FliW regulates biofilm formation in Geobacter sulfurreducens through interaction with CsrA.

CsrA is a post-transcriptional regulator that controls a wide range of bacterial phenotypes, including carbon metabolism, motility, quorum sensing, virulence, and biofilm formation. In Geobacter sulfurreducens, CsrA modulates both biofilm development and extracellular electron transfer in microbial fuel cells. In this study, we further investigated the regulatory mechanism of CsrA and its role in the formation of electroconductive biofilms in G. sulfurreducens. Bioelectrochemical analyses revealed that a ΔcsrA strain produces biofilms with enhanced electroconductivity compared with the wild-type strain. To identify the molecular basis of this regulation, we explore potential CsrA-binding partners, demonstrating that CsrA interacts with the FliW protein, as reported in other bacteria such as Bacillus subtilis. By utilizing site-directed mutagenesis, we identified that this interaction requires a conserved asparagine residue (N55) in CsrA, the disruption of which prevents the CsrA-FliW complex formation. Interestingly, fliW deletion resulted in reduced biofilm biomass and thickness contrasting with the enhanced phenotypes observed in the ΔcsrA strain. Furthermore, the ΔfliW mutant exhibited a differential expression of transcripts associated with the CsrA regulon in a pattern opposite to that of the ΔcsrA strain. These findings indicate that FliW antagonizes CsrA activity, and that the N55-mediated interaction is essential for this regulatory control. Collectively, these results allow us to propose a model in which the CsrA-FliW interaction acts as a molecular switch to control biofilm formation in G. sulfurreducens. Furthermore, this study expands our understanding of post-transcriptional regulation in electroactive bacteria and highlights the link between regulatory protein interactions, biofilm physiology, and extracellular electron transfer. KEY POINTS: • FliW regulates CsrA activity, thereby affecting biofilm formation. • CsrA regulation influences electroconductive biofilm development in G. sulfurreducens. • CsrA-FliW regulation offers targets to optimize bioenergy and bioremediation systems.