Marine Bacteria, XLVII - Psychrotolerant Bacteria from Extreme Antarctic Habitats as Producers of Rare Bis- and Trisindole Alkaloids.

From the gastrointestinal tract of a fish dredged near the South Orkney Islands in Antarctica, we isolated the psychrotolerant bacterial strain T262, which belongs to the species Vibrio splendidus. Investigation of this strain led to the isolation of a series of 15 bis- and trisindole derivatives. Among them, six new indole alkaloids, namely, turbomycin C [4'-n-butoxyphenyl-bis(1H-indol-3-yl)methane, 1a], turbomycin D [4'-n-propoxyphenyl-bis(1H-indol-3-yl)methane, 1b], turbomycin E [4'-ethoxyphenyl-bis(1H-indol-3-yl)methane, 1c], turbomycin F [4'-methoxy-3',5'-dinitrophenyl-bis(1H-indol-3-yl)methane, 2], trisindolal (3a), and 4-(1H-indol-3-yl-sulfanyl)phenol (4). Another new bisindole derivative elucidated as 2-(indol-3-ylmethyl)-indol-3-ylethanol (7a) was obtained together with six known compounds from the psychrotolerant Arthrobacter psychrochitiniphilus strain T406, isolated from the excrement of penguins. Some of the isolated compounds showed activity against both gram-positive and gram-negative bacteria at 10 µg/paper disk. Trisindolal (3a) was active against the peronosporomycetes Botrytis cinerea and Phytophthora infestans, and some of the indole derivatives indicated promising cytotoxicity towards human tumor cell lines. By exhibiting a mean IC50 of 0.45 µg/mL (1.17 µM), trisindolal (3a) showed pronounced potency and selectivity in a panel of 11 human tumor cell lines derived from 10 different tumor histotypes.

Drimane sesquiterpenoids from Marasmius sp. inhibiting the conidial germination of plant-pathogenic fungi.

From the basidiomycete Marasmius sp., strain IBWF 96046, three new sesquiterpenoids based on the drimane skeleton were isolated and named marasmene B and marasmals B and C. In this study, their isolation, structure elucidation, and biological evaluation are described. The compounds have a pronounced inhibitory effect on the conidial germination of several plant-pathogenic fungi.

Aqabamycins A-G: novel nitro maleimides from a marine Vibrio species: II. Structure elucidation.

The structures of secondary metabolites with antibacterial and cytotoxic activities produced by a marine Vibrio strain from the Red Sea were elucidated. Aqabamycin A (1a) and seven further nitro-substituted maleimide derivates named aqabamycins B-G (1b-f and 2) were obtained together with 12 known metabolites, 3-nitro-1H-indazole (3), indazole-3-carbaldehyde (4), 3-nitro-4-hydroxycinnamic acid, 4-hydroxycinnamic acid, 3-nitro-4-hydroxybenzaldehyde, phenyl-2-bis-indolylmethane (5a), turbomycin B (5b), vibrindole A (6), phenylacetic acid, 3-hydroxybenzoic acid, benzoic acid and 1,4-dithiane (7). Some of the known metabolites (for example, 3, 4 and 7) are described in this study for the first time as natural products. Their structures were elucidated based on 1D and 2D NMR, MS spectra and by comparison with synthetic material.

Aqabamycins A-G: novel nitro maleimides from a marine Vibrio species. I. Taxonomy, fermentation, isolation and biological activities.

