Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa

The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2]. Pseudomonas aeruginosa, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].
One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O₂ [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.
To establish these methods, the two known phosphodiesterases, PdeH from Escherichia coli [5] and RocR from P. aeruginosa [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an E. coli phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity in vivo.

 

 

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