Exopolysaccharides (EPSs) 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 unknown mechanism(s). Herein, we found that c-di-GMP modulated wrinkly colony morphology via regulating expression of
eppA
(
PP_5586
), a small individually transcribed gene with 177 base pairs, and this gene was adjacent to the upstream of
pea
cluster. Phenotype observation revealed that
eppA
was essential for Pea-dependent phenotypes. The deletion of
eppA
led to smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with the 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 blot revealed that EppA was located on cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on transcriptomic profile of
P. putida
. Bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in
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 regulating
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 possible pathway to construct genetically engineered strain for high Pea production.