A novel PAS –GGDEF -EAL protein involved in regulation of motility in Pseudomonas putida

HERRERA SEITZ, K 1 AND SHINGLER, V 2

Cuernavaca, México.

Congreso; X BLAST (Bacterial Locomotion and Signal Transduction),; 2009

BLAST

A NOVEL PAS-GGDEF-EAL PROTEIN INVOLVED IN REGULATION OF MOTILITY IN PSEUDOMONAS PUTIDA Herrera Seitz, K 1 and Shingler, V 2 1 IIB, FCEyN, Univ. Nac. del Mar del Plata, 2 Molecular Biology Department, Umeå University, Sweden. Chemotaxis allows motile bacteria to respond to chemical gradients to relocate themselves near the source of nutrients. Soil Pseudomonads are known to be able to grow in a wide variety of carbon sources, including many that are considered environmentally toxic. Unlike previously characterized chemotactic responses in Pseudomonas strains, taxis of P. putida CF600 and P. putida KT2440 towards methyl-phenols is dependent upon its ability to metabolize the compound, rather than on a classical ligand-binding chemoreceptor. E. coli metabolism-dependent taxis responses are mediated by the Aer receptor that is closely related to chemoreceptors, but which contains a FAD-binding PAS sensory domain. P. putida possesses three aer-like genes. During analysis of the Aer-like receptors of P. putida, Aer-1 was found to be encoded in a dicistonic operon with PP2258, a PAS-GGDEF-EAL domain protein. Our attention was drawn to PP2258 because a null mutant was found to exhibit a general motility defect on solid, but not in liquid, media (Sarand et al., 2008). GGDEF- and EAL-domains are associated with diguanylate cyclase and phosphodiesterase activities that are involved in turnover of the near ubiquitous bacterial second messenger c-di-GMP. The levels of c-di-GMP can modify cells behavior and motility; therefore we reasoned that PP2258 link to motility via c-di-GMP signaling. As a first approach to test this idea, the biochemical properties of wild type and mutant derivatives of PP2258 were studied using over expression of the protein both in E. coli and P. putida. When PP2258 was over expressed in E. coli or P. putida cells, c-di-GMP levels were markedly increased compared to those of control cells, although accumulation of c-di-GMP was much lower in E. coli than in P. putida extracts. Alanine substitutions of the GGDEF domain associated with c-di-GMP synthesis causes a major decrease c-di-GMP accumulation, while an alanine substitution in EAL domain associated with c-di-GMP hydrolysis led to a >7-fold increase in accumulation. Together, these results suggest that PP2258 could be one of the rare examples of a dual GGDEF-EAL domain protein where both domains are catalytically active. References: Sarand, I., Österberg, S., Holmqvist, S., Holmfeldt, P. Skärfstad, E., Parale, R. E., & Shingler, V. (2008) Metabolism-dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer-like proteins of Pseudomonas putida. Environ. Microbiology. 10:1320-1334