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.
The previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/ function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant DK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the DK mutant, the relative level of caesium was <30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.
Summary The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy‐filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co‐embedded mutant ΔK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP‐derived lead‐sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the ΔK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.
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