In a screening of marine bacteria, a Vibrio species isolated from the surface of the soft coral Sinularia polydactyla collected in the Red Sea was found to be a prolific producer of secondary metabolites with antibacterial and cytotoxic activities. Seven novel maleimide derivatives named aqabamycin A (1a), aqabamycin B (1b), aqabamycin C (1c), aqabamycin D (1d), aqabamycin E (1e and 1e'), aqabamycin F (1f) and aqabamycin G (2) were isolated together with several known metabolites such as 3-nitro-1H-indazole (3), indazole-3-carbaldehyde (4), phenyl-2-bis-indolylmethane (5a), turbomycin B (5b), vibrindole A (6), 1,4-dithiane (7), 3-(3-nitro-4-hydroxyphenyl)-2-propenoic acid (8), 3-nitro-4-hydroxybenzaldehyde (9), phenylacetic acid, benzoic acid, 3-hydroxybenzoic acid and 4-hydroxycinnamic acid. The aqabamycins, except aqabamycin A, bear a nitro group. Compounds 3, 4, 7 are described here for the first time from a natural source and vibrindole A was found to have cytotoxic activity.

Caripyrin, a new inhibitor of infection-related morphogenesis in the rice blast fungus Magnaporthe oryzae.

Caripyrin (trans-5-(3-methyloxiranyl)pyridincarboxylic acid methyl ester, 1), a new pyridyloxirane, was isolated from submerged cultures of the basidiomycete Caripia montagnei. The compound was found to inhibit conidial germination and appressorium formation in the rice blast fungus Magnaporthe oryzae, whereas the infection-related morphogenesis in several other phytopathogenic fungi was not affected. In plant assays on rice, 1 was found to protect plants more efficiently against fungal infection than the structurally related fungal secondary metabolite, fusaric acid. Contrary to the latter, 1 was neither cytotoxic, antibacterial, nor nematicidal.

Structure elucidation of hypocreolide A by enantioselective total synthesis.

The nonenolide hypocreolide A was isolated from culture filtrates of the ascomycete Hypocrea lactea. It exhibits moderate antimicrobial activity against various tested fungi and bacteria. Since neither the relative nor the absolute stereochemistry of the compound could be initially assigned, a stereochemically flexible total synthesis was developed. The two stereogenic centers were formed in high enantioselectivity and yield using transition metal catalyzed asymmetric reactions. While attempts to construct the ten-membered lactone in a ring-closing olefin metathesis gave disappointing results, a combination of cross metathesis and macrolactonization provided the title compound in nine steps and 12% overall yield.

Xanthepinone, an antimicrobial polyketide from a soil fungus closely related to Phoma medicaginis.

The isolation, biological characterization, and structure elucidation of xanthepinone, a novel antifungal metabolite isolated from the broth of submerged cultures of a soil fungus, are described. Xanthepinone inhibits the conidial germination of Magnaporthe grisea (2 microg/mL), Phytophthora infestans (5 microg/mL), and Botrytis cinerea (10 microg/mL) while showing only weak antibacterial activity; cytotoxicity was not observed up to 50 microg/mL. Molecular taxonomy revealed that the producing strain is close to species in the genus Phoma as well as to uncultured soil fungi and endophytes.

Nitro derivatives from the Arctic ice bacterium Salegentibacter sp. isolate T436.

Twenty-five aromatic nitro, dinitro and trinitro compounds were isolated in low yields of less than 1 mg l(-1) from a Salegentibacter sp. strain T436 derived from Arctic pack ice. Their structures were elucidated by MS and NMR techniques. Seven of these compounds, namely, 2-hydroxy-3-(4'-hydroxy-3'-nitrophenyl)-propionic acid methyl ester (6), 2-chloro-3- (4'-hydroxy-3'-nitrophenyl)propionic acid methyl ester (7), 3-(4'-hydroxy-3',5'-dinitrophenyl)-propionic acid methyl ester (14), 4'-hydroxy-3',5'-dinitrophenylethylchloride (16), (4'-hydroxy-3',5'-dinitrophenyl)-2-chloropropionic acid methyl ester (17), N-acetyl-3',5'-dinitrotyramine (18) and 2,6-dinitro-4-(2'-nitroethenyl)phenol (19) are new, and five are reported in this study from a natural source for the first time.

The fatty acid synthase of the basidiomycete Omphalotus olearius is a single polypeptide.

