Specific polar localization of ribosomes in Bacillus subtilis depends on active transcription

Abstract

The large subunit of ribosomes in Bacillus subtilis was tagged by generation of a fusion of ribosomal protein L1 to blue fluorescent protein (BFP). The fusion was fully active and localized around the nucleoids, predominantly close to the cell poles, in growing cells. However, in stationary phase cells, and in growing cells treated with rifampicin, L1-BFP was distributed throughout the cells, in contrast to cells treated with chloramphenicol, in which ribosomes still localized around nucleoids. These data show that specific localization of ribosomes is not due to nucleoid exclusion, but is a dynamic process due to active synthesis of RNA. Dual labelling of ribosomes and cold shock proteins (CSPs) tagged with green fluorescent protein (GFP) revealed colocalization of both protein classes. CSPs are implicated in coupling of transcription with translation and may bridge the spatial separation of ribosomes and nucleoid-associated RNA polymerase.

Fig. 1. Fluorescence microscopy of Bacillus subtilis cells. Blue panels show localization of L1-BFP, red panels shows SYTO59 DNA stain. Note that the gene encoding L1 is rplA. (A) Strain JM1 (rplA-bfp) growing at mid-exponential phase. (B) Deconvolution of Z-series of images taken through growing JM1 cells. (C) Growing cells of strain MW1 (amy::pxylgfp) expressing GFP only; upper panel, GFP; lower panel, Nomarski DIC. (D) JM1 cells during stationary phase; upper panel, Nomarski; lower panel, L1-BFP. (E) JM1 cells treated with chloramphenicol (inhibitor of translation) or (F) rifampicin (inhibitor of transcription) for 30 min. (G) Strain JM5 (rplA-bfp, origin GFP tag) during exponential growth. (H) Strain JM4 (rplA-bfp, cspB-gfp) expressing L1-BFP and CspB-GFP during exponential growth. (I) JM1 cells in blue or green filter. (J) MW2 (cspB-gfp) in blue or green filter sets. White lines show septa between cells, white bars indicate 2 µm.

Fig. 2. Chart showing number of chromosome origins found not coincident, partially overlapping or completely overlapping with spaces occupied by ribosomes.

Fig. 3. Model for coupling of spatially separated sites of transcription and translation in bacteria.