, 1991). Standard assay conditions were 50 mM Tris–HCl pH 7.5, 10 mM DTT, 2.5 mM ATP, 2.5 mM MgCl2, 3 mg mL−1 BSA, 0.5 mM CHAPS, and the indicated concentration of radio-labeled dN substrate in a final volume of 50 μL. The radioactive dNs (3H-dT, 3H-dA, 3H-dG, and 3H-dC) used in the assay were obtained from Moravek or PerkinElmer. When determining the activities in crude bacterial extracts, NaF (6 mM) was added to the reaction mixture to inhibit phosphatase activities, and when dC was used as the substrate, also 0.5 mM tetrahydrouridine (THUR) was added to inhibit possible cytidine deaminase activity. The activities were measured at 37 °C, except for PdTK1 and FpTK1, which were measured at 21 °C.

When necessary, the enzyme or crude extract was diluted in the enzyme dilution buffer (50 mM Tris–HCl pH 7.5, 1 mM CHAPS, 3 mg mL−1 BSA, and 5 mM DTT). One unit (u) of enzyme activity EX 527 is defined as the amount of kinase that can phosphorylate 1 nmol of nucleoside per minute under standard assay conditions (Munch-Petersen et al., 1998). Kinetic data were evaluated by fitting the data

to the Michaelis–Menten equation ν = Vmax*(S)/(Km + (S)) using nonlinear regression analysis using Graph prism software. In order to determine the effect of the temperature on the PdTK1 phosphorylating activity, mTOR inhibitor the activity of enzyme was measured at 5, 10, 15, 21, 25, 30, and 37 °C. In this case, all radio-assays were performed with 500 μM 3H-dT as substrate and ATP as phosphate donor. When measured at 21 and 25 °C, activities were determined by initial velocity measurements based on the four time samples, retrieved after 3, 6, 9, and 12 min. In the assays performed at 5, 10, and 15 °C, the four time samples were taken after 5, 15, 30, and 45 min. In order to determine the activity at 30 and 37 °C, CYTH4 the assays also had to be performed with the pro-longed time series, with time samples taken after 2, 5, 10, 20, 30, and 40 min, owing to the low

activities. In a separate experiment, thermostability at 0 and 37 °C was investigated by incubating the enzyme 1 h prior to the measurement of the activity at 21 °C. In this experiment, time samples were taken after 2, 5, 10, 20, 30, and 40 min. Also FpTK1 was initially found to exhibit the effect of temperature on the phosphorylation activity. Therefore, the assays were conducted at 21 °C. Several aquatic bacterial genome sequences were searched for genes homologous to the known, previously characterized bacterial and eukaryote dNKs. Two of the analyzed bacteria, F. psychrophilum JIP02/86 and Polaribacter sp. MED 152, both Gram-negative and both belonging to Bacteroidete class, served as model organisms in our studies. Putative genes encoding dNKs in the bacterial genomes of F. psychrophilum JIP02/86 (NC_009613) and Polaribacter sp. MED 125 (NZ_AANA00000000) are listed in Table S2. In each species, we identified one TK1-like kinase (FpTK1 and PdTK1, respectively; Table S2).