Fatty acids are essential components of almost all biological membranes. Additionally, they are important in energy storage, as second messengers during signal transduction, and in post-translational protein modification. De novo synthesis of fatty acids is essential for almost all organisms, and entails the iterative elongation of the growing fatty acid chain through a set of reactions conserved in all kingdoms. During our work on the biosynthesis of secondary metabolites, a 450-kDa protein was detected by SDS-PAGE of enriched fractions from mycelial lysates from the basidiomycete Omphalotus olearius. Protein sequencing of this protein band revealed the presence of peptides with homology to both alpha and beta subunits of the ascomycete fatty acid synthase (FAS) family. The FAS encoding gene of O. olearius was sequenced. The positions of its predicted 21 introns were verified. The gene encodes a 3931 amino acids single protein, with an equivalent of the ascomycetous beta subunit at the N-terminus and the a subunit at the C-terminus. This is the first report on an FAS protein from a homobasidiomycete and also the first fungal FAS which is comprised of a single polypeptide.

New naphthoquinone derivatives from the ascomycete IBWF79B-90A.

Bioactivity-guided fractionation of extracts from the fungus IBWF79B-90A resulted in the isolation of three known naphthoquinones, herbarin, dehydroherbarin, and O-methylherbarin and the azaanthraquinone scorpinone as well as three structurally related derivatives, O-phenethylherbarin and herbaridines A and B. All seven compounds exhibited cytotoxic activities against several cell lines.

Siderophore synthesis in Magnaporthe grisea is essential for vegetative growth, conidiation and resistance to oxidative stress.

The plant pathogenic fungus Magnaporthe grisea excretes siderophores of the coprogen-type for iron acquisition and uses ferricrocin for intracellular iron storage. In the present report we characterize mutants with defects in extracellular siderophore biosynthesis. Deletion of the M. grisea SSM2 gene, which encodes a non-ribosomal peptide synthetase, resulted in a loss of the production of all coprogens. The mutant strains had a reduced growth rate, produced fewer conidia and were more sensitive to oxidative stress. Ferricrocin production was not affected. Upon deletion of M. grisea OMO1, a gene predicted to encode an L-ornithine-N(5)-monooxygenase, no siderophores of any type were detected, the strain was aconidial, growth rate was reduced and sensitivity to oxidative stress was increased. Abundance of several proteins was affected in the mutants. The Deltassm2 and Deltaomo1 mutant phenotypes were complemented by supplementation of the medium with siderophores or reintroduction of the respective genes.

The Magnaporthe grisea class VII chitin synthase is required for normal appressorial development and function.

The plant pathogenic fungus Magnaporthe grisea is able to enter its host via appressorium-mediated penetration. Earlier investigations have shown that these infection structures are rich in the cell wall polysaccharide chitin. Previously, we have described how the transcription of a class VII chitin synthase-encoding gene CHS7 is completely dependent on the putative transcription factor Con7p during the germination of conidia, and how con7(-) mutants are unable to form appressoria under any conditions tested. Because of the pleiotropic effects of the con7(-) mutation, we examined the consequences of the targeted deletion of CHS7. The chs7(-) mutants generated were unable to form appressoria on artificial surfaces, except following the application of the exogenous inducers 1,16-hexadecanediol and cyclic adenosine monophosphate. The appressoria formed had a reduced chitin content and were often found to be smaller and misshapen compared with the wild-type. chs7(-) mutants were significantly reduced in their ability to enter rice plants, but growth in planta was not affected. Reverse transcriptase-polymerase chain reaction analysis demonstrated that CHS7 transcription was strongly induced on germination of spores, and a green fluorescent protein-tagged Chs7p protein was found to be produced abundantly during infection-related morphogenesis. Together, these data suggest that the class VII chitin synthase Chs7p of M. grisea is required for normal appressorium formation and function.

