A novel genetic system to direct programmed, high-level gene expression in natural environments.
C. Casavant, L. J. Halverson, G. A. Beattie and G. Phillips.

Many environmental applications of microorganisms require active microbial functions following introduction into natural habitats. Most current gene regulation strategies involve an inducer that must be continually present at significant levels to maintain maximal activity. Unfortunately, this condition cannot be guaranteed in nature, since inducers may not only be transiently present but also present in insufficient quantity for maximal activity. To address this problem, we have developed a novel genetic system that permits bacteria introduced into natural habitats to tightly repress target gene expression when it is not needed and rapidly and irreversibly induce high-level gene expression when the appropriate effector molecule is present. In this system, a target gene, which is controlled by the Lambda promoter PL, is strongly repressed by the Lambda cI repressor. Irreversible induction then occurs when an effector signal induces the synthesis of the excisionase/integrase (xis/int) genes, which are responsible for excision of the cI repressor gene from att or pseudo-att sites within the genome. We tested this system in E. coli by using the PBAD promoter of the arabinose operon as the signal sensitive promoter to direct expression of Xis/Int. Introduction of arabinose into the culture medium resulted in irreversible, high-level expression of GFP. Ultimately, we intend to use this system in a Pseudomonas strain for in situ detection of pollutant bioavailability in soil to assess whether the bacterial biosensor was ever exposed to the pollutant in its life history. However, based on preliminary work PL is a weak promoter in Pseudomonas fluorescens. To address this, we have developed a system where a strong constitutive promoter PnptII in P. fluorescens is placed upstream of the PL and is repressed by cI; we have shown this to work in E. coli. This genetic system may not only provide a unique means to direct programmed gene expression in natural habitats, but may also facilitate the identification of genes that are only expressed in situ.