BPS2026 – Droplets on the DNA: Can phase-separating transcription factors form a bacterial nucleolus-like hub?

Bacterial transcription is increasingly recognized as a spatially organized process, with transcriptional condensates forming to maximize ribosomal RNA (rRNA) synthesis, but the mechanisms of condensate formation and their role in modulating transcription remain unresolved. We report that NusG, the only transcription factor conserved across all domains of life, is a key organizer of these structures. Combining single-molecule and widefield studies in live cells, we show that NusG clusters persist across nutrient states, scale with DNA content, and track replication origins. Clustering resists diverse perturbations—including translation inhibition and temperature shifts—but it is abolished when transcription is halted by rifampicin or starvation, establishing rRNA anti-termination as the dominant cluster function. Blocking Rho termination reduced cluster brightness but did not abolish foci, suggesting Rho tunes condensate properties without being essential to formation. Mobility analyses then demonstrate that clusters are chromosome-anchored and move with the replication origin, positioning them close to rRNA operons throughout the cell cycle. We complement this with in vitro analyses to understand cluster formation. Purified NusG readily undergoes liquid-liquid phase separation at physiological salt and micromolar concentrations, producing dynamic droplets that fuse and dissolve with hexanediol. We show that NusG condensates can host active transcriptional assemblies, recruiting both transcription initiation and elongation complexes. These findings, together with NusG’s ability to form both specific and transient interactions—including non-specific contacts with DNA—establish NusG as a central organizer of transcriptional condensates. Our study supports a model in which “droplets on the DNA” act as nucleolus-like hubs that tune rRNA transcription and bacterial physiology.