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

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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

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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 P_ltetO-1 promoter, which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P_ltetO-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 K_d for ArdA and Ocr proteins with EcoKI enzyme differ 1700-fold: K_d (Ocr) = 10^-10 M, K_d (ArdA) = 1.7·10^-7 M.

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KEY WORDS: anti-restriction proteins, type I restriction-modification enzymes, ArdA, Ocr, luxCDABE, bioluminescence, luciferase

DOI: 10.1134/S0006297908080087

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