Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

Abstract

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR+ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10−10 M versus 10−7 M).