A molecular key for building hyphae aggregates: the role of the newly identified Streptomyces protein HyaS.
Bottom Line: The HyaS protein is dominantly associated with the substrate hyphae.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.These data present the first molecular basis for future manipulation of pellets, and concomitant production of secondary metabolites during biotechnological processes.
Affiliation: University of Osnabrück, FB Biology/Chemistry, Applied Genetics of Microorganisms, 49069 Osnabrück, Germany.Show MeSH
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Mentions: The upstream region of the hyaS gene was cloned in frame with the egfp gene (encoding the enhanced green fluorescent protein) into the modified bifunctional pWHM3 vector (see Experimental procedures). Subsequently, the resulting reporter construct (line G in Fig. 2) was transformed into S. lividans. Within spores, EGFP‐derived fluorescence was absent (Fig. 2A). However, after incubation on solid media, it occurred in germinating spores, within substrate hyphae (Fig. 2B), but not in arial mycelia (Fig. 2C). After incubation in liquid medium, EGFP‐derived fluorescence was present within germinating spores, the extending substrate hyphae (not shown), as well as within clumps of substrate hyphae (Fig. 2D). This result clearly showed that the regulatory region of hyaS had provoked transcription of the egfp gene (Fig. 2G), which subsequently led to the synthesis of the fluorescent EGFP protein. The control S. lividans transformant carried the control plasmid with a 550 bp PCR fragment without regulatory region in front of the egfp gene (see line H in Fig. 2 and Experimental procedures). This control lacked relevant EGFP‐derived fluorescence under all cultivation conditions including individual substrate hyphae (Fig. 2E) and pellets clumps of substrate hyphae (Fig. 2F).
Affiliation: University of Osnabrück, FB Biology/Chemistry, Applied Genetics of Microorganisms, 49069 Osnabrück, Germany.