Characterization and localization of prodiginines from Streptomyces lividans suppressing Verticillium dahliae in the absence or presence of Arabidopsis thaliana.

The ascomycete Verticillium dahliae causes worldwide vascular wilt of many field and horticultural plants. The melanized resting structures of this fungus, so-called microsclerotia, survive for many years in soils and continuously re-infect plants. Due to the absence of known fungicides, Verticillium wilt causes immense crop losses. We discovered that the Gram-positive, spore-forming soil bacterium Streptomyces lividans expresses members of the prodiginine family during co-cultivation with V. dahliae. Using HPLC and LC-MS analysis of cultures containing S. lividans alone or grown together with V. dahliae, we found that undecylprodigiosin [394.4 M+H](+) is highly abundant, and streptorubin B [392.4 M+H](+) is present in smaller amounts. Within co-cultures, the quantity of undecylprodigiosin increased considerably and pigment concentrated at and within fungal hyphae. The addition of purified undecylprodigiosin to growing V. dahliae hyphae strongly reduced microsclerotia formation. Undecylprodigiosin was also produced when S. lividans grew on the roots of developing Arabidopsis thaliana plants. Furthermore, the presence of the undecylprodigiosin producer led to an efficient reduction of V. dahliae hyphae and microsclerotia on plant-roots. Based on these novel findings and previous knowledge, we deduce that the prodiginine investigated leads to multiple cellular effects, which ultimately impair specific pathways for signal transduction and apoptosis of the fungal plant pathogen.

Extracellular Streptomyces vesicles: amphorae for survival and defence.

Blue-pigmented exudates arise as droplets on sporulated lawns of Streptomyces coelicolor M110 grown on agar plates. Our electron microscopical and biochemical studies suggest that droplets contain densely packed vesicles with large assemblies of different protein types and/or the polyketide antibiotic actinorhodin. Frozen-hydrated vesicles were unilamellar with a typical bilayer membrane, and ranged from 80 to 400 nm in diameter with a preferred width of 150-300 nm. By means of cryo-electron tomography, three types were reconstructed three-dimensionally: vesicles that were filled with particulate material, likely protein assemblies, those that contained membrane-bound particles, and a vesicle that showed a higher contrast inside, but lacked particles. Our LC/MS analyses of generated tryptic peptides led to the identification of distinct proteins that carry often a predicted N-terminal signal peptide with a twin-arginine motif or lack a canonical signal sequence. The proteins are required for a range of processes: the acquisition of inorganic as well as organic phosphate, iron ions, and of distinct carbon sources, energy metabolism and redox balance, defence against oxidants and tellurites, the tailoring of actinorhodin, folding and assembly of proteins, establishment of turgor, and different signalling cascades. Our novel findings have immense implications for understanding new avenues of environmental biology of streptomycetes and for biotechnological applications.

Streptomyces lividans inhibits the proliferation of the fungus Verticillium dahliae on seeds and roots of Arabidopsis thaliana.

Verticillium wilt, a vascular disease in more than 200 dicotyledonous plants, is due to the ascomycete fungus Verticillium dahliae. As documented by video-microscopy, the soil bacterium Streptomyces lividans strongly reduces the germination of V. dahliae conidia, and the subsequent growth of hyphae. Quantification by the use of DNA-intercalating dyes and Calcofluor-staining revealed that during prolonged co-cultivation, bacterial hyphae proliferate to a dense network, provoke a poor development of V. dahliae vegetative hyphae and lead to an enormous reduction of conidia and microsclerotia. Upon individual application to seeds of the model plant Arabidopsis thaliana, either the bacterial spores or the fungal conidia germinate at or within the mucilage, including its volcano-shaped structures. The extension of hyphae from each individual strain correlates with the reduction of the pectin-containing mucilage-layer. Proliferating hyphae then spread to roots of the emerging seedlings. Plants, which arise in the presence of V. dahliae within agar or soil, have damaged root cells, an atrophied stem and root, as well as poorly developed leaves with chlorosis symptoms. In contrast, S. lividans hyphae settle in bunches preferentially at the outer layer near tips and alongside roots. Resulting plants have a healthy appearance including an intact root system. Arabidopsis thaliana seeds, which are co-inoculated with V. dahliae and S. lividans, have preferentially proliferating bacterial hyphae within the mucilage, and at roots of the outgrowing seedlings. As a result, plants have considerably reduced disease symptoms. As spores of the beneficial S. lividans strain are obtainable in large quantity, its application is highly attractive.

A molecular key for building hyphae aggregates: the role of the newly identified Streptomyces protein HyaS.

Streptomycetes produce many metabolites with medical and biotechnological applications. During fermentations, their hyphae build aggregates, a process in which the newly identified protein HyaS plays an important role. The corresponding hyaS gene is present within all investigated Streptomyces species. Reporter fusions indicate that transcription of hyaS occurs within substrate hyphae of the Streptomyces lividans wild type (WT). The HyaS protein is dominantly associated with the substrate hyphae. The WT strain forms cylindrically shaped clumps of densely packed substrate hyphae, often fusing to higher aggregates (pellets), which remain stably associated during shaking. Investigations by electron microscopy suggest that HyaS induces tight fusion-like contacts among substrate hyphae. In contrast, the pellets of the designed hyaS disruption mutant ΔH are irregular in shape, contain frequently outgrowing bunches of hyphae, and fuse less frequently. ΔH complemented with a plasmid carrying hyaS resembles the WT phenotype. Biochemical studies indicate that the C-terminal region of HyaS has amine oxidase activity. Investigations of ΔH transformants, each carrying a specifically mutated gene, lead to the conclusion that the in situ oxidase activity correlates with the pellet-inducing role of HyaS, and depends on the presence of certain histidine residues. Furthermore, the level of undecylprodigiosin, a red pigment with antibiotic activity, is influenced by the engineered hyaS subtype within a strain. These data present the first molecular basis for future manipulation of pellets, and concomitant production of secondary metabolites during biotechnological processes.