In our study, we have examined the genetic and biochemical potential of several aquatic bacteria to phosphorylate native dNs. In two Gram-negative bacteria, F. psychrophilum JIP02/86 and Polaribacter sp. MED 152, we identified TK1-like kinase (FpTK1 and PdTK1, respectively, Table S2), which group together with the already characterized Gram-positive TK1-like dNKs (Fig. 1). MG-132 molecular weight The corresponding enzymes followed classical Michaelis–Menten kinetics and were strictly specific for dT and dU (Table 1, Fig. S2a,b,e,f ). The kinetics parameters for FpTK1 resemble those of the Gram-positive TK1s from Staphylococcus aureus TK1 (SaTK1) and

Bacillus cereus TK1 (BcTK1), which also supports the obtained phylogenetic relationship (Sandrini et al., 2007a,b). On the other hand, PdTK1 is from the kinetic point of view similar to Gram-negative TK1 from Salmonella Osimertinib nmr enterica (SeTK1) (Sandrini et al., 2007a). Surprisingly, while the previously characterized bacterial TK1s are relatively thermostable, both FpTK1 and PdTK1 were not active at 37 °C, and by measuring PdTK1 phosphorylating activity as a function

of temperature, it turned out that the activity of PdTK1 increased with temperature up to 21 °C (Fig. 3). When measured at higher temperatures, 25, 30, and 37 °C, the activity decreased over time. Furthermore, when pre-incubating the enzyme at 0 °C for one hour, the measured activity at 21 °C was 10-fold lower, while pre-incubation at 37 °C for one hour resulted in irreversible denaturation. These data can be important for the interpretation of the results obtained in the past studies, when the indirect activity of TK1 from aquatic bacteria was measured at 37 °C (Jeffrey & Paul, 1990). In short, when measuring the activity of bacteria isolated from cold niches by the incorporation of 3H-dT into newly synthesized DNA, one filipin should keep in mind that the activity should be measured at different temperatures. So far it has been thought that Gram-negative bacteria have only one dNK, TK1, while Gram-positive bacteria seem to have several dNKs (Sandrini et al., 2007a). The FpdNK and PddNK kinases followed classical Michaelis–Menten

kinetics and were able to phosphorylate dA and dC; however, both of them had dA as the preferred substrate (Table 1, Fig. S2c, d,g,h). None of them was able to efficiently phosphorylate dG; therefore, these two enzymes seem to act like dAK from S. aureus dAK (SadAK) or B. cereus dAK (BcdAK) (Sandrini et al., 2007a,b), but with much lower specificity for dC. The substrate preferences could be partially explained by the genome composition. Both bacteria have AT content of approximately 70%; therefore during DNA replication, they need more A and T than G and C. However, it is puzzling why the activity on dG is so low in both species (Table 1 and Table S2). We examined the sequenced genomes from several aquatic bacteria for the genes encoding dNKs, the key enzymes in the salvage of dNs.