title: Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria.
abstract: BACKGROUND
  The cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes.
  
  
  RESULTS
  We have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD* gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD* constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD* constructs. Furthermore, we have also implemented an inducible system to modulate pleD* expression and intracellular c-di-GMP rises "on demand".
  
  
  CONCLUSIONS
  mini-Tn7pleD* constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.
authors.affiliation: Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain. lorena.romero@eez.csic.es.
authors.forename: Lorena
authors.initials: L
authors.lastname: Romero-Jiménez
authors.affiliation: Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain. david.rodriguez@eez.csic.es.
authors.forename: David
authors.initials: D
authors.lastname: Rodríguez-Carbonell
authors.affiliation: Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain. maritrini.gallegos@eez.csic.es.
authors.forename: María Trinidad
authors.initials: MT
authors.lastname: Gallegos
authors.affiliation: Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain. Juan.Sanjuan@eez.csic.es.
authors.forename: Juan
authors.initials: J
authors.lastname: Sanjuán
authors.affiliation: Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain. dpmendoza@eez.csic.es.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pérez-Mendoza
mesh_terms: D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005827:Genetics, Microbial / Q000379:methods*; D042822:Genomic Instability; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D019761:Phosphorus-Oxygen Lyases / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP