Co-Expression of PA0290 and PelD Enhances Pseudomonas aeruginosa Biofilm Formation

How individual diguanylate cyclases generate specific outputs within bacterial c-di-GMP networks remains unclear. Here, we characterized PA0290, a PAS-PAC-GGDEF protein of Pseudomonas aeruginosa, and examined its relationship with the c-di-GMP receptor PelD. Deletion or overexpression of PA0290 alone did not significantly affect biofilm formation. A bacterial adenylate cyclase two-hybrid screen identified PelD as a candidate PA0290-interacting protein, and co-expression of PA0290 and PelD markedly enhanced static and flow-cell biofilm formation. Purified PA0290 generated an HPLC product peak with a retention time closely matching that of the authentic c-di-GMP standard, whereas substitution of the conserved GGEEF motif with GGAAF reduced product formation and weakened the biofilm-enhancing phenotype observed upon PA0290-PelD co-expression. PA0290-PelD co-expression did not produce sustained activation of the bulk c-di-GMP-responsive cdrA-lux reporter. Clinical isolates also displayed heterogeneous biofilm-forming capacity and variable PA0290 and pelD transcript abundance. Together, these findings support a functional association between PA0290 and PelD in biofilm regulation. The absence of sustained bulk c-di-GMP-responsive reporter activation suggests that this phenotype is not accompanied by a generalized increase in c-di-GMP-responsive transcription.