Spatial organization of the gene expression hardware in Pseudomonas putida

Area of Science:

• Microbiology
• Molecular Biology
• Cell Biology

Background:

• Gene expression involves transcription (DNA to RNA) and translation (RNA to protein).
• The spatial organization of these processes within bacterial cells is crucial for efficiency and regulation.
• Understanding the compartmentalization of cellular machinery provides insights into bacterial physiology.

Purpose of the Study:

• To investigate the spatial organization of ribosomes, RNA polymerase (RNAP), and the nucleoid in Pseudomonas putida.
• To determine if gene expression components segregate within the bacterial cytoplasm.
• To assess the stability of this organization under different growth conditions and perturbations.

Main Methods:

• Construction of Pseudomonas putida strains with fluorescently tagged ribosomal proteins and/or RNAP.
• Microscopy techniques to visualize the spatial distribution of tagged components and the nucleoid.
• Physiological approaches to perturb cellular conditions and observe effects on localization.

Main Results:

• Ribosomes and RNAP showed distinct localization patterns relative to the bacterial nucleoid.
• Ribosomes were separated from the nucleoid, while RNAP co-localized with the nucleoid.
• This spatial segregation was maintained under various growth conditions (exponential, stationary) and carbon sources.
• The segregation persisted despite cytoplasmic perturbations, such as polyhydroxyalkanoate granule accumulation.
• Inhibitors like chloramphenicol and rifampicin blurred, but did not eliminate, the observed phase separation.

Conclusions:

• Gene expression machinery in Pseudomonas putida exhibits a degree of physical separation, suggesting a phase separation phenomenon.
• This spatial division separates transcription (RNAP-nucleoid association) from translation (ribosome localization).
• mRNA likely translocates from transcription sites to ribosome-rich regions for efficient protein synthesis.