Toledo M A S, Schneider D R, Azzoni A R, Favaro M T P, Pelloso A C, Santos C A, Saraiva A M, Souza A P
Centro de Biologia Molecular e Engenharia Genética-CBMEG, Universidade Estadual de Campinas, Brazil.
Protein Expr Purif. 2011 Feb;75(2):204-10. doi: 10.1016/j.pep.2010.10.004. Epub 2010 Oct 14.
The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes.
OxyR氧化应激转录调节因子是一种DNA结合蛋白,属于LysR型转录调节因子(LTTR)家族。它能够感知细胞内的氧化物质并触发细胞反应,激活参与清除氧化物质的基因转录。在本研究中,我们对植物病原体木质部难养菌中预测的OxyR同源物(orf xf1273)进行了过表达、纯化和特性鉴定。这种细菌是由9a5c菌株引起的柑橘杂色黄化病(CVC)的病原体,会导致经济和社会损失。通过圆二色性分析了重组蛋白的二级结构。凝胶过滤显示XfoxyR在溶液中是二聚体。凝胶迁移分析表明,在体外条件下它确实能与其自身预测的启动子结合。然而,考虑到我们的对照实验,我们不能确定这种相互作用是否在体内发生。功能互补分析表明,xfoxyR能够恢复oxyR基因敲除的大肠杆菌菌株中的氧化应激反应。这些结果表明,预测的orfxf1273编码一种与大肠杆菌OxyR同源的转录调节因子,参与氧化应激反应。这对于木质部难养菌在感染和定殖过程中克服宿主的防御机制可能很重要。