Deletion of the zupT gene for a zinc importer influences zinc pools in Cupriavidus metallidurans CH34
Deletion of the zupT gene for a zinc importer influences zinc pools in Cupriavidus metallidurans CH34
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DOI：
10.1039/c3mt00267e
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发表时间：
2014-01-01
期刊：
影响因子：
3.4
通讯作者：
Nies, D. H.
中科院分区：
文献类型：
--
作者：
Herzberg, M.;Bauer, L.;Nies, D. H.
金属铜球菌菌株CH34通过相当非特异性的过渡金属进口和控制过剩金属的流出来实现高水平的过渡金属抗性。利用只具有有限数量金属外排系统的无质粒突变菌株AE104，细胞金属池被确定为这些转运反应的对应物。在低锌浓度下，菌株AE104在生长的指数期吸收Zn(II)，直到锌含量达到最佳水平（每细胞70 000 Zn(II)），而缺乏锌输入zupT的Delta zupT突变体只有20000 Zn(II)/细胞，可能是锌含量最低的突变体。突变体和亲本细胞在高（100 μ M）外部锌浓度（最佳锌含量）下每个细胞累积高达125000锌（II）。当缺失了所有已知锌外排系统基因的突变株Delta e4在接近其最高耐受水平（10 μ M）的锌浓度下培养时，这些细胞含有250000锌（II），可能是最大锌含量。除了锌，120000个钴或镉离子也可以填充这个锌池的一部分，这表明它实际上是一个未定义的二价过渡金属阳离子池，具有低底物特异性。即使当细胞含有足够数量的总锌时，重要的细胞过程也需要锌进口商ZupT，这表明存在紧密结合的锌离子池，锌离子池依赖于ZupT进行有效补充。zupT的缺失导致包涵体的形成，扰乱了RNA聚合酶的氧化应激抗性，降低了锌依赖亚基RpoC的合成效率，导致RpoC积累。此外，当用于锌输出跨包膜外排系统CzcCBA的czc等位基因在Delta zupT突变体中组成性表达时，会导致CzcA蛋白和该蛋白复合物的中心亚基消失，并导致金属抗性丧失。这种现象只有在Delta zupT细胞在固体培养基中多次转移培养时才会发生，而固体培养基会产生严重的锌饥饿。因此，C. metallidurans中似乎存在两个锌池：一个池中至少有20000个紧密结合的Zn（II）离子，另外一个池中有多达200000个松散结合且底物特异性低的阳离子。
Cupriavidus metallidurans strain CH34 accomplishes a high level of transition metal resistance by a combination of rather unspecific transition metal import and controlled efflux of surplus metals. Using the plasmid- free mutant strain AE104 that possesses only a limited number of metal efflux systems, cellular metal pools were identified as counterparts of these transport reactions. At low zinc concentrations strain AE104 took up Zn(II) until the zinc content reached an optimum level of 70 000 Zn(II) per cell in the exponential phase of growth, whereas a Delta zupT mutant lacking the zinc importer ZupT contained only 20000 Zn(II)/cell, possibly the minimum zinc content. Mutant and parent cells accumulated up to 125000 Zn(II) per cell at high (100 mu M) external zinc concentrations (optimum zinc content). When the mutant strain Delta e4, which has all the known genes for zinc efflux systems deleted, was cultivated in the presence of zinc concentrations close to its upper tolerance level (10 mu M), these cells contained 250000 Zn(II) per cell, probably the maximum zinc content. Instead of zinc, 120000 cobalt or cadmium ions could also fill-up parts of this zinc pool, showing that it is in fact an undefined pool of divalent transition metal cations bound with low substrate specificity. Even when the cells contained sufficient numbers of total zinc, the zinc importer ZupT was required for important cellular processes, indicating the presence of a pool of tightly bound zinc ions, which depends on ZupT for efficient replenishment. The absence of zupT led to the formation of inclusion bodies, perturbed oxidative stress resistance and decreased efficiency in the synthesis of the zinc-dependent subunit RpoC of the RNA polymerase, leading to RpoC accumulation. Moreover, when a czc allele for a zinc-exporting transenvelope efflux system CzcCBA was constitutively expressed in a Delta zupT mutant, this led to the disappearance of the CzcA protein and the central subunit of the protein complex, and to the loss of metal resistance. This phenomenon occurred only if the Delta zupT cells had been cultivated for several transfers in solid culture medium, which generated severe zinc starvation. Thus, two zinc pools appear to exist in C. metallidurans: one pool of at least 20000 tightly bound Zn(II) ions, in addition to a second pool of up to 200000 cations bound loosely and with low substrate specificity.