Two glycoside hydrolases decompose the sporangium matrix to release spores during sporangium dehiscence in <i>Actinoplanes missouriensis</i>

The actinomycete <i>Actinoplanes missouriensis</i> grows as a substrate mycelium and forms terminal sporangia containing a few hundred spores surrounded by an unidentified matrix called the sporangium matrix. Upon contact with water, sporangia release spores through a process called sporangium dehiscence, in which the opaque sporangium envelope becomes transparent, and sporangia swell with water, followed by spore release via the breakage of the outer envelope. In this study, we identified two paralogous hydrolases (GimA and GimB) required for sporangium dehiscence. Transcription of <i>gimA</i> and <i>gimB</i> is activated during sporangium dehiscence by a global transcriptional activator, TcrA. GimA and GimB are composed of an N-terminal signal peptide, glycoside hydrolase domain, and galactose-binding-like domain. No phenotypic changes were observed between the wild-type strain and single-gene null mutants of <i>gimA</i> and <i>gimB</i>. The double mutant (Δ<i>gimA</i>Δ<i>gimB</i> strain) formed normal sporangia, but spores were not released under dehiscence-inducing conditions; the sporangium envelope became transparent, but spores remained in the sporangium matrix. The release of spores from Δ<i>gimA</i>Δ<i>gimB</i> sporangia was immediately induced by the exogenous addition of recombinant GimA or GimB. Thin-layer chromatography revealed that GimA and GimB cleaved the substrate polysaccharide at different positions. Null mutants of genes within a putative polysaccharide biosynthesis gene cluster adjacent to <i>gimB</i> generated distorted sporangia, and no sporangium matrix was produced in one of the mutants. We conclude that a major component of the sporangium matrix is a polysaccharide consisting of repeating oligosaccharides and that GimA and GimB hydrolyze this polysaccharide to release spores during sporangium dehiscence.IMPORTANCEDispersal by means of zoospores is a common feature in the life cycle of many lower eukaryotes, including chytrid fungi, oomycetes, and some bacteria of actinomycetes. However, the molecular mechanisms underlying zoospore release from (zoo)sporangia remain largely unknown. Here, we revealed that two paralogous glycoside hydrolases (GimA and GimB) play essential roles in the release of spores, which can swim in aquatic environments as zoospores, from sporangia in <i>Actinoplanes missouriensis</i>, a filamentous soil-inhabiting bacterium. During sporangium dehiscence, GimA and GimB are produced and secreted extracellularly to hydrolyze the polysaccharide component of the sporangium matrix that encapsulates the spores. This study clarifies an unprecedented molecular mechanism in the process of zoospore release from the sporangia.