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

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Abstract
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 β‐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.

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