Components of the Clp complex are required for sporangium formation and dehiscence in Actinoplanes missouriensis

The actinomycete Actinoplanes missouriensis forms terminal sporangia that contain a few hundred spores. When immersed in water, the sporangium opens to release spores, which start swimming using flagella, via a process referred to as sporangium dehiscence. In this study, we conducted a functional analysis of genes encoding the components of the caseinolytic protease (Clp) complex, which comprises ATPase (chaperone) subunits (ClpX and ClpC) and proteolytic subunits (ClpP1–4). The clpX null mutant (Δ clpX ) strain formed sporangia smaller than the wild-type strain. The small sporangia scarcely opened under conditions that induce sporangium dehiscence in the wild-type strain. Consistently, the number of released spores was three orders of magnitude lower in the Δ clpX strain than in the wild-type strain. S1 nuclease mapping determined two transcriptional start points of clpX (TSS U and TSS D for upstream and downstream, respectively). A housekeeping sigma factor-dependent promoter and a FliA-family sigma factor-dependent promoter were found upstream of TSS U and TSS D , respectively. A gene complementation test showed that apparently normal sporangium formation in the Δ clpX strain was restored by the introduction of clpX with either of the two promoters, whereas both promoters were required for sporangium dehiscence. Meanwhile, mutant strains that produced ClpC with T30S or I33F/L34F replacements, which presumably reduced their substrate-binding activity, produced sporangia with irregular shapes. Furthermore, gene disruption experiments of four putative proteolytic subunit genes indicated that clpP3 is conditionally involved in sporangium dehiscence. We concluded that sporangium formation and dehiscence are regulated at the post-translational level via proteolysis by Clp complexes in A. missouriensis . IMPORTANCE Actinoplanes missouriensis has a complex life cycle, in which the sporangium containing a few hundred flagellated spores is the most characteristic structure. Spores are released from sporangia via a process called sporangium dehiscence. We have revealed that sporangium formation and dehiscence are regulated by several transcriptional regulators; however, post-transcriptional regulation of sporangium formation and dehiscence remains unknown. In the present study, we revealed that two ATPase components of the Clp complex, ClpC and ClpX, and a proteolytic component, ClpP3, are involved in sporangium formation and/or dehiscence. This study indicates that in addition to transcriptional regulation, proteolysis regulated by Clp complexes is another crucial factor in the morphological development of A. missouriensis .