Thompson Dorothea K, Beliaev Alexander S, Giometti Carol S, Tollaksen Sandra L, Khare Tripti, Lies Douglas P, Nealson Kenneth H, Lim Hanjo, Yates John, Brandt Craig C, Tiedje James M, Zhou Jizhong
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Appl Environ Microbiol. 2002 Feb;68(2):881-92. doi: 10.1128/AEM.68.2.881-892.2002.
The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems.
铁定向的基因协同调控依赖于fur（铁摄取调节因子）基因产物，它作为一种铁响应性转录阻遏蛋白发挥作用。为了研究异化金属还原菌——希瓦氏菌属MR-1中fur同源物的生物学功能，通过自杀质粒整合到该基因中构建了一个fur基因敲除菌株（FUR1），并使用表型分析、包含691个排列基因的DNA微阵列以及二维聚丙烯酰胺凝胶电泳对其进行表征。生理学研究表明，在比较FUR1和野生型菌株的厌氧生长及对各种电子受体的还原能力时，二者相似。然而，转录谱分析显示，在电子传递、能量代谢、转录调控和氧化应激保护方面具有预测功能的基因，在fur突变体中要么被抑制（ccoNQ、etrA、细胞色素b和c成熟编码基因、qor、yiaY、sodB、rpoH、phoB和chvI），要么被诱导（yggW、pdhC、prpC、aceE、fdhD和ppc）。fur的破坏还导致了推测参与铁摄取的基因（hxuC、alcC、fhuA、hemR、irgA和ompW）的去抑制。这与以下发现一致：在铁充足的条件下，fur突变体产生的铁载体水平比野生型菌株高两倍。对FUR1蛋白质组的一个子集（即主要是可溶性细胞质和周质蛋白）的分析表明，相对于野生型，11种主要蛋白质种类的丰度可重复地显示出显著（P < 0.05）差异。使用质谱法进行蛋白质鉴定表明，其中两种蛋白质（SodB和AlcC）的表达与微阵列数据相关。这些结果表明，希瓦氏菌属MR-1 Fur在能量代谢中可能具有调控作用，这扩展了传统的Fur作为铁摄取系统负调控因子的模型。
