Harvie Duncan R, Vílchez Susana, Steggles James R, Ellar David J
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Microbiology (Reading). 2005 Feb;151(Pt 2):569-577. doi: 10.1099/mic.0.27744-0.
A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a beta-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The Deltafur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.
在蜡样芽孢杆菌中鉴定并表征了枯草芽孢杆菌fur基因的同源物。发现克隆基因的预测氨基酸序列与转录调节因子Fur家族的其他成员高度相似。蜡样芽孢杆菌fur基因显示出部分互补大肠杆菌fur突变体的能力。纯化的蜡样芽孢杆菌Fur以金属依赖的方式特异性结合与枯草芽孢杆菌Fur框同源的19 bp DNA序列。对可用的蜡样芽孢杆菌基因组数据的分析确定了许多在启动子区域含有预测的Fur框序列的基因。这些基因中的许多预计在细菌铁摄取和代谢中起作用，但也有几个与毒力有关。通过β-半乳糖苷酶测定证实了铁载体生物合成操纵子的Fur和铁调节。通过用卡那霉素抗性盒破坏的拷贝对等位基因取代染色体基因构建了蜡样芽孢杆菌fur缺失菌株。发现Δfur突变体组成型表达铁载体，在细胞内积累铁的水平比野生型高约三倍，并且对过氧化氢高度敏感。在昆虫感染模型中，发现fur缺失菌株的毒力明显减弱，突出了Fur在该病原体毒力中所起的重要作用。
