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. Actinoplanessporangium formation regulator (AsfR) homologs are conserved among Actinoplanes bacteria. An asfR null mutant (Delta asfR) 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 Delta asfR 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 Delta asfR 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 Delta asfR 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.