Project description:The myxobacterial strain Stigmatella aurantiaca MYX-030 was selected as promising source for the discovery of new biologically active natural products by our screening methodology. The isolation, structure elucidation and initial biological evaluation of the myxocoumarins derived from this strain are described in this work. These compounds comprise an unusual structural framework and exhibit remarkable antifungal properties.
Project description:Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNA(Asp) and tRNA(Val), respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the t(Mx8) terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3' processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the t(Mx8) terminator sequence results in reduced levels of mature tRNA(Asp) and tRNA(Val) and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.
Project description:Innovative discovery approaches such as genome-mining and metabolomics-inspired methods have reshaped the natural product research field, complementing traditional bioactivity-based screens and allowing hitherto unseen compounds to be uncovered from previously investigated producers. In line with these trends, we report here imidacins, a novel class of secondary metabolites specific to the myxobacterial genus Stigmatella. A combination of secondary metabolome analysis, genome-mining techniques, spectroscopic analysis, and finally total synthesis was used to allow structure elucidation. Imidacins are urocanate-derived aliphatic acids with an adjacent cyclopropane moiety, structural features unprecedented in natural products to date.
Project description:Stigmatella aurantiaca is a gram-negative bacterium which forms, under conditions of starvation in a multicellular process, characteristic three-dimensional structures: the fruiting bodies. For studying this complex process, mutants impaired in fruiting body formation have been induced by transposon insertion with a Tn5-derived transposon. The gene affected (fbfB) in one of the mutants (AP182) was studied further. Inactivation of fbfB results in mutants which form only clumps during starvation instead of wild-type fruiting bodies. This mutant phenotype can be partially rescued, if cells of mutants impaired in fbfB function are mixed with those of some independent mutants defective in fruiting before starvation. The fbfB gene is expressed about 14 h after induction of fruiting body formation as determined by measuring beta-galactosidase activity in a merodiploid strain harboring the wild-type gene and an fbfB-delta trp-lacZ fusion gene or by Northern (RNA) analysis with the Rhodobacter capsulatus pufBA fragment fused to fbfB as an indicator. The predicted polypeptide FbfB has a molecular mass of 57.8 kDa and shows a significant homology to the galactose oxidase (GaoA) of the fungus Dactylium dendroides. Galactose oxidase catalyzes the oxidation of galactose and primary alcohols to the corresponding aldehydes.
