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Aquatic Microbial Ecology

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AME 85:121-129 (2020)  -  DOI:

Novel method for measuring aquatic bacterial productivity using D10-leucine based on protein synthesis rate

Kenji Tsuchiya1,*, Tomoharu Sano1, Noriko Tomioka1, Komatsu Kazuhiro1, Akio Imai1, Kazuhide Hayakawa2, Takamaru Nagata2, Takahiro Okamoto2, Victor S. Kuwahara3, Ayato Kohzu1

1National Institute for Environmental Studies, Ibaraki 305-8506, Japan
2Lake Biwa Environmental Research Institute, Shiga 520-0022, Japan
3Graduate School of Engineering, Soka University, Hachioji 192-8577, Japan
*Corresponding author:

ABSTRACT: The most widely used method for measuring bacterial production is tritium-labeled leucine (3H-Leu). Although this method provides methodological simplicity and high sensitivity, the employment of radioactive isotopes is often restricted by regulations, particularly in field settings. In this study, we developed a non-radioactive method for measuring bacterial productivity based on the protein synthesis rate, using deuterium-labeled leucine ((CD3)2CDCD2CD(NH2)COOH; D10-Leu); the proposed method was then compared and verified with the 3H-Leu method. The procedures of the proposed method are (1) incorporation of D10-Leu by bacteria, (2) acid hydrolysis (HCl) to amino acids and (3) quantification of D10-Leu (m/z 142.10) by liquid chromatography mass spectrometry (LC-MS/MS). In the LC-MS/MS analysis, we detected a larger amount of D9-Leu (m/z 141.10) and D8-Leu (m/z 140.10) than that of D10-Leu, suggesting that incorporated D10-Leu was rapidly metabolized such as in deamination and aminotransferase reactions. The incorporation rates of D10-Leu, D10-Leu + D9-Leu (D10+D9-Leu) and D10-Leu + D9-Leu + D8-Leu (D10+D9+D8-Leu) were significantly positively correlated to that of 3H-Leu, confirming the validity of the proposed method. Since D7-Leu (m/z 139.10) could not be detected, the amount of exogenous leucine incorporated into protein can be accurately estimated through D10+D9+D8-Leu measurement. The new compound-based quantification method using stable isotope-labeled leucine can be a powerful tool to estimate pure protein synthesis rate for measuring bacterial production.

KEY WORDS: Bacterial production · Protein synthesis · Leucine · Deoxyadenosine · Stable isotopes · Liquid chromatography mass spectrometry

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Cite this article as: Tsuchiya K, Sano T, Tomioka N, Kazuhiro K and others (2020) Novel method for measuring aquatic bacterial productivity using D10-leucine based on protein synthesis rate. Aquat Microb Ecol 85:121-129.

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