Gene replacement of adenylate kinase in the gram-positive thermophile Geobacillus stearothermophilus disrupts adenine nucleotide homeostasis and reduces cell viability
Gene replacement of adenylate kinase in the gram-positive thermophile Geobacillus stearothermophilus disrupts adenine nucleotide homeostasis and reduces cell viability
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DOI：
10.1007/s00792-004-0428-x
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发表时间：
2005-04-01
期刊：
影响因子：
2.9
通讯作者：
Shamoo, Y
中科院分区：
文献类型：
--
作者：
Couñago, R;Shamoo, Y
嗜热细菌对于工业界和研究界具有重要价值。不幸的是，这些生物体的细胞过程和机制在很大程度上仍未得到充分研究。在本研究中，我们研究了腺苷酸激酶 (AK) 失活如何影响革兰氏阳性中度嗜热菌、嗜热脂肪地芽孢杆菌菌株 NUB3621-R 的腺嘌呤核苷酸稳态。 AK 在活细胞的腺嘌呤核苷酸稳态中发挥着重要作用，并且已被证明对于革兰氏阴性嗜温大肠杆菌至关重要。为了研究 AK 在维持嗜热脂肪芽孢杆菌腺苷酸能量电荷 (EC) 和细胞活力中的作用，我们产生了该生物体的重组菌株，其中编码必需蛋白腺苷酸激酶 (AK) 的内源基因已被来自嗜温性枯草芽孢杆菌的 adk 基因取代。进行 PCR、DNA 测序和 Southern 分析以确认正确的基因替换和邻近基因的保存。重组细胞的最高生长温度比野生型细胞低近 20 摄氏度（56 摄氏度与 75 摄氏度）。酶活性测定和 EC 测量证明，这种温度敏感表型是枯草芽孢杆菌 AK 热灭活的继发因素。在较高温度（65 摄氏度）下，与野生型细胞 (0.45) 相比，重组细胞的 EC 值 (0.09) 也较低，这反映了 AK 失活后腺嘌呤核苷酸稳态的破坏。
Thermophilic bacteria are of great value for industry and research communities. Unfortunately, the cellular processes and mechanisms of these organisms remain largely understudied. In the present study, we investigate how the inactivation of adenylate kinase (AK) affects the adenine nucleotide homeostasis of a gram-positive moderate thermophile, Geobacillus stearothermophilus strain NUB3621-R. AK plays a major role in the adenine nucleotide homeostasis of living cells and has been shown to be essential for the gram-negative mesophile Escherichia coli. To study the role of AK in the maintenance of adenylate energy charge (EC) and cell viability of G. stearothermophilus, we generated a recombinant strain of this organism in which its endogenous gene coding for the essential protein adenylate kinase (AK) has been replaced with the adk gene from the mesophile Bacillus subtilis. PCR, DNA sequencing and Southern analysis were performed to confirm proper gene replacement and preservation of neighboring genes. The highest growing temperature for recombinant cells was almost 20 degrees C lower than for wild-type cells (56 vs. 75 degrees C). This temperature-sensitive phenotype was secondary to heat inactivation of B. subtilis AK, as evidenced by enzyme activity assays and EC measurements. At higher temperatures (65 degrees C), recombinant cells also had lower EC values (0.09) compared to wild-type cells (0.45), which reflects a disruption of adenine nucleotide homeostasis following AK inactivation.