Graham Alison I, Hunt Stuart, Stokes Sarah L, Bramall Neil, Bunch Josephine, Cox Alan G, McLeod Cameron W, Poole Robert K
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom.
J Biol Chem. 2009 Jul 3;284(27):18377-89. doi: 10.1074/jbc.M109.001503. Epub 2009 Apr 19.
Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn(2+) or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.
锌离子在生物化学中发挥着不可或缺的作用。然而，细菌从环境中获取锌离子（Zn²⁺）的能力很强，使得实现锌离子缺乏异常困难，因此尚未全面了解锌离子的重要性。本文首次报道了在不使用特异性较差的螯合剂的情况下，将大肠杆菌生长培养基中的锌离子含量降低至60纳米或更低。在缺锌培养基中生长的细胞生长速率降低，细胞内锌离子含量减少多达五倍。为了了解对锌离子缺乏的整体反应，对在恒化器培养中锌离子充足和锌离子缺乏条件下生长的细胞进行了微阵列分析。在来自缺锌恒化器的细胞中，有9个基因上调超过2倍（p < 0.05），包括zinT（yodA）。已证明zinT受Zur（锌摄取调节因子）调控。缺乏zinT的突变体在锌离子限制条件下表现出生长缺陷，细胞内锌离子水平降低了3倍。纯化的ZinT蛋白具有一个单一的高亲和力金属结合位点，可容纳锌离子（Zn²⁺）或镉离子（Cd²⁺）。另一个上调的基因ykgM被认为编码一种不含锌离子指结构的锌离子指核糖体蛋白L31的旁系同源物。编码周质锌离子结合蛋白znuA的基因表达增加。在分批培养和恒化器培养过程中，细胞“获取”的锌离子比最初添加到培养基中的更多，可能是因为从培养容器中浸出。与使用螯合剂N,N,N',N'-四（2-吡啶甲基）乙二胺相比，锌离子去除被证明是一种更精确的耗尽锌离子的方法。
