ABSTRACT
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.
Purification and characterization of a cell wall hydrolase encoded by the cw1A gene ofBacillus subtilis