title: Creation of stable Pseudomonas aeruginosa promoter-reporter fusion mutants using linear plasmid DNA transformation.
abstract: BACKGROUND
  Pseudomonas aeruginosa is an important opportunistic human pathogen that is commonly encountered clinically in different types of infections. Reporter-gene systems and construction of mutants defective in specific functions are useful tools for studying the cellular physiology and virulence of this organism. The common mutant construction process requires constructing target alleles into large size suicide vector(s) for transformations, and extra steps involved in resolving merodiploids. Here we describe a new approach using linearized plasmid transformation for creating a green fluorescent protein (GFP) reporter gene system to study promoter activities in P. aeruginosa.
  
  
  RESULTS
  We successfully created promoter-reporter fusion plasmids for studying the promoter activity of virulence genes in P. aeruginosa. The promoter of exoenzyme S (a virulence factor) was used in preparation of these fusion plasmids. These fusion plasmids were linearized and used directly to transform P. aeruginosa. Stable P. aeruginosa chromosomally integrated promoter-reporter fusion mutants were obtained. We demonstrated that the promoter of Exoenzyme S gene was activated when P. aeruginosa was grown in a biofilm state, as evidenced by the expression of GFP in these biofilm cells.
  
  
  CONCLUSIONS
  Direct transformation with linearized plasmid DNA provides another avenue to create P. aeruginosa mutants. This new approach eliminates the use of suicide vector(s) for creating P. aeruginosa mutants, and thus speeds up the process mutant construction.
authors.affiliation: Dental and Craniofacial Trauma Research and Tissue Regeneration, US Army Institute of Surgical Research, JB Fort Sam Houston, 3650 Chambers Pass, Bldg 3610, San Antonio, TX, 78234-6315, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.affiliation: Dental and Craniofacial Trauma Research and Tissue Regeneration, US Army Institute of Surgical Research, JB Fort Sam Houston, 3650 Chambers Pass, Bldg 3610, San Antonio, TX, 78234-6315, USA. kai.p.leung.civ@mail.mil.
authors.forename: Kai P
authors.initials: KP
authors.lastname: Leung
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D018441:Biofilms / Q000254:growth & development; D004269:DNA, Bacterial / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D036002:ADP Ribose Transferases; C444820:exoenzyme S
keywords: Green fluorescent protein; Linear DNA transformation; Promoter activity; Pseudomonas aeruginosa