Neisseria gonorrhoeae bacterioferritin:: structural heterogeneity, involvement in iron storage and protection against oxidative stress
Neisseria gonorrhoeae bacterioferritin:: structural heterogeneity, involvement in iron storage and protection against oxidative stress
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DOI：
10.1099/00221287-145-10-2967
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发表时间：
1999-10-01
期刊：
影响因子：
2.8
通讯作者：
Morse, SA
中科院分区：
文献类型：
--
作者：
Chen, CY;Morse, SA
在无细胞提取物中鉴定出来自淋病奈瑟菌 F62 菌株的储铁蛋白细菌铁蛋白 (Bfr)，并随后通过柱色谱进行纯化。通过凝胶过滤估计淋球菌 Bfr 的分子量为 400 kDa；然而，SDS-PAGE 分析表明它由 18 kDa (BfrA) 和 22 kDa (BfrB) 亚基组成。使用源自BfrB的N端氨基酸序列和大肠杆菌Bfr的C端氨基酸序列的简并引物通过PCR扩增编码BfrB的DNA。随后使用单特异性引物 PCR 通过基因组步移获得 bfrA 的 DNA 序列。两个 Bfr 基因串联定位，中间有 27 bp 的间隙。潜在的毛皮结合序列（19 bp 中的 12 个与共有奈瑟氏球菌毛皮序列相同）位于 bfrA 的 5' 侧翼区域内，位于推定的 -35 六聚体前面。 bfrA和bfrB的DNA序列之间的同源性为55.7%；推导的BfrA（154个残基）和BfrB（157个残基）的氨基酸序列显示出39.7%的同一性，并且与大肠杆菌Bfr分别显示出41.3%和56.1%的同一性。用多克隆抗大肠杆菌探针进行蛋白质印迹检测，检测到重组 BfrA 和 BfrB 在大肠杆菌菌株 DH5 α 中的表达。大肠杆菌 Bfr 抗血清。大多数 Bfr 是具有相同亚基的均聚物；然而，此处提供的证据表明淋球菌 Bfr 由两个相似但不相同的亚基组成，这两个亚基似乎都是形成功能性 Bfr 所必需的。通过将 Omega 片段插入 BfrB 基因构建了 Bfr 缺陷突变体。在铁限制条件下，复合培养基中 BfrB 缺陷型突变体的生长减少。与同基因亲本菌株相比，BfrB 缺陷突变体对 H2O2 和百草枯的杀伤作用也更敏感。这些结果表明淋球菌 Bfr 在铁储存和防止铁介导的氧化应激中发挥重要作用。
The iron-storage protein bacterioferritin (Bfr) from Neisseria gonorrhoeae strain F62 was identified in cell-free extracts and subsequently purified by column chromatography. Gonococcal Bfr had an estimated molecular mass of 400 kDa by gel filtration; however, analysis by SDS-PAGE revealed that it was composed of 18 kDa (BfrA) and 22 kDa (BfrB) subunits. DNA encoding BfrB was amplified by PCR using degenerate primers derived from the N-terminal amino acid sequence of BfrB and from a C-terminal amino acid sequence of Escherichia coli Bfr. The DNA sequence of bfrA was subsequently obtained by genome walking using single-specific-primer PCR. The two Bfr genes were located in tandem with an intervening gap of 27 bp. A potential Fur-binding sequence (12 of 19 bp identical to the consensus neisserial fur sequence) was located within the 5' flanking region of bfrA in front of a putative -35 hexamer. The homology between the DNA sequences of bfrA and bfrB was 55.7 %; the deduced amino acid sequences of BfrA (154 residues) and BfrB (157 residues) showed 39.7 % identity, and showed 41.3 % and 56.1 % identity, respectively, to E. coli Bfr. Expression of recombinant BfrA and BfrB in E. coli strain DH5 alpha was detected on Western blots probed with polyclonal anti-E. coli Bfr antiserum. Most Bfrs are homopolymers with identical subunits; however, the evidence presented here suggests that gonococcal Bfr was composed of two similar but not identical subunits, both of which appear to be required for the formation of a functional Bfr. A Bfr-deficient mutant was constructed by inserting the Omega fragment into the BfrB gene. The growth of the BfrB-deficient mutant in complex medium was reduced under iron-limited conditions. The BfrB-deficient mutant was also more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These results demonstrate that gonococcal Bfr plays an important role in iron storage and protection from iron-mediated oxidative stress.