Redox-active compound generated by bacterial crosstalk induces hypha branching in Streptomyces species

Chemical cross talks between Mycolicibacterium septicum HEK138M and Bacillus subtilis 168 affect the bacterial morphology of Streptomyces variegatus HEK138A. We found that S. variegatus exhibits unusual hyphae branching by the bacterial interaction. We aimed to elucidate the mechanism by performing activity guided purification of substances that induce the unusual cell morphology. We found that pyrogallol, a redox active aromatic small molecule induced significant hyphae branching in S. variegatus and the activity was also observed in some of other Streptomyces species. Interestingly, the pyrogallol activity was diminished by adding catalase, which broke down H2O2. To further confirm the involvement, H2O2 was tested and similar activity which induced hyphal branching was observed. This indicates that reactive oxygen species (ROS) generated by redox-active compound (RAC) is the inducing factor of hyphae branching. Further investigation revealed that pyrogallol was generated by NahG enzyme homolog of M. septicum using 2,3-dihydroxybenzoic acid as substrate by heterologous expression in E. coli. Moreover, co-culture with gene knock-out mutants revealed that 2,3-dihydroxybenzoic acid was supplied by B. subtilis produced as intermediate of bacterial siderophore bacillibactin. Since the hyphae branching of vegetative mycelium can increase the density of filamentous network and consequently help secure the milieu in soil, our results suggested that those filamentous soil bacteria use ROS which can be supplied from plant derived RAC as a signal. As those RAC ubiquitously exist in soil environment, the system will be beneficial for sensing the nutrient sources in addition to the generally considered defensive response to oxidative stress. ImportanceThe characterization of interactions between three or more bacteria are lacking as these interactions are visually imperceptible in general. Our current study revealed changes of morphological behavior by the bacterial interaction. This study showed that hydrogen peroxide generated by redox-active compound derived from a breakdown product of siderophore can significantly increase the number of hyphae tip extension in filamentous bacteria. Our result implies the existence of oxidative response system using a low amount of reactive oxygen species as an integrated signal to sense the plant-derived carbon source by the filamentous soil bacteria. As a result of sensing, filamentous soil bacteria may decide whether the hypha tip should be extended to further explore the area or increase the tips to densify filamentous network to monopolize the nutrients in the milieu.