Phase separation of mycobacterial Rho factor is associated with acid stress

Area of Science:

• Bacterial transcription regulation
• Protein biophysics
• Microbial stress response

Background:

• Rho is a bacterial transcription termination factor.
• Some mycobacterial Rho proteins possess unique additional regions.
• These regions are predicted to be intrinsically disordered and involved in Liquid-Liquid Phase Separation (LLPS).

Purpose of the Study:

• To investigate LLPS in mycobacterial Rho proteins.
• To determine the role of additional regions in Rho-mediated LLPS.
• To explore the in vivo relevance of Rho LLPS in mycobacteria.

Main Methods:

• Bioinformatic analysis of Rho proteins from mycobacterial species.
• In vitro characterization of Rho condensate formation.
• In vivo observation of Rho phase separation in *Mycolicibacterium smegmatis*.
• Genetic manipulation of Rho to assess the function of additional regions.

Main Results:

• Rho from *Mycolicibacterium smegmatis* forms in vitro and in vivo condensates via LLPS.
• LLPS is dependent on the additional regions present in mycobacterial Rho.
• Intrinsically disordered regions alone are sufficient for in vitro droplet formation.
• Rho phase separation occurs in *M. smegmatis* during acid stress and impacts cell fitness.

Conclusions:

• LLPS is a widespread mechanism for mycobacterial Rho assembly into functional condensates.
• The additional regions of Rho are critical for its phase separation properties.
• Rho-mediated LLPS may play a role in regulating transcription and enabling stress tolerance in mycobacteria.