title: FleQ of Pseudomonas putida KT2440 is a multimeric cyclic diguanylate binding protein that differentially regulates expression of biofilm matrix components.
abstract: The intracellular signal molecule cyclic di-GMP (c-di-GMP) is an important element in regulation of biofilm formation by bacteria. In Pseudomonas aeruginosa, FleQ functions as a c-di-GMP-dependent transcriptional regulator of expression of flagellar genes and the exopolysaccharide (EPS) Pel, a component of the biofilm extracellular matrix. In the plant-beneficial bacterium Pseudomonas putida KT2440, a mutation in fleQ reduces biofilm formation and colonization of plant surfaces. Using isothermal titration calorimetry and electrophoretic mobility shift assays, we show in this work that FleQ of P. putida interacts with c-di-GMP and directly binds the promoter regions of flagellar and EPS genes. Data obtained by analytical gel filtration and ultracentrifugation indicate that FleQ is in multiple oligomeric states in solution (dimers, tetramers and hexamers), which do not show altered equilibrium in the presence of c-di-GMP. DNA binding is independent of c-diGMP, although it is favored by the second messenger in the case of the promoter of the operon responsible for synthesis of the species-specific EPS Pea. Analysis of expression using transcriptional fusions showed an influence of FleQ upon two of the four EPS operons under regular growth conditions. Finally, a consensus sequence for promoter recognition by FleQ in P. putida is also proposed.
authors: Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, Granada 18008, Spain. Electronic address: nene.molina@eez.csic.es.
authors: María Antonia
authors: MA
authors: Molina-Henares
authors: Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, Granada 18008, Spain. Electronic address: maribel.ramos@eez.csic.es.
authors: María Isabel
authors: MI
authors: Ramos-González
authors: Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, Granada 18008, Spain. Electronic address: abdelali.daddaoua@eez.csic.es.
authors: Abdelali
authors: A
authors: Daddaoua
authors: Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, Granada 18008, Spain. Electronic address: anamfe@eez.csic.es.
authors: Ana María
authors: AM
authors: Fernández-Escamilla
authors: Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, Granada 18008, Spain. Electronic address: manuel.espinosa@eez.csic.es.
authors: Manuel
authors: M
authors: Espinosa-Urgel
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018441:Biofilms / Q000254:growth & development*; D002151:Calorimetry; D002850:Chromatography, Gel; D016384:Consensus Sequence; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005407:Flagella / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011135:Polysaccharides, Bacterial; C062025:bis(3',5')-cyclic diguanylic acid; D000251:Adenosine Triphosphatases; D006152:Cyclic GMP
keywords: Biofilm regulation; Electrophoretic mobility shift assay; Exopolysaccharide; Isothermal titration microcalorimetry