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

Abstract

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.

Bioinformatic and functional characterization of cyclic-di-GMP metabolic proteins in Vibrio alginolyticus unveils key diguanylate cyclases controlling multiple biofilm-associated phenotypes

“…We identified 4 c-di-GMP metabolic genes, including VA4033 , VA1591 , VA0356 , and VA0088 , all with intact active sites for the GGDEF domain ( Table 1 ), involved in the enhanced rugose colony phenotypes on LB agar plates ( Figure 2 ). It is well known that there is a strong correlation between the phenotypes of colony rugosity, biofilm formation and EPS production, and moreover, these phenotypes are often accompanied by elevated intracellular c-di-GMP levels ( Nakhamchik et al, 2008 ; Romling et al, 2013 ; Wu et al, 2020 ; Xiao et al, 2022 ). In general, enhanced EPS production often results in rugose colony phenotypes ( Casper-Lindley and Yildiz, 2004 ; Beyhan et al, 2008 ), a phenomenon also demonstrated in the present work, for example, overexpression strains ∆ VA1591 p1591, ∆ VA4033 p4033, ∆ VA0356 p0356, and ∆ VA0088 p0088 produced rugose colonies that also had strong CR binding capacities ( Figures 2 , 5 ).…”

Becoming settlers: Elements and mechanisms for surface colonization by Pseudomonas putida

“…The designation ‘cfc’ comes from a ‘crinkle‐free colony’, which is the characteristic phenotype of the P. putida reference strain KT2440. The nearly species‐specific Pea EPS is specifically required for the acquisition of the crinkle phenotype induced by increased c‐di‐GMP (Matilla et al, 2011; Xiao et al, 2022).…”