Arginine kinase McsB and ClpC complex impairs the transition to biofilm formation in Bacillus subtilis.

Robust biofilm formation on host niches facilitates beneficial Bacillus to promote plant growth and inhibit plant pathogens. Arginine kinase McsB is involved in bacterial development and stress reaction by phosphorylating proteins for degradation through a ClpC/ClpP protease. Conversely, cognate arginine phosphatase YwlE counteracts the process. Regulatory pathways of biofilm formation have been studied in Bacillus subtilis, of which Spo0A∼P is a master transcriptional regulator, which is transcriptionally activated by itself in biofilm formation. Previous studies have shown that Spo0A∼P transcript regulation controls biofilm formation, where MecA binds ClpC to inhibit Spo0A∼P-dependent transcription without triggering degradation. It remains unclear whether McsB and ClpC regulate biofilm formation together and share a similar non-proteolytic mechanism like MecA/ClpC complex. In this study, we characterized McsB and ClpC as negative regulators of biofilm formation and matrix gene eps expression. Our genetic and morphological evidence further indicates that McsB and ClpC inhibit eps expression by decreasing the spo0A and sinI expression, leading to the release of SinR, a known repressor of eps transcription. Given that the spo0A and sinI expression is transcriptionally activated by Spo0A∼P in biofilm formation, we next demonstrate that McsB interacts with Spo0A directly by bacterial two-hybrid system and Glutathione transferase pull-down experiments. Additionally, we present that McsB forms a complex with ClpC to dampen biofilm formation in vivo. Finally, we show that YwlE acts as a positive regulator of biofilm formation, counteracting the function of McsB. These findings suggest that McsB, ClpC, and YwlE play vital roles in the transition to biofilm formation in Bacillus subtilis, providing new insights into the regulatory mechanisms underlying biofilm development and sharing a similar non-proteolytic mechanism in biofilm formation as MecA/ClpC complex.