Thymidine Uptake in Bacteria: The Effect of Purine Nucleosides

Abstract

The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 pg/ml while the initial concentration of purine nucleosides ranged from 25 to 250 pg/ml. Adenine nucleosides when present at a concentration more than 50 pg/ml strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial concentration of deoxyadenosine (or adenosine) of 250 pg/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase.Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 pg/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides.It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides.Exogenous thymidine is incorporated into prototrophic bacteria in a very inefficient way: the process stops after a few minutes [l-41. This is due to the interaction of thymidine phosphorylase with external thymidine and its conversion to thymine [2]. Boyce and Setlow [I] showed that simultaneous addition of deoxyadenosine to a growing Escherichia coli culture leads to a more efficient uptake of the supplied thymidine. The authors concluded that in the presence of deoxyadenosine the exogenous thymidine is preserved from the degradative action of thymidine phosphorylase and thus can be taken up by cells for a longer time. This interpretation of deoxyadenosine action was accepted in subsequent papers deoxyadenosine even increased the rate of thymidine incorporation.In this work we used a broad range of adenosine and deoxyadenosine concentrations to study their effect on thymidine uptake by cells of E. coli and 3. subtilis. Our results show that adenosine and deoxyadenosine do inhibit the thymidine uptake in both species. The inhibition is practically complete provided an adenine nucleoside is present in sufficiently high excess.Guanosine and deoxyguanosine do not compete with thymidine for transport into the cell [9,10] but according to the data presented both show a definite protective action for thymidine against its phosphorolysis by the thymidine phosphorylase. MATERIALS AND METHODS ChemicalsThymine, thymidine, adenosine, deoxyadenosine, guanosine, deoxyadenosine, and 2-deoxyribose 1 -phosphate (all of A grade) were purchased from Calbiochem (U.S.A.). Casein hydrolysate and Bacto

Use of Bromodeoxyuridine Immunocapture To Identify Active Bacteria Associated with Arbuscular Mycorrhizal Hyphae

“…and Bacillus spp.) were found, which is consistent with previous studies in which primarily gram-positive, rod-shaped bacteria associated with AM (8,43) and ectomycorrhizal (47) (22,36,53,58,59) or BrdU (57) into their genomes. It has previously been shown by using other approaches that gram-negative bacteria, such as pseudomonads, are predominantly active in rhizospheres or upon nutrient addition in soil (10,26), and thus they often behave as opportunistic r strategists (35).…”