Methodological approaches to help unravel the intracellular metabolome of Bacillus subtilis

Microbial Cell Factories

Research

Hendrikje Weidmann¹  Hanna Meyer²  Michael Lalk² 

[1] Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Str. 15a, 17487, Greifswald, Germany;Oncotest GmbH, Am Flughafen 12-14, 79108, Freiburg, Baden-Württemberg, Germany;Institute of Biochemistry, Ernst-Moritz-Arndt-University Greifswald, Felix-Hausdorff-Strasse 4, 17487, Greifswald, Germany;

关键词: Bacillus subtilis;    Metabolomics;    Sampling;    Energy charge;    Quenching;    Filtration;   

DOI  :  10.1186/1475-2859-12-69

 received in 2012-12-06, accepted in 2013-07-01,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundBacillus subtilis (B. subtilis) has become widely accepted as a model organism for studies on Gram-positive bacteria. A deeper insight into the physiology of this prokaryote requires advanced studies of its metabolism. To provide a reliable basis for metabolome investigations, a validated experimental protocol is needed since the quality of the analytical sample and the final data are strongly affected by the sampling steps. To ensure that the sample analyzed precisely reflects the biological condition of interest, outside biases have to be avoided during sample preparation.ResultsProcedures for sampling, quenching, extraction of metabolites, cell disruption, as well as metabolite leakage were tested and optimized for B. subtilis. In particular the energy status of the bacterial cell, characterized by the adenylate energy charge, was used to evaluate sampling accuracy. Moreover, the results of the present study demonstrate that the cultivation medium can affect the efficiency of the developed sampling procedure.ConclusionThe final workflow presented here allows for the reproducible and reliable generation of physiological data. The method with the highest qualitative and quantitative metabolite yield was chosen, and when used together with complementary bioanalytical methods (i.e., GC-MS, LC-MS and 1H-NMR) provides a solid basis to gather information on the metabolome of B. subtilis.

【 授权许可】

Unknown   
© Meyer et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.