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Comparative Analysis of Anti-restriction Activities of ArdA (ColIb-P9) and Ocr (T7) Proteins

G. B. Zavilgelsky*, V. Yu. Kotova, and S. M. Rastorguev

State Research Institute of Genetics and Selection of Industrial Microorganisms, 1-yi Dorozhnyi Proezd 1, 117545 Moscow, Russia; fax: (495) 315-0501; E-mail: zavilgel@genetika.ru

* To whom correspondence should be addressed.

Received January 30, 2008
Anti-restriction proteins ArdA and Ocr are specific inhibitors of type I restriction-modification enzymes. The IncI1 transmissible plasmid ColIb-P9 ardA and bacteriophage T7 0.3(ocr) genes were cloned in pUC18 vector. Both ArdA (ColIb-P9) and Ocr (T7) proteins inhibit both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. ColIb-P9 ardA, T7 0.3(ocr), and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the PltetO-1 promoter, which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the PltetO-1 promoter can be regulated over an up to 5000-fold range by supplying anhydrotetracycline to the E. coli MG1655Z1 tetR+ cells. Effectiveness of the anti-restriction activity of the ArdA and Ocr proteins depended on the intracellular concentration. It is shown that the dissociation constants Kd for ArdA and Ocr proteins with EcoKI enzyme differ 1700-fold: Kd (Ocr) = 10-10 M, Kd (ArdA) = 1.7·10-7 M.
KEY WORDS: anti-restriction proteins, type I restriction-modification enzymes, ArdA, Ocr, luxCDABE, bioluminescence, luciferase

DOI: 10.1134/S0006297908080087