title: A GFP-lacZ bicistronic reporter system for promoter analysis in environmental gram-negative bacteria.
abstract: Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods.
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnología, CSIC, Cantoblanco, Madrid, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Silva-Rocha
authors.forename: Victor
authors.initials: V
authors.lastname: de Lorenzo
mesh_terms: D001565:Benzoates / Q000494:pharmacology; D002876:Chromosomes, Bacterial / Q000235:genetics; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D007763:Lac Operon; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D001616:beta-Galactosidase
chemical_list: D001565:Benzoates; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase