Energy sensing and cyclic di-GMP signalling in P. putida

ANNA ÅBERG, KARINA HERRERA SEITZ, SOFIA ÖSTERBERG AND VICTORIA SHINGLER.

Hannover, Germany

Conferencia; XII International Conference “Pseudomonas”,; 2009

Energy sensing and cyclic di-GMP signalling in P. putida Anna Åberg, Karina Herrera Seitz*, Sofia Österberg and Victoria Shingler Department of Molecular Biology, Umeå University, Umeå, Sweden. *Present address: University Nacional de Mar del Plata, Mar del Plata, Argentina Anna Åberg anna.aberg@molbiol.umu.se Abstract Bacteria use extensive regulatory networks to co-ordinate their physiology to prevailing nutritional and stress conditions. One of the most recently identified, and least understood, involves the nucleotide second messenger cyclic di-GMP (c-di-GMP). This near ubiquitous bacterial signalling molecule has profound effects on numerous bacterial life-style and adaptive process, but in many cases mechanistic information is lacking. C-di-GMP turn-over is mediated by GGDEF- and EAL-domain proteins that mediate opposing diguanylate cyclase and phosphodiesterase activities, respectively. Bacterial genome sequences have shown that GGDEF- and EAL-domains are highly abundant, widely distributed, and often occur within the same protein. However, in most dual GGDEF-EAL-domain proteins only one of the two domains is catalytically active, with the other domain adapted to serve a regulatory role. The experimental system under scrutiny involves the Pseudomonas putida KT2440 derived PP2258 protein that is encoded in a bi-cistronic operon along with the energy sensor Aer1. PP2258 is a tri-domain (PAS-GGDEF-EAL) protein that appears to be one of the rare examples of a c-di-GMP turnover protein in which both catalytic domains are active. Alanine substitutions within the PP2258-SGDEF motif reduce c-di-GMP production, whereas an alanine substitution within the PP2258-EGL domain resulted in > 7-fold increase in c-di-GMP levels as measured by two-dimensional thin layer chromatography. Within the bacterial cell, PP2258 is involved in regulating motility of P. putida since a PP2258 null mutant is defective in motility on plate. However, electron microscopic analysis has revealed that the null mutant possesses the same number of flagella in its polar bundles as the wild-type strain. This suggests that PP2258 regulates motility via c-di-GMP signalling to an adaptor protein, rather than regulating the expression of the flagella components. Possession of a PAS domain combined with co-regulation with an energy-sensor further implicates PP2258 and c-di-GMP signalling in coupling control of motility to the energy status of the cell. We will present our most recent mutagenesis analysis of the PP2258 protein aimed to test this idea.