Creation of stable Pseudomonas aeruginosa promoter-reporter fusion mutants using linear plasmid DNA transformation.
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.


FINDINGS
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.


CONCLUSION
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.
['Chen|Ping|P|', 'Leung|Kai P|KP|']
D036002:ADP Ribose Transferases D001426:Bacterial Proteins D001427:Bacterial Toxins D018441:Biofilms D004269:DNA, Bacterial D017930:Genes, Reporter D049452:Green Fluorescent Proteins D006801:Humans D018613:Microscopy, Confocal D009154:Mutation D010957:Plasmids D016133:Polymerase Chain Reaction D011401:Promoter Regions, Genetic D011552:Pseudomonas Infections D011550:Pseudomonas aeruginosa D011993:Recombinant Fusion Proteins D014169:Transformation, Bacterial D014774:Virulence
Pseudomonas aeruginosa Green fluorescent protein Promoter activity Linear DNA transformation