pseudopterosins are active against several clinically relevant pathogens. Both compound classes exhibit low cytotoxicity and accessibility to targeted synthesis, yet their antibacterial mechanisms remain elusive. In this study, we investigated the modes of action of Ptx and pseudopterosin G (PsG) in Bacillus subtilis employing an unbiased approach that combines gel-based proteomics with a mathematical similarity analysis of response profiles. Proteomic responses to sublethal concentrations of Ptx and PsG were compared to a library of antibiotic stress response profiles revealing that both induce a stress response characteristic for agents targeting the bacterial cell envelope by interfering with membrane-bound steps of cell wall biosynthesis. Microscopy-based assays confirmed that both compounds compromise the integrity of the bacterial cell wall without disrupting the membrane potential. Furthermore, LC-MSE analysis showed that the greater potency of PsG against B. subtilis, reflected in a lower MIC and a more pronounced proteomic response, may be rooted in a more effective association with and penetration of B. subtilis cells. We conclude that Ptx and PsG target the integrity of the gram-positive cell wall.

Metabolomic Comparison and Assessment of Co-cultivation and a Heat-Killed Inducer Strategy in Activation of Cryptic Biosynthetic Pathways.

Co-cultivation has been used as a promising tool to turn on or up-regulate cryptic biosynthetic pathways for microbial natural product discovery. Recently, a modified culturing strategy similar to co-cultivation was investigated, where heat-killed inducer cultures were supplemented to the culture medium of producer fermentations to induce cryptic pathways. In the present study, the repeatability and effectiveness of both methods in turning on cryptic biosynthetic pathways were unbiasedly assessed using UHPLC-HRESIMS-based metabolomics analysis. Both induction methods had good repeatability, and they resulted in very different induced metabolites from the tested producers. Co-cultivation generated more induced mass features than the heat-killed inducer cultures, while both methods resulted in the induction of mass features not observed using the other induction method. As examples, pathways leading to two new natural products, N-carbamoyl-2-hydroxy-3-methoxybenzamide (1) and carbazoquinocin G (5), were induced and up-regulated through co-culturing a producer Streptomyces sp. RKND-216 with inducers Alteromonas sp. RKMC-009 and M. smegmatis ATCC 120515, respectively.

Guanahanolide A, a Meroterpenoid with a Sesterterpene Skeleton from Coral-Derived Streptomyces sp.

A meroterpenoid, guanahanolide A (1), was purified from a fermentation extract of Streptomyces sp. RKBH-B7. The planar structure of guanahanolide A (1) was elucidated by NMR spectroscopy, revealing a meroterpenoid comprised of an unprecedented sesterterpene skeleton. Upon determination of the relative configuration of 1 through X-ray crystallography, its absolute configuration was unambiguously assigned using Mosher ester analysis. Guanahanolide A (1) showed moderate cytotoxicity against human cancer cell lines MCF-7, HTB-26, and HCT-116.

Discovery of an Isothiazolinone-Containing Antitubercular Natural Product Levesquamide.

Antitubercular agent levesquamide is a new polyketide-nonribosomal peptide (PK-NRP) hybrid marine natural product isolated from Streptomyces sp. RKND-216. The structure contains a rare isothiazolinone moiety which has only been reported in collismycin SN. Structure elucidation by NMR spectroscopy was a significant challenge due to a deficiency of protons in this aromatic moiety. Therefore, the genome of Streptomyces sp. RKND-216 was sequenced to identify the levesquamide biosynthetic gene cluster (BGC). Analysis of the BGC provided structural insights and guided stable-isotope labeling experiments, which led to the assignment of the fused pyridine-isothiazolinone moiety. The BGC and the labeling experiments provide further insights into the biosynthetic origin of isothiazolinones. Levesquamide exhibited antimicrobial activity in the microplate alamarBlue assay (MABA) and low oxygen recovery assay (LORA) against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 9.65 and 22.28 µM, respectively. Similar activity was exhibited against rifampicin- and isoniazid-resistant M. tuberculosis strains with MIC values of 9.46 and 9.90 µM, respectively. This result suggests levesquamide has a different mode of action against M. tuberculosis compared to the two first-line antitubercular drugs rifampicin and isoniazid. Furthermore, levesquamide shows no cytotoxicity against the Vero cell line, suggesting it may have a useful therapeutic window.