Ascomycones A-C, heptaketide metabolites from an unidentified ascomycete.

The ascomycones A-C (1-3), three novel heptaketide-derived secondary metabolites, have been isolated from cultures of an unknown ascomycete. While 1 and 2 are closely related pyranonaphthoquinones, 3 has a bicyclic dihydronaphthoquinone core. Compounds 2 and 3 exhibited significant activity against several phytopathogenic fungi.

Tetracyclic terpenoids from Dasyscyphus niveus, dasyscyphins D and E.

Cultures of the ascomycete Dasyscyphus niveus have yielded two new tetracyclic dasyscyphin-type terpenoids (1 and 2), and their structures were elucidated by NMR spectroscopy and X-ray crystallography. The absolute configuration of dasyscyphin D (1) was determined by synthesis and NMR spectroscopy of diastereomeric MTPA esters. Both compounds inhibited the germination of conidia of Magnaporthe grisea at 25 microg/mL.

Sterelactones: new isolactarane type sesquiterpenoids with antifungal activity from Stereum sp. IBWF 01060.

Four members of a new family of tetracyclic sesquiterenoids possessing the isolactarane skeleton have been isolated from mycelial cultures of Stereum sp. IBWF 01060. Their structure elucidation and their antifungal activity against several plant pathogens as well as other microorganisms are reported.

Intermediates in the oxidative pathway from torulene to torularhodin in the red yeasts Cystofilobasidium infirmominiatum and C. capitatum (Heterobasidiomycetes, Fungi).

Two red Cystofilobasidium spp. isolated from spring sap-flows of Betula pendula were analysed for their carotenoid content. In Cystofilobasidium infirmominiatum, three unusual pigments were detected and identified by structure elucidation as oxidised torulene derivatives. These included 16'-hydroxytorulene and torularhodinaldehyde, two carotenoids known so far only from chemical synthesis or as postulated biosynthetic intermediates en route to torularhodin. Unprecedented formation of beta-apo-2'-carotenal was also observed. The production of these pigments in pure culture was dependent on enhanced oxidative stress caused by cultivation in well-aerated (indented) flasks with or without 2% ethanol (16'-hydroxytorulene), or with 100 microM duroquinone (torularhodinaldehyde and beta-apo-2'-carotenal). Among these three pigments, only 16'-hydroxytorulene was detected in C. capitatum. Torularhodin, a common end product of carotenoid oxidation in red yeasts, was not produced by either species under any incubation conditions. Biosynthetic aspects of incomplete oxidation of torulene by these Cystofilobasidium spp. are discussed.

Cytotoxic alpha-pyrones from Xylaria hypoxylon.

Two new alpha-pyrone derivatives, xylarone (1) and 8,9-dehydroxylarone (2) possessing cytotoxic activities, were isolated from the culture fluid of submerged cultures of the ascomycete Xylaria hypoxylon, strain A27-94. Their structures were elucidated by spectroscopic methods.

New aromatic nitro compounds from Salegentibacter sp. T436, an Arctic Sea ice bacterium: taxonomy, fermentation, isolation and biological activities.

Nineteen aromatic nitro compounds were isolated from the culture broth of an Arctic sea ice bacterium. Four of these compounds are new and six compounds are reported from a natural source for the first time. The new natural products showed weak antimicrobial and cytotoxic activities. 2-nitro-4-(2'-nitroethenyl)-phenol was the most potent antimicrobial and cytotoxic substance. Some of the compounds exhibit plant growth modulating activities. Based on its biochemical properties and the 16S rRNA gene sequence, the producing strain can be described as a distinct species within the genus Salegentibacter.

A marine Mesorhizobium sp. produces structurally novel long-chain N-acyl-L-homoserine lactones.

Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C(10) to C(16). Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C(12)-homoserine lactone (5-cis-3-oxo-C(12)-HSL) (compound 1) and 5-cis-C(12)-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.