Biochemical and molecular characterization of a restriction endonuclease Tvu2HI from Thermoactinomyces vulgaris 2H and study of its R-M system

Abstract

A bacterial strain 2H isolated from soil and identified as Thermoactinomyces vulgaris produce a potent Type II restriction endonuclease activity that has been extracted by a PEG/dextran aqueous two-phase system. Optimal temperature for the restriction endonuclease activity was 55–65°C. Specific DNA cleavage was obtained at pH range 7–10 and 10–20mM MgCl2. Restriction cleavage analysis followed by sequencing confirms GG^CC as the recognition sequence. This enzyme, named Tvu2HI, is a thermostable isoschizomer of the mesophilic prototype restriction endonuclease HaeIII. Sequencing of the complete Thermoactinomyces vulgaris 2H genome revealed the presence of two adjacent ORFs coding for the restriction endonuclease Tvu2HI and the corresponding methyltransferase; an ORF coding for a putative Vsr nicking enzyme was found close to those coding for the Tvu2HI restriction-modification system. Phylogenetic analysis based on sequence alignment suggests a common origin of Tvu2HI R-M system with HaeIII-like R-M systems. This is the first investigation dealing with a Type II restriction endonuclease identified in a natural isolate of the genus Thermoactinomyces.