Second Messenger c-di-GMP Modulates Exopolysaccharide Pea-Dependent Phenotypes via Regulation of eppA Expression in Pseudomonas putida.

The exopolysaccharide (EPS) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida. The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through an unknown mechanism(s). Herein, we found that c-di-GMP modulates wrinkly colony morphology via the regulation of expression of eppA (PP_5586), a small individually transcribed gene of 177 bp, and this gene was adjacent to the upstream region of the pea cluster. Phenotype observation revealed that eppA was essential for Pea-dependent phenotypes. The deletion of eppA led to a smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with loss of the entire pea operon. eppA expression was positively regulated by c-di-GMP via the transcriptional effector FleQ, and eppA was essential for the c-di-GMP-induced wrinkly colony morphology. Structure prediction results implied that EppA had two transmembrane regions, and Western blotting revealed that EppA was located on the cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on the transcriptomic profile of P. putida. A bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in the pea cluster and adjacent operons. Overall, these findings reveal that EppA is essential for Pea-dependent phenotypes and that c-di-GMP modulates Pea-dependent phenotypes via regulation of eppA expression in P. putida. IMPORTANCE Microbe-secreted EPSs are high-molecular-weight polysaccharides that have the potential to be used as industrially important biomaterials. The EPS Pea in P. putida is essential for wrinkly colony morphology and pellicle formation. Here, we identified a function-unknown protein, EppA, which was also essential for Pea-dependent wrinkly colony morphology and pellicle formation, and EppA was probably involved in Pea secretion. Meanwhile, our results indicated that the second messenger c-di-GMP positively regulated the expression of EppA, resulting in Pea-dependent wrinkly colony morphology. Our results reveal the relationship of c-di-GMP, EppA, and Pea-dependent phenotypes and provide a possible pathway to construct genetically engineered strains for high Pea production.