An Ichip-Domesticated Sponge Bacterium Produces an N-Acyltyrosine Bearing an α-Methyl Substituent.

The ichip (isolation chip) was employed for the first time in a marine sponge (Xestospongia muta), and a putatively new bacterial species, Alteromonas sp. RKMC-009, was isolated. Strain RKMC-009 produces a novel N-acyltyrosine (1) that is appended with a rare α-methyl substituent within the aminoacyl moiety and also exhibits Gram-positive antibacterial activity. We determined through an SAR experiment that the α-methyl is necessary for Staphylococcus activity of 1 and that it enhances Enterococcus activity.

Discovery of Primarolides A and B from Marine Fungus Asteromyces cruciatus Using Osmotic Stress and Treatment with Suberoylanilide Hydroxamic Acid.

Advances in whole-genome sequencing of many fungal species has revealed the presence of numerous "silent" biosynthetic genes, highlighting their potential to produce a wide variety of natural products. These silent biosynthetic genes are regulated in part by their highly condensed chromatin structure, which can be modified to allow transcription in response to external stimuli. In this study, Asteromyces cruciatus was subjected to both epigenetic modification and osmotic stress to enhance the production of new natural products. This "cooperative induction" strategy led to the isolation and characterization of two new polyketides from a fermentation of A. cruciatus treated with suberoylanilide hydroxamic acid and sodium chloride. The metabolic profiles of the control and treated samples were assessed using ultra-high performance liquid chromatography high-resolution electrospray ionization mass spectrometry (UHPLC-HRESIMS) metabolomic analysis, highlighting the upregulation of two new polyketides, primarolides A and B. These compounds were purified using reversed-phase flash chromatography followed by high-performance liquid chromatography, and their planar structures were established using NMR spectroscopy.

Fusaristatin C, a Cyclic Lipodepsipeptide from Pithomyces sp. RKDO 1698.

A new cyclic lipodepsipeptide, fusaristatin C (1), was obtained from the fungus Pithomyces sp. RKDO 1698, which was isolated from the Caribbean octocoral Eunicea fusca. The 2D structure of fusaristatin C was elucidated using NMR spectroscopy and mass spectrometry, while the absolute configuration of the sole chiral amino acid residue (l-serine) was determined using Marfey's method. 3-Hydroxy-2,11-dimethyltetradecanoic acid (HDMT) was cleaved from 1, and the absolute configuration at the C-3 position was determined using Mosher's ester analysis. Subsequent J-based configuration analysis of 1 allowed for assignment of the C-2 configuration. Fusaristatin C exhibited no antimicrobial activity or cytotoxicity.

Isolation of Imaqobactin, an Amphiphilic Siderophore from the Arctic Marine Bacterium Variovorax Species RKJM285.

The amphiphilic siderophore imaqobactin was isolated from the Arctic bacterium Variovorax sp. RKJM285, a strain isolated from marine sediment collected from an inlet near Clyde River, Nunavut, Canada. The 2D structure of imaqobactin was determined by a combination of LC-HRMS, MS/MS, and NMR spectroscopic methods. The absolute configuration of the depsipeptide core was determined by Marfey's analysis, and the relative configuration of the 4,7-diamino-3-hydroxy-2-methylheptanoic acid moiety was determined by NOESY and selective NOE experiments. The photoreductive properties of imaqobactin were tested and are discussed. Initial tests for antimicrobial and cytotoxic activity of imaqobactin were also performed, identifying moderate antimicrobial activity.

The secreted metabolome of Streptomyces chartreusis and implications for bacterial chemistry.

