SummaryBlue‐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.

SummaryStreptomycetes 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.

In a previous study, we reported that the novel annexin XX1 (annexin E1), identical to alpha14-giardin, is specifically localized to the flagella and to the median body of the trophozoites. However, the mode of interaction and the direct partners involved remained unclear. In the present study, we show that alpha4-giardin obviously does not evenly distribute over the full length of the axonemes, but rather, resides at local slubs near the proximal part and the ends of the flagella. In immunocytochemical co-localization studies, the anti-giardin primary antibody exclusively reacted with distinct regions of the flagella in permeabilized cells, whereas the anti-tubulin antibody bound to all areas of the axonemes in the cells and to isolated cytoskeletons. Isolated cytoskeletons did not react with anti-giardin antibodies. alpha14-Giardin itself is able to assemble to multimeric structures. Taken together, our findings suggest that alpha14-giardin adheres to microtubules of the flagella via self-assembly that may regulated by Ser/Thr-phosphorylation.

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