K38 Succinylation of CsrA Regulates Biofilm Formation in Pseudomonas fluorescens PF08 by Modulating c-di-GMP Levels

Pseudomonas fluorescens PF08 is a significant food spoilage organism belonging to a species widely utilized for agricultural biocontrol, yet the post-translational regulation of its biofilm formation remains poorly understood. This work explored the role of lysine succinylation (Ksucc) in regulating CsrA activity, a global post-transcriptional regulator. NanoHPLC-MS/MS identified Lys38 (K38), a highly conserved residue within the CsrA RNA-binding interface, as a functional succinylation site. Site-directed mutagenesis revealed that K38 succinylation significantly inhibits biofilm formation by reducing c-di-GMP levels, exopolysaccharide production, and surface hydrophobicity, while enhancing flagellar-mediated motility. Colonization assays on foods, contact surfaces, and the rhizosphere confirmed that CsrA succinylation restricts biofilm formation across diverse ecological niches. These results identify K38 succinylation of CsrA as a regulatory modification that modulates the transition between motile and sessile lifestyles in P. fluorescens PF08. These findings expand the understanding of post-translational control of biofilm formation in this ecologically versatile bacterium relevant to food spoilage and agricultural environments.