biofilms9-89, updated on 27 Jul 2020
https://doi.org/10.5194/biofilms9-89
biofilms 9 conference
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License.

Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa

Anna Scherhag, Martina Rüger, Katrin Gerbracht, Jaqueline Rehner, Susanne Zehner, and Nicole Frankenberg-Dinkel
Anna Scherhag et al.
  • TU Kaiserslautern, Biology, Microbiology, Germany (ascherha@bio.uni-kl.de)

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.

 

 

[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.

[2] Römling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629–639.

[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.

[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17–23.

[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434–2446.

[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.  (2012). Signaling. J. Bacteriol. 194: 4837–4846

 

How to cite: Scherhag, A., Rüger, M., Gerbracht, K., Rehner, J., Zehner, S., and Frankenberg-Dinkel, N.: Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa, biofilms 9 conference, Karlsruhe, Germany, 29 September–1 Oct 2020, biofilms9-89, https://doi.org/10.5194/biofilms9-89, 2020