Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array
Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array
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DOI：
10.1271/bbb.66.1337
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发表时间：
2002-06-01
影响因子：
1.6
通讯作者：
Ikeda, M
中科院分区：
文献类型：
--
作者：
Hayashi, M;Mizoguchi, H;Ikeda, M
在确定了谷氨酸棒杆菌的全基因组序列后，我们开发了一种DNA阵列来广泛研究与碳代谢相关的基因表达和调控。为此，总共120 ℃。谷氨酸基因，包括那些在中枢代谢和氨基酸生物合成，通过PCR扩增和打印在玻璃载玻片上。所得阵列，称为“代谢阵列”，用于与通过从总RNA样品逆转录产生的荧光标记的cDNA探针杂交。作为在这种工业上重要的微生物中的转录组分析的第一个演示，我们应用代谢阵列来研究在葡萄糖和乙酸盐作为唯一碳源上生长的细胞之间的差异转录谱。已知的乙酸调控基因（aceA、aceB、pta和ack）的基因表达变化与北方分析和酶分析的文献数据一致，表明代谢阵列在C.谷氨酸。除了已知的反应，许多以前未被识别的共调节基因被确定。例如，几个TCA循环基因，如gltA、sdhA、sdhB、fumH和mdh，以及促凋亡基因pck在乙酸盐培养基中上调。另一方面，一些参与糖酵解和戊糖磷酸途径的基因，以及许多氨基酸生物合成基因，在乙酸下调。此外，两个缺口基因，gapA和gapB，被发现是反向调节，这表明糖酵解和异生之间的碳代谢存在一个新的调节步骤。
Following the determination of the whole-genome sequence of Corynebacterium glutamicum, we have developed a DNA array to extensively investigate gene expression and regulation relevant to carbon metabolism. For this purpose, a total of 120 C. glutamicum genes, including those in central metabolism and amino acid biosyntheses, were amplified by PCR and printed onto glass slides. The resulting array, designated a "metabolic array", was used for hybridization with fluorescently labeled cDNA probes generated by reverse transcription from total RNA samples. As the first demonstration of transcriptome analysis in this industrially important microorganism, we applied the metabolic array to study differential transcription profiles between cells grown on glucose and on acetate as the sole carbon source. The changes in gene expression observed for the known acetate-regulated genes (aceA, aceB, pta, and ack) were well consistent with the literature data of northern analyses and enzyme assays, indicating the utility of the metabolic array in transcriptome analysis of C. glutamicum. In addition to the known responses, many previously unrecognized co-regulated genes were identified. For example, several TCA cycle genes, such as gltA, sdhA, sdhB, fumH, and mdh, and the gluconeogenic gene pck were up-regulated in the acetate medium. On the other hand, a few genes involved in glycolysis and the pentose phosphate pathway, as well as many amino acid biosynthetic genes, were down-regulated in acetate. Furthermore, two gap genes, gapA and gapB, were found to be inversely regulated, suggesting the presence of a new regulatory step for carbon metabolism between glycolysis and gluconeogenesis.