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Extraction and detection of guanosine 5′-diphosphate-3′-diphosphate in amino acid starvation cells of Clavibacter michiganensis

  • Fungal and Bacterial Physiology - Research Paper
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Abstract

Guanosine 5′-diphosphate-3′-diphosphate (ppGpp) is a small molecule nucleotide alarmone that can accumulate under the amino acid starvation state and trigger the stringent response. This study reported the extraction of ppGpp from the Gram-positive bacteria Clavibacter michiganensis through methods using formic acid, lysozyme, or methanol. Following extraction, ppGpp was detected through ultra-high-performance liquid chromatography (UHPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methanol method showed the highest extraction efficiency for ppGpp among the three methods tested. C. michiganensis cells in exponential growth phase was induced in amino acid starvation by serine hydroxamate (SHX) and used for ppGpp extraction and detection. When using the methanol extraction method, the results showed that ppGpp concentrations in SHX-treated samples were 15.645 nM, 17.656 nM, 20.372 nM, and 19.280 nM at 0 min, 15 min, 30 min and 1 h, respectively, when detected using LC-MS/MS. This is the first report on ppGpp extraction and detection in Clavibacter providing a new idea and approach for nucleotide detection and extraction in bacteria.

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Acknowledgements

We thank Weihua Wang and Lili Li (Tsinghua University) for their valuable technical assistance and advice for detection of ppGpp by LC-MS/MS.

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Funding

This work was supported by the Natural Science Foundation of China (No. 31571972) and the National Key Research and Development Program of China (No. 2017YFD0201601).

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Authors and Affiliations

  1. Beijing Key Laboratory of Seed Disease Testing and Control, Department of Plant Pathology, China Agricultural University, Beijing, 100193, People’s Republic of China

    Kaihong Bai, Xing Chen, Na Jiang, Jianqiang Li & Laixin Luo

  2. Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, People’s Republic of China

    Qingyang Lyu

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  1. Kaihong Bai
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  2. Xing Chen
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Contributions

LL, JL, and NJ conceived and designed the research. KB wrote this manuscript and analyzed the data. LL and QL revised the manuscript. KB and XC conducted the experiments. All authors read and approved the final manuscript.

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Correspondence to Laixin Luo.

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Bai, K., Chen, X., Jiang, N. et al. Extraction and detection of guanosine 5′-diphosphate-3′-diphosphate in amino acid starvation cells of Clavibacter michiganensis. Braz J Microbiol 52, 1573–1580 (2021). https://doi.org/10.1007/s42770-021-00488-1

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