Abstract
Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular thymidine into their DNA, suggesting their biomass production might be overlooked when using the conventional method. In the present study, to evaluate appropriateness of deoxyribonucleosides for evaluating bacterial production of natural bacterial communities from the viewpoint of DNA synthesis, incorporation rates of four deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine) labeled by nitrogen stable isotope (15N) into bacterial DNA were examined in both ocean (Sagami Bay) and freshwater (Lake Kasumigaura) ecosystems in July 2015 and January 2016. In most stations in Sagami Bay and Lake Kasumigaura, we found that incorporation rates of deoxyguanosine were the highest among those of the four deoxyribonucleosides, and the incorporation rate of deoxyguanosine was approximately 2.5 times higher than that of thymidine. Whereas, incorporation rates of deoxyadenosine and deoxycytidine were 0.9 and 0.2 times higher than that of thymidine. These results clearly suggest that the numbers of bacterial species which can incorporate exogenous deoxyguanosine into their DNA are relatively greater as compared to the other deoxyribonucleosides, and measurement of bacterial production using deoxyguanosine more likely reflects larger numbers of bacterial species productions.
Highlights
In aquatic ecosystems, bacteria play key roles to drive the microbial loop using dissolved/particulate organic matter as their substrates, and to transfer energy and materials to higher trophic levels as well as conventional grazing food chains [1]
In the procedure of 15N-dA method, bacterial DNA is extracted after incubation, the extracted DNA is enzymatically hydrolyzed to nucleosides, and the incorporated 15N-dA is directly quantified by using liquid chromatography mass spectrometry (LC-MS)
A in Sagami Bay, the highest incorporation rates of deoxyribonucleosides were found for dA in July and dT in January, and the second was dG in both of July and January (Fig 1)
Summary
Bacteria play key roles to drive the microbial loop using dissolved/particulate organic matter as their substrates, and to transfer energy and materials to higher trophic levels as well as conventional grazing food chains [1]. The conventional methods of measuring bacterial production in seawater and freshwater ecosystems are tritium-thymidine method (3H-dT method; [3, 4]) and tritiumleucine method (3H-Leu method; [5]). In this method, bacteria incorporate radioisotope (tritium; 3H) labeled thymidine or leucine into their DNA or protein, respectively, and the incorporation rate is measured to estimate bacterial production. Bacteria incorporate radioisotope (tritium; 3H) labeled thymidine or leucine into their DNA or protein, respectively, and the incorporation rate is measured to estimate bacterial production This method provides methodological simplicity and high sensitivity, the use of radioisotopes is often restricted by regulation, in field settings. The direct quantification of the incorporated nucleosides can obtain pure DNA synthesis rates, which should solve a problem of radio-isotopic non-specific labeling in 3H-dT method resulting in overestimation of DNA synthesis rate [7]
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