Christopher A. Kauffman

Widespread and Persistent Populations of a Major New Marine Actinomycete Taxon in Ocean Sediments

ABSTRACT A major taxon of obligate marine bacteria within the order Actinomycetales has been discovered from ocean sediments. Populations of these bacteria (designated MAR 1) are persistent and widespread, spanning at least three distinct ocean systems. In this study, 212 actinomycete isolates possessing MAR 1 morphologies were examined and all but two displayed an obligate requirement of seawater for growth. Forty-five of these isolates, representing all observed seawater-requiring morphotypes, were partially sequenced and found to share characteristic small-subunit rRNA signature nucleotides between positions 207 and 468 (Escherichia coli numbering). Phylogenetic characterization of seven representative isolates based on almost complete sequences of genes encoding 16S rRNA (16S ribosomal DNA) yielded a monophyletic clade within the family Micromonosporaceae and suggests novelty at the genus level. This is the first evidence for the existence of widespread populations of obligate marine actinomycetes. Organic extracts from cultured members of this new group exhibit remarkable biological activity, suggesting that they represent a prolific resource for biotechnological applications.

Marineosins A and B, cytotoxic spiroaminals from a marine-derived actinomycete.

Two novel spiroaminals, marineosins A and B (1, 2), containing two pyrrole functionalities, were isolated from cultures of a marine sediment-derived actinomycete related to the genus Streptomyces. The marineosins, which appear to be derived from unknown modifications of prodigiosin-like pigment pathways, showed significant inhibition of human colon carcinoma (HCT-116) in an in vitro assay (IC50 = 0.5 microM for marineosin A) and selective activities in diverse cancer cell types.

Aspergillamides A and B: Modified cytotoxic tripeptides produced by a marine fungus of the genus Aspergillus

Abstract Two isomeric linear peptides, aspergillamides A and B ( 1, 2 ), were isolated from the mycelium of a cultured marine fungus of the genus Aspergillus . The producing strain (designated CNC-120), was obtained from a saline lake sediment sample collected from Acklins Island, the Bahamas. The structures of the new peptides were elucidated using comprehensive 2D NMR methods. At 25°C, in both acetone and dimethylsulfoxide, aspergillamide A exists as a 1:1 mixture of trans - and cis -amide rotational isomers ( 1a and 1b ). Under identical conditions, aspergillamide B is predominantly in the cis -amide form. The absolute stereochemistries of the amino acids in aspergillamide A were assigned as L by hydrolysis and comparison with commercial standards. Aspergillamide A showed modest in vitro cytotoxicity [IC 50 = 16 μg/ml) toward the human colon carcinoma cell line HCT-116.

High recovery of culturable bacteria from the surfaces of marine algae

The culturability of heterotrophic marine bacteria obtained from the surfaces of two species of marine algae (Lobophora variegata andHalimeda copiosa) was assessed by comparing total DAPI-stained cell counts to colony-forming bacterial counts on two agar media. The colony-forming bacterial counts on a low-nutrient medium (LN) consisting of seawater and agar were significantly greater for both algal species than counts obtained on a high-nutrient medium (HN) similar in composition to that typically used for the isolation of heterotrophic marine bacteria. On average, 14 and 58%, respectively, of the total bacteria fromL. variegata andH. copiosa were culturable on LN. These recovery rates far exceed those typically reported for marine bacteria. Of 119 LN strains obtained in pure culture, 55% failed to grow on HN. The yeast extract component of HN (1.5 gl-1) was responsible for the majority of the observed inhibition, suggesting that this nutrient can be highly toxic to marine bacteria. Eighty-nine percent of the strains inhibited by HN were capable of growth when the nutrients in this medium were diluted by a factor of 100 with seawater. Of 66 epiphytic strains, 30 (45%) initially inhibited by HN showed the ability to adapt to this medium after a period of laboratory handling. The initial inability of low-nutrient-adapted bacteria to grow on high-nutrient media may be due to nutrient shock. The results presented here indicate that the culturability of specific populations of marine bacteria can be dramatically improved by the use of low-nutrient media. Further, the importance of developing new medium formulations that eliminate traditional nutrients, some of which are clearly toxic to bacteria, is demonstrated.

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.