Actinomycetes are known for producing diverse secondary metabolites. Combining genomics with untargeted data-dependent tandem MS and molecular networking, we characterized the secreted metabolome of the tunicamycin producer Streptomyces chartreusis NRRL 3882. The genome harbors 128 predicted biosynthetic gene clusters. We detected >1,000 distinct secreted metabolites in culture supernatants, only 22 of which were identified based on standards and public spectral libraries. S. chartreusis adapts the secreted metabolome to cultivation conditions. A number of metabolites are produced iron dependently, among them 17 desferrioxamine siderophores aiding in iron acquisition. Eight previously unknown members of this long-known compound class are described. A single desferrioxamine synthesis gene cluster was detected in the genome, yet different sets of desferrioxamines are produced in different media. Additionally, a polyether ionophore, differentially produced by the calcimycin biosynthesis cluster, was discovered. This illustrates that metabolite output of a single biosynthetic machine can be exquisitely regulated not only with regard to product quantity but also with regard to product range. Compared with chemically defined medium, in complex medium, total metabolite abundance was higher, structural diversity greater, and the average molecular weight almost doubled. Tunicamycins, for example, were only produced in complex medium. Extrapolating from this study, we anticipate that the larger part of bacterial chemistry, including chemical structures, ecological functions, and pharmacological potential, is yet to be uncovered.

Discovery of a New Natural Product and a Deactivation of a Quorum Sensing System by Culturing a "Producer" Bacterium With a Heat-Killed "Inducer" Culture.

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of "inducer" microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS.

Anaerobic digestion of paunch in a CSTR for renewable energy production and nutrient mineralization.

A laboratory study investigated the anaerobic digestion of paunch in a continuous stirred tank reactor (CSTR) for the recovery of biogas and mineralization of nutrients. At an organic loading rate (OLR) of 2.8gVSL(-1)day(-1) with a 30-day hydraulic retention time (HRT), a CH4 yield of 0.213Lg(-1)VS and CH4 production rate of 0.600LL(-1)day(-1) were obtained. Post-anaerobic digestion of the effluent from the CSTR for 30days at 40°C recovered 0.067Lg(-1)VS as CH4, which was 21% of the batch CH4 potential. Post-digestion of the effluent from the digestate obtained at this OLR is needed to meet the stable effluent criteria. Furthermore, low levels of soluble ions such as K(+), Ca(2+) and Mg(2+) were found in the liquid fraction of the digestate and the remainder could have been retained in the solid digestate fraction. This study demonstrates the potential of biogas production from paunch in providing renewable energy. In addition, recovery of plant nutrients in the digestate is important for a sustainable agricultural system.

Evaluation of pseudopteroxazole and pseudopterosin derivatives against Mycobacterium tuberculosis and other pathogens.

Pseudopterosins and pseudopteroxazole are intriguing marine natural products that possess notable antimicrobial activity with a commensurate lack of cytotoxicity. New semi-synthetic pseudopteroxazoles, pseudopteroquinoxalines and pseudopterosin congeners along with simple synthetic mimics of the terpene skeleton were synthesized. In order to build structure-activity relationships, a set of 29 new and previously reported compounds was assessed for in vitro antimicrobial and cytotoxic activities. A number of congeners exhibited antimicrobial activity against a range of Gram-positive bacteria including Mycobacterium tuberculosis H37Rv, with four displaying notable antitubercular activity against both replicating and non-replicating persistent forms of M. tuberculosis. One new semi-synthetic compound, 21-((1H-imidazol-5-yl)methyl)-pseudopteroxazole (7a), was more potent than the natural products pseudopterosin and pseudopteroxazole and exhibited equipotent activity against both replicating and non-replicating persistent forms of M.tuberculosis with a near absence of in vitro cytotoxicity. Pseudopteroxazole also exhibited activity against strains of M. tuberculosis H37Rv resistant to six clinically used antibiotics.

Eunicidiol, an anti-inflammatory dilophol diterpene from Eunicea fusca.

A new dilophol diterpene, eunicidiol (1), has been isolated from the crude extract of Eunicea fusca, a gorgonian coral collected from Hillsboro Ledge, Florida. This compound was purified, along with fuscol (2) and eunicol (3), using a combination of normal- and reversed-phase chromatography methods. The structure of eunicidiol (1) was elucidated by 1D and 2D NMR spectroscopic analysis, and the absolute configuration was assigned using Mosher's method. The anti-inflammatory activity of 1-3 was evaluated by measuring their ability to reduce phorbol myristate acetate (PMA)-induced edema in a mouse ear model. Topical application of a 100 µg/ear dose of diterpenes 1-3 significantly reduced edema by 44%, 46%, and 54%, respectively. This activity was superior to indomethacin, a known anti-inflammatory used as a control.