Zaigler Alexander, Schuster Stephan C, Soppa Jörg
J. W. Goethe-Universität, Biozentrum Niederursel, Institut für Mikrobiologie, Marie-Curie-Str 9, D-60439 Frankfurt, Germany.
Mol Microbiol. 2003 May;48(4):1089-105. doi: 10.1046/j.1365-2958.2003.03497.x.
Haloferax volcanii is a moderately halophilic archaeon that can grow aerobically and anaerobically with a variety of substrates. We undertook a novel approach for the characterization of metabolic adaptations, i.e. transcriptome analysis with a onefold-coverage shotgun DNA microarray. A genomic library was constructed and converted into a polymerase chain reaction (PCR) product library, which was used to print two DNA microarrays, a 960-spot test array used for optimization of microarray analysis and a 2880-spot onefold-coverage array. H. volcanii cultures were shifted from casamino acid-based metabolism to glucose-based metabolism, and the transcriptome changes were analysed with the onefold-coverage array at five time points covering the transition phase and the onset of exponential growth with the new carbon source. About 10% of all genes were found to be more than 2.5-fold regulated at at least one time point. The genes fall into five clusters of kinetically co-regulated genes. For members of all five clusters, the results were verified by Northern blot analyses. The identity of the regulated genes was determined by sequencing. Many co-regulated genes encode proteins of common functions. Expected as well as a variety of unexpected findings allowed predictions about the central metabolism, the transport capacity and the cellular composition of H. volcanii growing on casamino acids and on glucose. The microarray analyses are in accordance with the growth rates and ribosome contents of H. volcanii growing on the two carbon sources. Analysis of the results revealed that onefold-coverage shotgun DNA microarrays are well suited to characterize the regulation of metabolic pathways as well as protein complexes in response to changes in environmental conditions.
嗜盐栖热菌是一种中度嗜盐古菌，能够利用多种底物进行有氧和无氧生长。我们采用了一种全新的方法来表征代谢适应性，即使用单倍覆盖鸟枪法DNA微阵列进行转录组分析。构建了一个基因组文库，并将其转化为聚合酶链反应（PCR）产物文库，该文库用于打印两个DNA微阵列，一个960点的测试阵列用于微阵列分析的优化，另一个2880点的单倍覆盖阵列。将嗜盐栖热菌培养物从基于酪蛋白氨基酸的代谢转换为基于葡萄糖的代谢，并在涵盖转换阶段和新碳源指数生长起始阶段的五个时间点，使用单倍覆盖阵列分析转录组变化。发现所有基因中约10%在至少一个时间点的调控倍数超过2.5倍。这些基因分为五个动力学共调控基因簇。对于所有五个基因簇的成员，结果通过Northern印迹分析进行了验证。通过测序确定了受调控基因的身份。许多共调控基因编码具有共同功能的蛋白质。预期的以及各种意外的发现使得能够对嗜盐栖热菌在酪蛋白氨基酸和葡萄糖上生长时的中心代谢、转运能力和细胞组成进行预测。微阵列分析与嗜盐栖热菌在两种碳源上生长的生长速率和核糖体含量一致。结果分析表明，单倍覆盖鸟枪法DNA微阵列非常适合表征代谢途径以及蛋白质复合物在环境条件变化时的调控情况。
