Molecular cloning, sequencing, and expression of the heat-labile uracil-DNA glycosylase from a marine psychrophilic bacterium, strain BMTU3346

Abstract

The gene encoding a heat-labile uracil-DNA glycosylase (UDG) from a psychrophilic, gram-positive marine strain (BMTU3346) has been cloned, sequenced, and expressed in Escherichia coli. The UDG is a cold-active enzyme with an apparent temperature optimum of 35 degrees C and a half-life of 2min at 40 degrees C. The amino acid sequence shows an identity of 39.1%-46.2% to UDGs from mesophilic bacteria. The primary structure was examined for features that could be related to the thermolability of the enzyme. The amino acid sequence of the heat-labile UDG shows 22 differences with respect to the consensus sequence derived from bacterial UDGs. Features previously recognized in cold-active enzymes such as extended surface loops or a decrease in the number of arginine residues or proline residues in loops were not observed. Because dominant features that could be related to the thermolability of the UDG from BMTU3346 cannot be identified, more subtle modifications of the conformation seem to be responsible for its thermolability.

Characterization of heat‐labile uracil‐DNA glycosylase from Psychrobacter sp. HJ147 and its application to the polymerase chain reaction

“…entomophila L48 (51.2% identity, YP_609783), Ps. aeruginosa UCBPP‐PA14 (51.2% identity, YP_792535), E. coli (45.4% identity, BAA10923), H. influenzae Rd KW20 (45.1% identity, NP_438191), V. parahaemolyticus AQ3810 (43.9% identity, EDM56312) and a marine psychrophilic bacterium known as strain BMTU 3346 (37.7% identity; [14]).…”

“…The Psp HJ147 UDG has an apparent temperature optimum of approx. 25 °C, a value that is significantly lower than the optimum (35 °C) for the UDG from a marine psychrophilic bacterium, strain BMTU 3346 [14], that (41 °C) of a cold‐adapted UNG from Atlantic cod [15], that (37 °C) of Chlamydia pneumoniae UDG [25] and that (50 °C) of E. coli UNG [14]. At 10 °C the Psp HJ147 UDG showed about 32% of its apparent maximal activity (Figure 3A) as compared with 10% for the UDG of the marine psychrophilic bacterium strain BMTU 3346.…”

“…Besides their scientific interest, some heat‐labile UDGs are valuable because of their applications in PCR. Recently, some heat‐labile UDGs were isolated, characterized and commercialized from strain BMTU 3346, a marine psychrophilic bacterium [14], and from the Atlantic cod ( Gadus morhua ) [15]. The availability of a heat‐labile form of UDG permits use of a one tube, one‐step RT–PCR (reverse transcription–PCR) format, with UTP/UDG contamination control [16].…”

Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays

“…The potential problem with residual UDG activity after PCR has led some investigators to use a heat-labile form of the enzyme (5,6). Incubations with heat-labile UDG (Roche Applied Science) at the manufacturer's recommended conditions of 25C for 10 min prevented detectable amplification in only 6 of 20 samples containing the TSPY PCR product (Table 1).…”

“…Similarly, amplification of U2 was not significantly affected by the presence of UDG (data not shown). It should be noted that the annealing temperature used for these amplifications was 58C, and additional tests may be warranted in cases where annealing temperatures are closer to 50C, at which UDG has maximal activity (5). …”