Involvement of an orphan response regulator of the two-component regulatory system in the formation of physiologically mature sporangia in Actinoplanes missouriensis.

The actinomycete Actinoplanes missouriensis forms terminal sporangia that contain dormant sporangiospores. Upon contact with water, sporangia release zoospores through a process called sporangium dehiscence. In this study, we characterized asfR (AMIS_76070), which encodes an orphan response regulator receiver domain protein of the two-component regulatory system, as one of 136 genes whose transcription was highly activated during sporangium formation. Actinoplanes sporangium formation regulator (AsfR) homologs are conserved among Actinoplanes bacteria. An asfR null mutant (ΔasfR) strain formed normally shaped sporangia containing apparently normal dormant spores, but they exhibited defective sporangium dehiscence; the number of spores released from the sporangia of the ΔasfR strain was four orders of magnitude lower than that from the sporangia of the wild-type strain. This phenotypic change was recovered by introducing asfR with its own promoter into the ΔasfR strain. Based on the amino acid sequence and predicted structure, the function of AsfR appeared to be controlled by the phosphorylation of Asp-72. Consistently, the phenotypic change observed in the ΔasfR strain was not restored by introducing a mutated asfR (D72N) gene. Three orphan histidine kinases (HKs) in A. missouriensis were found to interact with AsfR by screening using a bacterial two-hybrid assay. However, gene disruption experiments revealed that these three HKs were not required for sporangium dehiscence in A. missouriensis. Although the molecular functions of AsfR remain to be elucidated, this study shows that AsfR is involved in the formation of physiologically mature sporangia that are fully prepared to release spores under sporangium dehiscence-inducing conditions.IMPORTANCEActinoplanes missouriensis undergoes a life cycle involving complex morphological development, including mycelial growth, sporangium formation and dehiscence, swimming as zoospores, germination, and outgrowth to mycelial growth. In this study, we revealed that a stand-alone response regulator receiver domain protein, AsfR, is required for the formation of physiologically mature sporangia that can release spores under sporangium dehiscence-inducing conditions. A. missouriensis seems to express genes that are involved in sporangium dehiscence during sporangium formation, considering that an asfR null mutant produced normally shaped sporangia, but these sporangia were deficient in sporangium dehiscence. Although the molecular functions of AsfR, as well as the histidine kinase(s) that phosphorylates AsfR, remain to be elucidated, identification of AsfR as a possible key regulator for the preparation of the onset and progression of sporangium dehiscence is significant, because almost no proteins involved in the early stages of sporangium dehiscence have been identified in A. missouriensis.