Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química & Centro de Biotecnología, Universidad Técnica Federico Santa María, Valparaíso, Chile.
PLoS One. 2019 Aug 30;14(8):e0221881. doi: 10.1371/journal.pone.0221881. eCollection 2019.
Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth β-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.
黄素蛋白是一种含有黄素单核苷酸 (FMN) 的辅基的小型电子转移蛋白，在氧化应激或铁限制期间发挥重要作用。本研究的目的是鉴定和表征模式芳香降解菌 Paraburkholderia xenovorans LB400 中的黄素蛋白，并分析它们在百草枯和 H2O2 诱导的氧化应激过程中的保护作用。在 LB400 主染色体和次染色体上鉴定出两个编码黄素蛋白（此后称为 P. xenovorans 的 fldX 用于黄素蛋白）的基因（BxeA0278 和 BxeB0391）。黄素蛋白编码基因的基因组结构表明，编码膜蛋白、转运蛋白以及参与氧化还原过程和大分子生物合成的蛋白质的基因。对两种 LB400 黄素蛋白的二级结构预测表明，它们具有五个β-折叠与五个α-螺旋交错的典型黄素蛋白结构。FldX1 含有一个插入第五个β-链的环，表明它属于长链黄素蛋白，而 FldX2 是短链黄素蛋白。对来自 43 个细菌属的 73 种黄素蛋白的系统发育分析显示了 8 个簇（I-VIII），而 FldX1 和 FldX2 分别属于长链和短链黄素蛋白簇。FldX1 和 FldX2 在 P. xenovorans 中过表达。有趣的是，过表达长链黄素蛋白 FldX1（p2-fldX1）的菌株在葡萄糖中的生长速度比对照菌株快。与对照菌株相比，过表达长链黄素蛋白 FldX1（p2-fldx1）的重组菌株在平板上对百草枯（20 mM）的敏感性降低，在液体培养基中的存活率更高。过表达黄素蛋白 FldX1 和 FldX2 的菌株在暴露于 1 mM 百草枯时的存活率（>95%）高于对照菌株（68%）。与对照菌株相比，过表达 FldX1 和 FldX2 的菌株在暴露于 1 mM 百草枯后脂质过氧化（>20%）和暴露于 1 mM H2O2 后蛋白质羰基化（~30%）较低。在暴露于百草枯时，菌株 p2-fldx1 下调了 katG4、hpf、trxB1 和 ohr 基因（>2 倍），而菌株 p2-fldx2 上调了 oxyR 和 ahpC1 基因（>2 倍）。总之，P. xenovorans LB400 的黄素蛋白 FldX1 和 FldX2 赋予细胞对百草枯和 H2O2 等氧化剂的保护作用。
