Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography-mass spectrometry
Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography-mass spectrometry
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DOI：
10.1007/s10142-004-0117-y
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发表时间：
2004-10-01
影响因子：
2.9
通讯作者：
Niehaus, K.
中科院分区：
文献类型：
--
作者：
Barsch, A.;Patschkowski, T.;Niehaus, K.
为了更好地了解紫花苜蓿与其寄主紫花苜蓿的共生关系，建立了对土壤细菌Sinorhizobium Meliloti的代谢产物分析。由于细菌代谢物的组成变化很快，因此开发快速收集和提取细菌代谢物的方法是至关重要的一步。在微量培养基中生长的紫花苜蓿1021细胞培养物通过离心、过滤或在液氮中立即冷冻然后进行冷冻干燥步骤来收获。细菌在甲醇中机械裂解，经甲氧基化和硅烷化后，用GC-MS分析亲水性化合物。通过与NIST 98数据库和现有标准的比较，鉴定了不同的化合物。从每个色谱图的大约200个峰中，到目前为止已经鉴定出65种化合物。比较不同的提取方法，得出代谢物的组成，发现几种氨基酸和氨基酸前体发生了明显的变化。主成分分析(PCA)能够根据不同碳源上生长的紫花苜蓿细胞的代谢产物谱来区分它们。通过对紫花苜蓿亮氨酸营养缺陷型突变株与野生型的代谢产物组成的比较，发现突变株中明显积累了2-异丙基苹果酸。有趣的是，积累的代谢产物并不是突变酶3-异丙基苹果酸脱氢酶的直接底物，而是苹果酸异构酶的底物，该异构酶在亮氨酸的生物合成途径中进一步上游作用。这一发现进一步强调了将代谢数据整合到后基因组研究中的重要性。
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.