Regulation of Sporangium Formation, Spore Dormancy, and Sporangium Dehiscence by a Hybrid Sensor Histidine Kinase in Actinoplanes missouriensis: Relationship with the Global Transcriptional Regulator TcrA.

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.

Identification and Characterization of a Cell Wall Hydrolase for Sporangiospore Maturation in Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis grows as substrate mycelium and forms terminal sporangia containing a few hundred spores as dormant cells. Upon contact with water, the sporangia open up and release spores to external environments. Here, we report a cell wall hydrolase, GsmA, that is required for sporangiospore maturation in A. missouriensis The gsmA gene is conserved among Actinoplanes species and several species of other rare actinomycetes. Transcription of gsmA is activated in the late stage of sporangium formation by the global transcriptional activator TcrA, which is involved in sporangium formation and dehiscence. GsmA is composed of an N-terminal signal peptide for the twin arginine translocation pathway, two tandem bacterial SH3-like domains, and a glucosaminidase domain. Zymographic analysis using a recombinant C-terminal glucosaminidase domain protein showed that GsmA is a hydrolase able to digest cell walls extracted from the vegetative mycelia of A. missouriensis and Streptomyces griseus A gsmA deletion mutant (ΔgsmA) formed apparently normal sporangia, but they released chains of 2 to 20 spores under sporangium dehiscence-inducing conditions, indicating that spores did not completely mature in the mutant sporangia. From these results, we concluded that GsmA is a cell wall hydrolase for digesting peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in A. missouriensis Unexpectedly, flagella were observed around the spore chains of the ΔgsmA mutant by transmission electron microscopy. The flagellar formation was strictly restricted to cell-cell interfaces, giving an important insight into the polarity of the flagellar biogenesis in a spherical spore.IMPORTANCE In streptomycetes, an aerial hypha is compartmentalized by multiple septations into prespores, which become spores through a series of maturation processes. However, little is known about these maturation processes. The rare actinomycete Actinoplanes missouriensis produces sporangiospores, which are assumed to be formed also from prespores generated by the compartmentalization of intrasporangium hyphae via septation. The identification of GsmA as a cell wall hydrolase for the separation of adjacent spores sheds light on the almost unknown processes of sporangiospore formation in A. missouriensis Furthermore, the fact that GsmA orthologues are conserved within the genus Actinoplanes but not in streptomycetes indicates that Actinoplanes has developed an original strategy for the spore maturation in a specific environment, that is, inside a sporangium.

Characterization of Zoospore Type IV Pili in Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis produces terminal sporangia containing a few hundred flagellated spores. After release from the sporangia, the spores swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and begin to germinate in niches for vegetative growth. Here, we report the characterization and functional analysis of zoospore type IV pili in A. missouriensis The pilus gene (pil) cluster, consisting of three apparently σFliA-dependent transcriptional units, is activated during sporangium formation similarly to the flagellar gene cluster, indicating that the zoospore has not only flagella but also pili. With a new method in which zoospores were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed relatively easily using transmission electron microscopy, showing 6 ± 3 pili per zoospore (n = 37 piliated zoospores) and a length of 0.62 ± 0.35 µm (n = 206), via observation of fliC-deleted, nonflagellated zoospores. No pili were observed in the zoospores of a prepilin-encoding pilA deletion (ΔpilA) mutant. In addition, the deletion of pilT, which encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the frequency of pilus retraction. Several adhesion experiments using wild-type and ΔpilA zoospores indicated that the zoospore pili are required for the sufficient adhesion of zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes conserve the pil cluster, which indicates that the zoospore pili yield an evolutionary benefit in the adhesion of zoospores to hydrophobic materials as footholds for germination in their mycelial growth.IMPORTANCE Bacterial zoospores are interesting cells in that their physiological state changes dynamically: they are dormant in sporangia, show temporary mobility after awakening, and finally stop swimming to germinate in niches for vegetative growth. However, the cellular biology of a zoospore remains largely unknown. This study describes unprecedented zoospore type IV pili in the rare actinomycete Actinoplanes missouriensis Similar to the case for the usual bacterial type IV pili, zoospore pili appeared to be retractable. Our findings that the zoospore pili have a functional role in the adhesion of zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella properly according to their different purposes provide an important insight into the cellular biology of the zoospore.