Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph N1G 2W1, ON, Canada.
J Fish Dis. 2010 Apr;33(4):331-40. doi: 10.1111/j.1365-2761.2009.01125.x. Epub 2010 Jan 12.
Signature-tagged mutagenesis was used to identify genes essential for survival of Yersinia ruckeri in its natural host, rainbow trout, Oncorhynchus mykiss. A mini-Tn5-Km2 signature-tagged mutant, C6-1, was missing from rainbow trout kidney at 7 days after an immersion challenge. The transposon insertion in C6-1 was in a homologue of the znuA gene of Escherichia coli that encodes ZnuA, a zinc-binding periplasmic protein of the high-affinity zinc transporter ZnuABC. Further sequencing of the C6-1 locus in Y. ruckeri identified homologues of two other genes: znuB, encoding a putative inner membrane permease, and znuC, encoding a putative ATPase. When present on a low-copy plasmid, the znuABC locus of Y. ruckeri fully restored growth of a zinc transport-deficient DeltaznuABC mutant of E. coli. Unlike DeltaznuABC mutants of E. coli and Salmonella typhimurium, the DeltaznuABC mutant of Y. ruckeri did not demonstrate significantly slower growth in zinc-deficient M9 minimal medium or in Luria-Bertani (LB) medium supplemented with the metal chelators EDTA and tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). In LB medium, the znuA::lacZ and znuCB::lacZ transcriptional fusions of Y. ruckeri were derepressed by addition of EDTA and TPEN and were repressed by addition of zinc and manganese. In a competitive challenge by immersion, the DeltaznuABC mutant was unable to compete with the parental strain and survived poorly in rainbow trout kidney, indicating that the ZnuABC transporter has a role in establishing and maintaining a rainbow trout infection by Y. ruckeri.
利用标记突变技术鉴定了与维氏气单胞菌在其自然宿主虹鳟鱼中的生存相关的必需基因。在虹鳟鱼浸泡攻毒 7 天后,突变体 C6-1 从虹鳟鱼肾脏中缺失。C6-1 中的转座子插入位于大肠杆菌 znuA 基因的同源物中,该基因编码 ZnuA,一种高亲和力锌转运体 ZnuABC 的锌结合周质蛋白。进一步测序发现 Y. ruckeri 的 C6-1 基因座的另外两个基因的同源物:znuB,编码一种假定的内膜渗透酶;和 znuC,编码一种假定的 ATP 酶。当存在于低拷贝质粒上时,Y. ruckeri 的 znuABC 基因座完全恢复了锌转运缺陷型 E. coli DeltaznuABC 突变体的生长。与大肠杆菌和鼠伤寒沙门氏菌的 DeltaznuABC 突变体不同,Y. ruckeri 的 DeltaznuABC 突变体在缺锌的 M9 基础培养基或补充金属螯合剂 EDTA 和四(2-吡啶基甲基)乙二胺(TPEN)的 LB 培养基中生长速度没有明显变慢。在 LB 培养基中,Y. ruckeri 的 znuA::lacZ 和 znuCB::lacZ 转录融合体被 EDTA 和 TPEN 诱导表达,被锌和锰抑制表达。在浸泡竞争挑战中,DeltaznuABC 突变体无法与亲本菌株竞争,在虹鳟鱼肾脏中存活不良,表明 ZnuABC 转运体在建立和维持维氏气单胞菌对虹鳟鱼的感染中发挥作用。