Abstract
The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of an hhkA deletion mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in a tcrA deletion mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but this was not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity for the TcrA box sequences in the electrophoretic mobility shift assay. Taking these observations together with other results, we proposed that HhkA and TcrA compose a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development, including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensisIMPORTANCEActinoplanes missouriensis goes through complex morphological differentiation, including formation of flagellated spore-containing sporangia, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unknown. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system is employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes.