Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

Abstract

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence. IMPORTANCE BldC is a global transcriptional regulator that acts as a "brake" in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.