Barriuso-Iglesias Mónica, Schluesener Daniela, Barreiro Carlos, Poetsch Ansgar, Martín Juan F
Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, Av. Real, 1, 24006. León, Spain.
BMC Microbiol. 2008 Dec 17;8:225. doi: 10.1186/1471-2180-8-225.
C. glutamicum has traditionally been grown in neutral-pH media for amino acid production, but in a previous article we reported that this microorganism is a moderate alkaliphile since it grows optimally at pH 7.0-9.0, as shown in fermentor studies under tightly controlled pH conditions. We determined the best pH values to study differential expression of several genes after acidic or basic pH conditions (pH 6.0 for acidic expression and pH 9.0 for alkaline expression). Thus, it was interesting to perform a detailed analysis of the pH-adaptation response of the proteome of C. glutamicum ATCC 13032 to clarify the circuits involved in stress responses in this bacterium. In this paper we used the above indicated pH conditions, based on transcriptional studies, to confirm that pH adaptation results in significant changes in cytoplasmatic and membrane proteins.
The cytoplasmatic and membrane proteome of Corynebacterium glutamicum ATCC 13032 at different pH conditions (6.0, 7.0 and 9.0) was analyzed by classical 2D-electrophoresis, and by anion exchange chromatography followed by SDS-PAGE (AIEC/SDS-PAGE). A few cytoplasmatic proteins showed differential expression at the three pH values with the classical 2D-technique including a hypothetical protein cg2797, L-2.3-butanediol dehydrogenase (ButA), and catalase (KatA). The AIEC/SDS-PAGE technique revealed several membrane proteins that respond to pH changes, including the succinate dehydrogenase complex (SdhABCD), F0F1-ATP synthase complex subunits b, alpha and delta (AtpF, AtpH and AtpA), the nitrate reductase II alpha subunit (NarG), and a hypothetical secreted/membrane protein cg0752. Induction of the F0F1-ATP synthase complex beta subunit (AtpD) at pH 9.0 was evidenced by Western analysis. By contrast, L-2.3-butanediol dehydrogenase (ButA), an ATPase with chaperone activity, the ATP-binding subunit (ClpC) of an ATP-dependent protease complex, a 7 TMHs hypothetical protein cg0896, a conserved hypothetical protein cg1556, and the dihydrolipoamide acyltransferase SucB, were clearly up-regulated at pH 6.0.
The observed protein changes explain the effect of the extracellular pH on the growth and physiology of C. glutamicum. Some of the proteins up-regulated at alkaline pH respond also to other stress factors suggesting that they serve to integrate the cell response to different stressing conditions.
传统上谷氨酸棒杆菌是在中性pH培养基中培养以生产氨基酸，但在之前的一篇文章中我们报道，这种微生物是一种中度嗜碱菌，因为在严格控制pH条件下的发酵罐研究表明，它在pH 7.0 - 9.0时生长最佳。我们确定了在酸性或碱性pH条件（酸性表达pH 6.0，碱性表达pH 9.0）后研究几个基因差异表达的最佳pH值。因此，对谷氨酸棒杆菌ATCC 13032蛋白质组的pH适应反应进行详细分析，以阐明该细菌应激反应所涉及的途径很有意思。在本文中，我们基于转录研究使用上述指定的pH条件，以确认pH适应导致细胞质和膜蛋白发生显著变化。
通过经典的二维电泳以及阴离子交换色谱随后进行SDS - PAGE（AIEC/SDS - PAGE）分析了谷氨酸棒杆菌ATCC 13032在不同pH条件（6.0、7.0和9.0）下的细胞质和膜蛋白质组。一些细胞质蛋白在这三个pH值下通过经典的二维技术显示出差异表达，包括一个假定蛋白cg2797、L - 2,3 - 丁二醇脱氢酶（ButA）和过氧化氢酶（KatA）。AIEC/SDS - PAGE技术揭示了几种对pH变化有反应的膜蛋白，包括琥珀酸脱氢酶复合物（SdhABCD）、F0F1 - ATP合酶复合物亚基b、α和δ（AtpF、AtpH和AtpA）、硝酸还原酶IIα亚基（NarG）以及一个假定的分泌/膜蛋白cg0752。通过蛋白质免疫印迹分析证明了在pH 9.0时F0F1 - ATP合酶复合物β亚基（AtpD）的诱导。相比之下，L - 2,3 - 丁二醇脱氢酶（ButA），一种具有伴侣活性的ATP酶、ATP依赖性蛋白酶复合物的ATP结合亚基（ClpC）、一个具有7个跨膜螺旋的假定蛋白cg0896、一个保守的假定蛋白cg1556以及二氢硫辛酰胺酰基转移酶SucB，在pH 6.0时明显上调。
观察到的蛋白质变化解释了细胞外pH对谷氨酸棒杆菌生长和生理的影响。一些在碱性pH上调的蛋白质也对其他应激因素有反应，这表明它们有助于整合细胞对不同应激条件的反应。
