Bereswill Stefan, Waidner Uta, Odenbreit Stefan, Lichte Flavia, Fassbinder Frank, Bode G Nter, Kist Manfred
University of Freiburg, Institute of Medical Microbiology and Hygiene, Department of Microbiology and Hygiene,Hermann-Herder-Str. 11, D-79104 Freiburg,Germany.
Max-von-Pettenkofer-Institute of Hygiene and Medical Microbiology, Department of Bacteriology,Pettenkoferstr. 9a, D-80336 Munich,Germany.
Microbiology (Reading). 1998 Sep;144 ( Pt 9):2505-2516. doi: 10.1099/00221287-144-9-2505.
The function of the pfr gene encoding the ferritin from Helicobacter pylori was investigated using the Fur titration assay (FURTA) in Escherichia coli, and by characterization of a pfr-deficient mutant strain of H. pylori. Nucleotide sequence analysis revealed that the pfr region is conserved among strains (> 95% nucleotide identity). Two transcriptional start sites, at least one of them preceded by a sigma 70-dependent promoter, were identified. Provision of the H. pylori pfr gene on a multicopy plasmid resulted in reversal of the Fur-mediated repression of the fhuF gene in E. coli, thus enabling the use of the FURTA for cloning of the ferritin gene. Inactivation of the pfr gene, either by insertion of a resistance cassette or by deletion of the up- and downstream segments, abolished this function. Immunoblot analysis with a Pfr-specific antiserum detected the Pfr protein in H. pylori and in E. coli carrying the pfr gene on a plasmid. Pfr-deficient mutants of H. pylori were generated by marker-exchange mutagenesis. These were more susceptible than the parental strain to killing by various metal ions including irons, copper and manganese, whereas conditions of oxidative stress or iron deprivation were not discriminative. Analysis by element-specific electron microscopy revealed that growth of H. pylori in the presence of iron induces the formation of two kinds of cytoplasmic aggregates: large vacuole-like bodies and smaller granules containing iron in association with oxygen or phosphorus. Neither of these structures was detected in the pfr-deficient mutant strain. Furthermore, the ferritin accumulated under iron overload and the pfr-deficient mutant strains lacked expression of a 12 kDa protein which was negatively regulated by iron in the parental strain. The results indicate that the nonhaem-iron ferritin is involved in the formation of iron-containing subcellular structures and contributes to metal resistance of H. pylori. Further evidence for an interaction of ferritin with iron-dependent regulation mechanisms is provided.
利用大肠杆菌中的Fur滴定法（FURTA）以及幽门螺杆菌pfr缺陷突变株的特性研究了编码幽门螺杆菌铁蛋白的pfr基因的功能。核苷酸序列分析表明，pfr区域在各菌株中保守（核苷酸同一性>95%）。鉴定出两个转录起始位点，其中至少一个之前有一个依赖于σ70的启动子。在多拷贝质粒上提供幽门螺杆菌pfr基因导致大肠杆菌中Fur介导的fhuF基因抑制作用的逆转，从而使得能够利用FURTA克隆铁蛋白基因。通过插入抗性盒或缺失上下游片段使pfr基因失活，消除了该功能。用Pfr特异性抗血清进行免疫印迹分析，在幽门螺杆菌和携带质粒上pfr基因的大肠杆菌中检测到了Pfr蛋白。通过标记交换诱变产生了幽门螺杆菌的pfr缺陷突变体。这些突变体比亲本菌株更容易受到包括铁、铜和锰在内的各种金属离子的杀伤，而氧化应激或铁缺乏条件并无区分作用。通过元素特异性电子显微镜分析发现，在铁存在的情况下幽门螺杆菌的生长诱导形成两种细胞质聚集体：大的液泡样体和较小的含有与氧或磷结合的铁的颗粒。在pfr缺陷突变株中未检测到这些结构中的任何一种。此外，铁过载时积累的铁蛋白以及pfr缺陷突变株缺乏亲本菌株中受铁负调控的12 kDa蛋白的表达。结果表明，非血红素铁铁蛋白参与含铁亚细胞结构的形成，并有助于幽门螺杆菌的金属抗性。提供了铁蛋白与铁依赖性调节机制相互作用的进一步证据。