Structure and biosynthesis of the marine streptomycete ansamycin ansalactam A and its distinctive branched chain polyketide extender unit

Reported is the structure and biosynthesis of ansalactam A, an ansamycin class polyketide produced by an unusual modification of the polyketide pathway. This new metabolite, produced by a marine sediment-derived bacterium of the genus Streptomyces , possesses a novel spiro γ-lactam moiety and a distinctive isobutyryl polyketide fragment observed for the first time in this class of natural products. The structure of ansalactam A was defined by spectroscopic methods including X-ray crystallographic analysis. Biosynthetic studies with stable isotopes further led to the discovery of a new, branched chain polyketide synthase extender unit derived from (E)-4-methyl-2-pentenoic acid for polyketide assembly observed for the first time in this class of natural products.

Halovirs A-E, new antiviral agents from a marine-derived fungus of the genus Scytalidium.

Marine micro-organisms represent an under explored resource for the discovery of novel antiviral agents. Here, we describe a series of peptides designated halovirs A-E (1-5) that are produced during the saline fermentation of a marine-derived fungus of the genus Scytalidium. These lipophilic, linear peptides are potent in vitro inhibitors of the herpes simplex viruses 1 and 2. Evidence is presented that the halovirs directly inactivate herpes viruses, a mechanism of action that could be applicable in the prevention of HSV transmission. The total structures of these new compounds were established by a combination of spectral and chemical techniques. Salient structural features of the halovir hexapeptides include a nitrogen terminus acylated by myristic (C14) or lauric (C12) acid, an unusual Aib-Hyp dipeptide segment, and a carboxyl terminus reduced to a primary alcohol. A qualitative analysis of the secondary structures of these molecules using variable temperature NMR experiments and NOE analyses is also reported.

Anthracimycin, a Potent Anthrax Antibiotic from a Marine‐Derived Actinomycete

Licensed to kill: A new antibiotic, anthracimycin (see scheme), produced by a marine-derived actinomycete in saline culture, shows significant activity toward Bacillus anthracis, the bacterial pathogen responsible for anthrax infections. Chlorination of anthracimycin gives a dichloro derivative that retains activity against Gram-positive bacteria, such as anthrax, but also shows activity against selected Gram-negative bacteria.

Marinisporolides, Polyene-Polyol Macrolides from a Marine Actinomycete of the New Genus Marinispora

Two new polyene macrolides, marinisporolides A and B (1, 2), were isolated from the saline culture of the marine actinomycete, strain CNQ-140, identified as a member of the new marine genus Marinispora. The marinisporolides are 34-membered macrolides composed of a conjugated pentaene and several pairs of 1,3-dihydroxyl functionalities. Marinisporolide A (1) contains a bicyclic spiro-bis-tetrahydropyran ketal functionality, while marinisporolide B (2) is the corresponding hemiketal. The structures of these new compounds were assigned by combined spectral and chemical methods including extensive 2D NMR experiments and correlations of (13)C NMR data with Kishis Universal NMR Database. Chemical modifications, including methanolysis, acetonide formation, and application of the modified Mosher method, provided the full stereostructures of these molecules. Three additional macrolides, marinisporolides C-E (3-5), which are olefin geometric isomers of marinisporolide A (1), were also isolated and their structures defined. Under room light, marinisporolides A and B readily photoisomerize to C-E indicating that they are most likely produced by photochemical conversion during the cultivation or isolation procedures. Although polyenes, marinisporolides A (1) and B (2) showed weak to no antifungal activity against Candida albicans.

Fijiolides A and B, Inhibitors of TNF-α-Induced NFκB Activation, from a Marine-Derived Sediment Bacterium of the Genus Nocardiopsis

Fijiolide A, a potent inhibitor of TNF-alpha-induced NFkappaB activation, along with fijiolide B, were isolated from a marine-derived bacterium of the genus Nocardiopsis. The planar structures of fijiolides A (1) and B (2) were elucidated by interpretation of 2D NMR spectroscopic data, while the absolute configurations of these compounds were defined by interpretation of circular dichroism and 2D NMR data combined with application of the advanced Moshers method. Fijiolides A and B are related to several recently isolated chloroaromatic compounds, which appear to be the Bergman cyclization products of enediyne precursors. Fijiolide A reduced TNF-alpha-induced NFkappaB activation by 70.3%, with an IC(50) value of 0.57 micro-M. Fijiolide B demonstrated less inhibition, only 46.5%, without dose dependence. The same pattern was also observed with quinone reductase (QR) activity: fijiolide A was found to induce quinone reductase-1 (QR1) with an induction ratio of 3.5 at a concentration of 20 microg/mL (28.4 microM). The concentration required to double the activity was 1.8 microM. Fijiolide B did not affect QR1 activity, indicating the importance of the nitrogen substitution pattern for biological activity. On the basis of these data, fijiolide A is viewed as a promising lead for more advanced anticancer testing.