Isolation and characterization of the dut gene of Escherichia coli. I. Cloning in thermoinducible plasmids

Abstract

We have constructed thermoinducible plasmids carrying the gene (dut) for the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli. A 9.4-kb BamHI restriction enzyme fragment carrying the dut gene was inserted into the runaway-replication plasmid pKN402A (Uhlin et al., Gene 6 (1979) 91-106). Strains carrying such plasmids increased their dUTPase activity considerably. In minimal medium a 200-fold increase was demonstrated. A smaller (1.5-kb) SacI-BamHI fragment from the dut region was also cloned into pKN402A. The dUTPase production in dut mutant strains carrying this plasmid (pKK141) was only at about wild-type level after temperature shift. To test the hypothesis that the SacI cleavage used affects a control region for the dut gene, we recloned the dut fragment by transferring it from pKK141 into pHUB2 (Bernard et al., Gene 5 (1979) 59-76), a plasmid carrying the phage lambda pL promoter. A 3.6-kb EcoRI-BamHI fragment from pKK141, including the 1.5-kb SacI-BamHI segment from the dut region, was inserted downstream from the pL promoter. When this plasmid was present in a strain containing a thermosensitive lambda repressor gene, thermoinduction of dUTPase was negligible, apparently due to the presence of some termination signals between pL and dut. Therefore, we removed a 1.9-kb EcoRI-SacI fragment from the region between pL and the dut gene and replaced it with a 0.22-kb EcoRI-SacI fragment, obtained from the b2 region of lambda. Strains carrying such a shortened dut-pHUB2 derivative and a temperature-sensitive lambda repressor overproduced dUTPase very dramatically after heat induction. The final level reached was 300-400 times the wild-type level, corresponding to 10% of the total soluble protein. The information obtained, together with analysis of plasmid-directed polypeptide products described by Lundberg et al. (Gene 22 (1983) 127-131) shows that the SacI site is indeed on the promoter-proximal side of the dut gene.