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

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.

Genomes of the Symbiotic Nitrogen-Fixing Bacteria of Legumes

“…This is perhaps best exemplified in studies involving indeterminate root nodules, where the issue of analyzing expression patterns at various stages of infection has been addressed through the use of bacterial and plant developmental mutants. The poor overlap that has been documented between transcriptomic and proteomic data emphasizes the importance of developing additional functional genomic approaches, such as studies focusing upon the metabolomics of stem-and root-nodule bacteria (Barsch et al, 2004;Colebatch et al, 2004). Such initiatives are necessary to extend our knowledge of the fascinating partnership that exists between legumes and their microsymbionts.…”

The Interplay of Proton, Electron, and Metabolite Supply for Photosynthetic H2 Production in Chlamydomonas reinhardtii

“…The samples were incubated with 100 l of methoxylamine hydrochloride (Sigma-Aldrich) in pyridine (20 mg/ml) for 90 min at 37°C while stirring. N-Methyl-N-(trimethylsilyl)trifluoroacetamide (Macherey & Nagel) was then added, and the mixture was incubated for 30 min at 37°C while stirring (32).…”

“…Mass spectra were recorded at 20 scans s Ϫ1 using a scanning range of 50 -750 m/z. GC/MS Metabolite Identification and Quantification-Metabolite and fatty acids identification was performed according to a previous study (32). The evaluation of the chromatograms was performed with Xcalibur software (ThermoFinnigan).…”

Metabolite Profiling of Chlamydomonas reinhardtii under Nutrient Deprivation

“…Whereas plant material can be flash-frozen in liquid nitrogen directly, the situation is different for suspension cultures. Although freezing of entire microbial cultures with subsequent analysis of the metabolite composition have been reported (Barsch et al, 2004), this approach suffers from severe drawbacks, as no distinction can be made between intracellular and excreted compounds and the large excess of constituents of the growth medium is not compatible with the use of highly sensitive chromatographic separation methods. Separation of the cells from the medium is usually achieved by centrifugation or filtration.…”