Genetic sensor for strong methylating compounds.

ABSTRACT: Methylating chemicals are common in industry and agriculture and are often toxic, partly due to their propensity to methylate DNA. The Escherichia coli Ada protein detects methylating compounds by sensing aberrant methyl adducts on the phosphoester backbone of DNA. We characterize this system as a genetic sensor and engineer it to lower the detection threshold. By overexpressing Ada from a plasmid, we improve the sensor’s dynamic range to 350-fold induction and lower its detection threshold to 40 ?M for methyl iodide. In eukaryotes, there is no known sensor of methyl adducts on the phosphoester backbone of DNA. By fusing the N-terminal domain of Ada to the Gal4 transcriptional activation domain, we built a functional sensor for methyl phosphotriester adducts in Saccharomyces cerevisiae. This sensor can be tuned to variable specifications by altering the expression level of the chimeric sensor and changing the number of Ada operators upstream of the Gal4-sensitive reporter promoter. These changes result in a detection threshold of 28 ?M and 5.2-fold induction in response to methyl iodide. When the yeast sensor is exposed to different SN1 and SN2 alkylating compounds, its response profile is similar to that observed for the native Ada protein in E. coli, indicating that its native function is retained in yeast. Finally, we demonstrate that the specifications achieved for the yeast sensor are suitable for detecting methylating compounds at relevant concentrations in environmental samples. This work demonstrates the movement of a sensor from a prokaryotic to eukaryotic system and its rational tuning to achieve desired specifications.

SUBMITTER: Moser F 

PROVIDER: S-EPMC3859139 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature