Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography-mass spectrometry

Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography-mass spectrometry
October 2004
Received: 10 March 2004 Revised: 12 May 2004 Accepted: 11 June 2004 Published online: 24 July 2004
Aiko Barsch1, Thomas Patschkowski1 and Karsten Niehaus1
Functional & Integrative Genomics
Volume 4, Number 4
Springer-Verlag GmbH
(1) Lehrstuhl f?r Genetik, Fakult?t f?r Biologie, Universit?t Bielefeld, Postfach 100131, 33501 Bielefeld, Germany
Abstract A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula. A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition. S. meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step. Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS. The different compounds were identified by comparison with the NIST 98 database and available standards. From about 200 peaks in each chromatogram 65 compounds have been identified so far. A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools. A principal component analysis (PCA) was able to distinguish S. meliloti cells grown on different carbon sources based on their metabolite profile. A comparison of the metabolite composition of a S. meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant. Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine. This finding further emphasises the importance of integrating metabolic data into post-genomic research.
Keywords GC-MS - Metabolomics - Sinorhizobium meliloti - leuB
Karsten Niehaus
Email: karsten.niehaus@genetik.uni-bielefeld.de
Phone: +49-521-1065631
Fax: +49-521-1065626
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