Toes Ann-Charlotte M, Daleke Maria H, Kuenen J Gijs, Muyzer Gerard
Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
Microbiology (Reading). 2008 Sep;154(Pt 9):2709-2718. doi: 10.1099/mic.0.2008/016857-0.
Copper homeostasis is tightly regulated in all living cells as a result of the necessity and toxicity of this metal in free cationic form. In Gram-negative bacteria CPx-type ATPases (e.g. CopA in Escherichia coli) and heavy-metal efflux RND proteins (e.g. CusA in E. coli) play an important role in transport of copper across the cytoplasmic and outer membrane. We investigated the expression of CusA- and CopA-like proteins in Shewanella oneidensis MR1 and Shewanella strain MB4, a Mn(IV)-reducing isolate from a metal-polluted harbour sediment. Q-PCR analysis of total mRNA extracted from cultures grown under aerobic conditions with 25 microM copper showed significantly increased expression of cusA (Student's t-test: MR1, P<0.0001; MB4, P=0.0006). This gene was also induced in the presence of 100 microM copper and 10 or 25 microM cadmium in both tested strains. In the absence of oxygen, with fumarate as final electron acceptor and 100 microM copper, a prolonged lag phase (5 h) was observed and general fitness decreased as evidenced by twofold lower copy numbers of 16S rRNA compared to aerobic conditions. cusA expression in cells grown under these conditions remained at comparable levels (MR1) or was slightly decreased (MB4), compared to aerobic copper challenges. A gene homologous to the copA gene of S. oneidensis was not detected in strain MB4. Although low copA copy numbers were observed in strain MR1 under conditions with 25 and 100 microM copper, copA was not detected in mRNA from cultures grown in the presence of 10 microM cadmium, or in the absence of added heavy metals. However, copA was highly induced under anaerobic conditions with 100 microM copper (P=0.0011). These results suggest essentially different roles for the two proteins CopA and CusA in the copper response in S. oneidensis MR1, similar to findings in more metal-resistant bacteria such as Escherichia coli and Cupriavidus metallidurans.
由于游离阳离子形式的铜具有必要性和毒性，所有活细胞中的铜稳态都受到严格调控。在革兰氏阴性细菌中，CPx型ATP酶（如大肠杆菌中的CopA）和重金属外排RND蛋白（如大肠杆菌中的CusA）在铜跨细胞质膜和外膜的转运中起重要作用。我们研究了嗜铁钩端螺旋菌MR1和希瓦氏菌MB4（一种从金属污染的港口沉积物中分离出的锰（IV）还原菌）中CusA和CopA样蛋白的表达。对在含有25微摩尔铜的有氧条件下培养的细菌提取的总mRNA进行Q-PCR分析，结果显示cusA的表达显著增加（学生t检验：MR1，P<0.0001；MB4，P=0.0006）。在两种受试菌株中，该基因在存在100微摩尔铜以及10或25微摩尔镉的情况下也被诱导表达。在无氧条件下，以富马酸作为最终电子受体且含有100微摩尔铜时，观察到延长的滞后期（5小时），并且总体适应性下降，这可通过与有氧条件相比16S rRNA拷贝数降低两倍来证明。与有氧铜刺激相比，在这些条件下生长的细胞中cusA的表达保持在相当水平（MR1）或略有下降（MB4）。在菌株MB4中未检测到与嗜铁钩端螺旋菌copA基因同源的基因。尽管在含有25和100微摩尔铜的条件下，在菌株MR1中观察到copA拷贝数较低，但在含有10微摩尔镉的培养物的mRNA中或在未添加重金属的情况下未检测到copA。然而，在含有100微摩尔铜的厌氧条件下，copA被高度诱导（P=0.0011）。这些结果表明，CopA和CusA这两种蛋白在嗜铁钩端螺旋菌MR1对铜的反应中起着本质上不同的作用，这与在更具金属抗性的细菌如大肠杆菌和金属耐铜菌中的发现相似。
