Two new heptaketides, corynesporol (1) and 1-hydroxydehydroherbarin (2), along with herbarin (3) were isolated from an endolichenic fungal strain, Corynespora sp. BA-10763, occurring in the cavern beard lichen Usnea cavernosa. The structures of 1−3 were elucidated from their spectroscopic data. Aerial oxidation of corynesporol (1) yielded herbarin (3). Acetylation of 1 afforded the naphthalene derivative 4, whereas acetylation of 3 gave the corresponding naphthoquinone 6 and dehydroherbarin (5). All compounds were evaluated for their cytotoxicity and ability to inhibit migration of human metastatic breast and prostate cancer cell lines MDA-MB-231 and PC-3M, respectively. Dehydroherbarin (5) inhibited migration of both cell lines at concentrations not toxic to these cell lines. This is the first report of metabolites from an endolichenic fungus.

Numerous plant pathogens, rhizosphere symbionts, and endophytic bacteria and yeasts produce the important phytohormone indole-3-acetic acid (IAA), often with profound effects on host plants. However, to date IAA production has not been documented among foliar endophytes -- the diverse guild of primarily filamentous Ascomycota that live within healthy, above-ground tissues of all plant species studied thus far. Recently bacteria that live within hyphae of endophytes (endohyphal bacteria) have been detected, but their effects have not been studied previously. Here we show not only that IAA is produced in vitro by a foliar endophyte (here identified as Pestalotiopsis aff. neglecta, Xylariales), but that IAA production is enhanced significantly when the endophyte hosts an endohyphal bacterium (here identified as Luteibacter sp., Xanthomonadales). Both the endophyte and the endophyte/bacterium complex appear to rely on an L-tryptophan dependent pathway for IAA synthesis. The bacterium can be isolated from the fungus when the symbiotic complex is cultivated at 36°C. In pure culture the bacterium does not produce IAA. Culture filtrate from the endophyte-bacterium complex significantly enhances growth of tomato in vitro relative to controls and to filtrate from the endophyte alone. Together these results speak to a facultative symbiosis between an endophyte and endohyphal bacterium that strongly influences IAA production, providing a new framework in which to explore endophyte-plant interactions.

Five new isopimarane diterpenes, smardaesidins A – E (1 – 5) and two new 20-nor-isopimarane diterpenes, smardaesidins F (6) and G (7) together with sphaeropsidins A (8), and C – F (10 – 13) were isolated from an endophytic fungal strain, Smardaea sp. AZ0432, occurring in living photosynthetic tissue of the moss Ceratodon purpureus. Of these, smardaesidins B (2) and C (3) were obtained as an inseparable mixture of isomers. Chemical reduction of sphaeropsidin A (8) afforded sphaeropsidin B (9) whereas catalytic hydrogenation of 8 yielded 7-O-15,16-tetrahydrosphaeropsidin A (14), and its new derivative, 7-hydroxy-6-oxo-isopimara-7-en-20-oic acid (15). Acetylation and diazomethane reaction of sphaeropsidin A (8) afforded two of its known derivatives, 6-O-acetylsphaeropsidin A (16) and 8,14-methylenesphaeropsidin A methyl ester (17), respectively. Methylation of 10 yielded sphaeropsidin C methyl ester (18). The planar structures and relative configurations of the new compounds 1 – 7, and 15 were elucidated using MS, and 1D and 2D NMR experiments while the absolute configurations of the stereocenters of 4 and 6 – 8 were assigned using modified Mosher’s ester method, CD spectra, and comparison of specific rotation data with literature values. Compounds 1 – 18 were evaluated for their potential anticancer activity using several cancer cell lines and cells derived from normal human primary fibroblasts. Of these, compounds 8, 11, and 16 showed significant cytotoxic activity. More importantly, sphaeropsidin A (8) showed cell-type selectivity in cytotoxicity assay and inhibited migration of metastatic breast adenocarcinoma (MDA-MB-231) cells at sub-cytotoxic concentrations.

In several diazotrophic species of Proteobacteria, P II signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH 4 ؉ by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the P II -like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP in A. vinelandii were studied to determine whether the Nif ؊ phenotype of glnD strains was due to an inability to modify GlnK, an effort previously hampered because glnK is an essential gene in this organism. A glnKY51F mutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH 4 ؉ , suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation.

Four new ent-kaurane diterpenoids, geopyxins A–D (1–4) were isolated from Geopyxis aff. majalis, a fungus occurring in the lichen Pseudevernia intensa, whereas Geopyxis sp. AZ0066 inhabiting the same host afforded two new ent-kaurane diterpenoids, geopyxins E and F (5 and 6) together with 1 and 3. The structures of 1–6 were established on the basis of their spectroscopic data while the absolute configurations were assigned using modified Mosher’s ester method. Methylation of 1–3, 5, and 6 gave their corresponding methyl esters 7–11. On acetylation, 1 and 7 yielded their corresponding monoacetates 12 and 14, and diacetates 13 and 15. All compounds were evaluated for their cytotoxic and heat-shock induction activities. Compounds 2, 7–10, 12, 14, and 15 showed cytotoxic activity in the low micromolar range against all five cancer cell lines tested, but only compounds 7–9, 14, and 15 were found to activate the heat-shock response at similar concentrations. From a preliminary structure-activity perspective, the electrophilic α,β-unsaturated ketone carbonyl motif present in all compounds except 6 and 11 was found to be necessary but not sufficient for both cytotoxicity and heat-shock activation.