Spatial organization of the gene expression hardware in Pseudomonas putida

Abstract

The localization of ribosomes, RNA polymerase (RNAP) and the nucleoid of Pseudomonas putida cells has been inspected with genetic, microscopical and physiological approaches. To this end, strains of P. putida were constructed with fluorescent tags to either ribosomal proteins or the RNAP or both. Their relative positions in respect to the bacterial DNA revealed the separation of the ribosomal pool from the nucleoid, which however co-localized entirely with the tridimensional distribution of RNAP. This split was kept under all growth conditions: exponential versus stationary and different carbon sources. To test the robustness of what appeared to be a phase separation phenomenon, different types of perturbations were entered. In one case, cells were grown under conditions known to accumulate polyhydroxyalkanoate granules that caused a mechanical impact in the cytoplasm - which failed to destroy the split between the translation versus transcription machineries. However, both chloramphenicol and rifampicin blurred - but not eliminated - the boundaries between the phases. The picture that emerges from all these data is not only that the different components of the gene expression hardware are physically divided but also that a large share of the mRNAs ought to move from their site of transcription towards ribosome-rich regions of the cell.