Site-Specific Recombination-Based Genetic System for Reporting Transient or Low-Level Gene Expression

Thumbnail Image
Date
2002-07-01
Authors
Casavant, N. Carol
Beattie, Gwyn
Phillips, Gregory
Halverson, Larry
Major Professor
Advisor
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Authors
Person
Beattie, Gwyn
Professor
Research Projects
Organizational Units
Organizational Unit
Microbiology
Microbiology allows you to learn about the microorganisms that affect us every day and how they interact with their surroundings. Through the program, you will be equipped with the knowledge to work in areas related to agriculture, the environment and medicine.
Organizational Unit
Journal Issue
Is Version Of
Versions
Series
Department
MicrobiologyAgronomy
Abstract

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 gfpafter exposure to 6.5 to 65 μM 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.

Comments

This is an article from Applied and Environmental Microbiology 68 (2002): 3588, doi: 10.1128/AEM.68.7.3588–3596.2002. Posted with permission.

Description
Keywords
Citation
DOI
Copyright
Tue Jan 01 00:00:00 UTC 2002
Collections