Comparative analysis of anti-restriction activities of ArdA (ColIb-P9) and Ocr (T7) proteins.
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.
Zavilgelsky
G B
GB
State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia. zavilgel@genetika.ru
Kotova
V Yu
VY
Rastorguev
S M
SM
D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013754:Tetracyclines / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C096738:ArdA protein, E coli; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C440255:Ocr protein, bacteriophage T7; D012097:Repressor Proteins; D013754:Tetracyclines; D014764:Viral Proteins; C016229:4-epianhydrotetracycline