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GrainGenes Reference Report: AEM-68-3588

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Reference	AEM-68-3588
Title	Site-specific recombination-based genetic system for reporting transient or low-level gene expression
Journal	Applied and Environmental Microbiology
Year	2002
Volume	68
Pages	3588-3596
Author	Casavant NC Beattie GA Phillips GJ Halverson LJ
Abstract	Summary: 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 micromolar 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
Keyword	[ Hide all but 1 of 53 ] application arabad promoter arabinose availability bacterial bacterial genes bacteriophage barley barley rhizosphere barley seedlings biological control cell cell wall enterobacter enterobacter cloacae environment escherichia coli excision exposure gene germination gfp high-level host in vivo induction legume localization microbiology microcosm molecule monitoring plasmid population production progeny promoter recombination reporter reporter gene repression rhizosphere seed seedling selection site-specific recombination soil subsequent target tips transient vectors washington

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