Site-specific recombination-based genetic system for reporting transient or low-level gene expression.
We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene gfp in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent Enterobacter cloacae JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the cI repressor gene, which is flanked by att sites, and the subsequent irreversible expression of gfp in the original cell and in its progeny. In culture, nearly 100% of an E. cloacae JL1157(pAraLHB) population expressed gfp after exposure to 6.5 to 65 microM arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment.
Casavant
N Carol
NC
Department of Agronomy, Iowa State University, Ames, IA 50011, USA.
Beattie
Gwyn A
GA
Phillips
Gregory J
GJ
Halverson
Larry J
LJ
D001089:Arabinose / Q000096:biosynthesis*; D015374:Biosensing Techniques / Q000379:methods*; D016972:Enterobacter cloacae / Q000378:metabolism*; D015870:Gene Expression; D005821:Genetic Techniques; D001467:Hordeum / Q000382:microbiology; D011995:Recombination, Genetic
D001089:Arabinose