Mfd interacts with RNA polymerase to modulate flagellum-dependent motility, chemotaxis, and population heterogeneity in the stationary phase of Bacillus subtilis

ABSTRACT Bacillus subtilis is a model for cell differentiation, capable of transitioning between distinct states: a sessile, chained state (often associated with biofilm formation), and a motile, planktonic state. This transition is governed by a complex regulatory network that includes alternative sigma factor-D (σ D ), which drives the expression of genes for autolysins, flagellar biosynthesis, and chemotaxis, resulting in separated, flagellated, motile cells. Although the conserved transcription-coupled repair factor m utation f requency d ecline (Mfd) is best known for resolving stalled RNA polymerase (RNAP) during DNA repair, its involvement in cell differentiation during the stationary phase remains poorly understood. This research shows that in the absence of Mfd translocation or RNAP recruitment, RNAP completes transcription of motility genes more frequently, despite unchanged σ D transcript levels. Increased transcription of the σ D -dependent motility regulon led to greater flagellation; however, swimming motility and pH taxis were paradoxically reduced, indicating that Mfd is required to translate flagellar gene expression into functional motility. Notably, Mfd-deficient cells failed to maintain chained subpopulations, indicating an additional role in sustaining population heterogeneity. These findings reveal a previously unrecognized function of Mfd as an RNAP-modulating factor that coordinates motility gene expression, thereby expanding our understanding of how transcription-coupled repair proteins influence bacterial physiology and behavior under non-proliferative conditions. IMPORTANCE The m utation f requency d ecline (Mfd) enzyme mediates transcription-coupled repair in transcribed genes by directly interacting with RNA polymerase (RNAP) during transcription of coding sequences. However, whether the Mfd factor regulates gene expression associated with adaptations unrelated to DNA repair and mutagenesis remains vague. Here, we show that Mfd regulates the completion of full transcripts of genes that confer swimming motility, chemotaxis, and cell heterogeneity in Bacillus subtilis . Furthermore, we identified the Mfd’s translocase activity and its interaction with RNAP as key elements of this regulation. Therefore, Mfd’s importance in bacterial physiology and adaptation goes beyond DNA repair.