A stringently controlled expression system for analysing lateral gene transfer between bacteria

The lateral transfer of genetic information among microorganisms is a major force driving the outstanding adaptability of microbial communities to environmental changes. Until now little information has been obtained on gene transfer in natural ecosystems, We present here a genetic circuit for detecting and quantifying horizontal gene transfer from a defined donor microorganism to recipient organisms in the absence of selection for a recipient-specific phenotype, The system consists of an engineered lacZ (encoding beta-galactosidase) reporter gene whose expression is controlled by a synthetic regulatory element based on a fusion between the Pr promoter-operator from lambda bacteriophage and the 5' non-coding leader region of the tnp gene encoding the IS10 transposase function, Expression of this reporter cassette in the recombinant microorganism is completely shut down by two chromosomally encoded trans-acting repressors working at the level of transcription (the CI-EK117 protein from the lambda phage), and at the level of translation (the antisense RNA-OUT of the IS10 element), When the reporter element is transferred to a different host by any mechanism, it escapes repression and becomes expressed, The system was validated with Pseudo-monas putida, and conjugational transfer frequencies of the reporter element as low as 10(-6) were detected, The modular design and broad host range of the genetic circuit, in combination with biomarkers which permit real-time in situ detection, will facilitate the monitor-ing of gene flow in a non-disruptive manner within the environment.