Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation [Ribo-seq]

Translational control during the intricate process of sporulation in Bacillus subtilis as a response to nutrient limitation is still underexplored. Here, we employed a comprehensive approach including RNA-seq, ribosome profiling and fluorescence microscopy to dissect the translational landscape of B. subtilis during sporulation. We identified two events of translation silencing and described the spatiotemporal changes in the subcellular location of translational machinery during sporulation. Using a triple knock-out strain (3KO) of zinc-independents paralogs of three zinc-dependent ribosomal proteins L31, L33 and S14, we investigated the potential regulatory role of ribosome during sporulation. The 3KO strain exhibited delayed sporulation, reduced germination efficiency, and dysregulated translation including expression of key metabolic and sporulation-related genes as well as disruptions in translation silencing, particularly in late sporulation. RNA was isolated from sporulating Bacillus subtilis WT and 3KO (delta RpmEB, RpmGC, RpsNB) strains at eight timepoints. Cultures were treated with chloramphenicol, and mRNA and ribosomal footprints were purified. rRNA depletion was performed, and cDNA libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina. Sequencing was carried out on an Illumina NextSeq500, generating single-end 50bp reads.