施氏假单胞菌A1501铁吸收调节蛋白Fur的表达特性与功能鉴定

doi:10.19586/j.2095-2341.2022.0061

生物技术进展 ›› 2022, Vol. 12 ›› Issue (3): 387-395.DOI: 10.19586/j.2095-2341.2022.0061

施氏假单胞菌A1501铁吸收调节蛋白Fur的表达特性与功能鉴定

刘一超(), 陆超, 战嵛华, 柯秀彬, 陆伟, 燕永亮()

中国农业科学院生物技术研究所，北京 100081

收稿日期:2022-04-22 接受日期:2022-05-05 出版日期:2022-05-25 发布日期:2022-05-26
通讯作者: 燕永亮
作者简介:刘一超 E-mail：yichao.liu@kcl.ac.uk；
基金资助:
国家重点研发计划项目(2019YFA0904700);国家自然科学基金项目(31770067);中国科学院战略先导计划项目(XDA28030201)

Expressional and Functional Characterization of the Ferric Uptake Regulator Fur in Pseudomonas stutzeri A1501

Yichao LIU(), Chao LU, Yuhua ZHAN, Xiubin KE, Wei LU, Yongliang YAN()

Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2022-04-22 Accepted:2022-05-05 Online:2022-05-25 Published:2022-05-26
Contact: Yongliang YAN

摘要/Abstract

摘要：

铁是细胞生理代谢的重要金属元素，是多种氧化还原酶类、过氧化物酶类的重要组成部分。革兰氏阴性菌中，胞内铁转运、储存和利用主要由铁吸收调节蛋白Fur（ferric uptake regulator）调控。水稻根际联合固氮菌——施氏假单胞菌（Pseudomonas stutzeri）A1501基因组中含有一个fur同源基因，但其具体功能尚不清楚。比较了A1501中fur基因及其编码蛋白的序列特征，并通过实时荧光定量PCR（real-time quantitative，qRT-PCR）和Western blotting杂交方法测定了fur基因和Fur蛋白的表达特性，结果发现fur基因表达水平与环境中铁浓度呈负相关，在铁限制条件下高表达，铁过量条件下表达受抑制。构建了fur基因突变株和功能回补株，表型测定发现fur突变影响了A1501的碳源代谢谱，并导致A1501对过氧化氢胁迫敏感。胞内铁含量测定表明，fur突变株内的铁含量为野生型的1.3倍，进一步qRT-PCR测定表明fur突变影响了铁离子转运系统及过氧化物酶编码基因的表达。研究结果为解析根际微生物胞内铁平衡调节及根际环境适应机制奠定了理论基础。

关键词: 施氏假单胞菌A1501, 铁吸收调节蛋白, 生物固氮, 氧化胁迫, 铁转运系统

Abstract:

Iron is one of the inevitable metal elements for cell physiological metabolisms， and an important co-factor for a variety of oxidoreductases and peroxidases. In Gram-negative bacteria， the intracellular iron transport， storage and utilization are mainly regulated by the ferric uptake regulator （Fur）. A fur homologous gene was identified in the genome of Pseudomonas stutzeri A1501 which is a root-associated nitrogen-fixing bacterium， but its function was still unclear. In this study， the biological information， especially the sequence difference of both the fur genes and the Fur proteins among A1501 and other typical bacteria were compared. The expression characteristics of the fur gene and the Fur protein in A1501 were explored via the application of both real-time quantitative PCR （qRT-PCR） and Western blotting. The results showed that both the Fur protein and the fur gene were highly expressed under iron-limited conditions but the expression inhibited under iron-exceed conditions. The fur knockout mutant and functional complementary strains were constructed. It was observed that deletion of fur affected the carbon source metabolic spectrum of A1501 and also caused higher sensitivity to hydrogen peroxide stress. Meanwhile， the level of intracellular iron in the fur mutant was 1.3 times of that in wild type. Further qRT-PCR analysis indicated that Fur regulated the expression of the intracellular iron transport system and superoxide dismutase genes. This study provided a theoretical basis for the analysis of the intracellular iron homeostatic regulation and rhizosphere environment adaptation of rhizosphere microorganisms.

Key words: Pseudomonasstutzeri A1501, ferric uptake regulator, biological nitrogen fixation, oxidative stress, iron transport system

中图分类号:

Q939.11+1

刘一超, 陆超, 战嵛华, 柯秀彬, 陆伟, 燕永亮. 施氏假单胞菌A1501铁吸收调节蛋白Fur的表达特性与功能鉴定[J]. 生物技术进展, 2022, 12(3): 387-395.

Yichao LIU, Chao LU, Yuhua ZHAN, Xiubin KE, Wei LU, Yongliang YAN. Expressional and Functional Characterization of the Ferric Uptake Regulator Fur in Pseudomonas stutzeri A1501[J]. Current Biotechnology, 2022, 12(3): 387-395.

图/表 8

表1 本研究所用引物

Table 1 Primers used in this study

引物名称	引物序列（5´→3´）	功能
furKOF1	5´-ATAAGCTTCTGGACACCATCTACACCCT-3´	fur上游同源臂扩增引物，用于构建缺失突变株
furKOR1	5´-AGCTCGAATTCGATATCCCTTTTTTATGCCGCTTT-3´	fur上游同源臂扩增引物，用于构建缺失突变株
furKOF2	5´-AAATAAGGATCCTCTAGACTCATGTGTCGACGGT-3´	fur下游同源臂扩增引物，用于构建缺失突变株
furKOR2	5´-ATGCTAGCTTTTTTCATTCGGTTTCC-3´	fur下游同源臂扩增引物，用于构建缺失突变株
furF	5´-AAGCTTACCTGACCGAACAATCCCT-3´	fur扩增引物，用于构建功能回补株
furR	5´-GGTACCGGTCGTTGGCATTGAATA-3´	fur扩增引物，用于构建功能回补株
PST_0819-F	5′-ATAGCCTGCTCAATGCCCA-3′	qRT-PCR引物
PST_0819-R	5′-TGCCCACCACTGGAAGTTG-3′
PST_0820-F	5′-TGGTTGCGATGTATCCGCT-3′
PST_0820-R	5′-GCCAGACTGAACAATCCCGT-3′
PST_0821-F	5′-ACCAGCAGGAAGAAGACACC-3′
PST_0821-R	5′-CGATGAGGATGTTGCCCTT-3′
PST_0822-F	5′-AAGCTTGCCTTCACCATCAA-3′
PST_0822-R	5′-TTTAACGGCTTCTCGGGATAC-3′
PST_2271-F	5′-TTCTGGTTGTAGGCGGAGGA-3′
PST_2271-R	5′-GTTTCCGGACTGGTATTCGC-3′
PST_4066-F	5′-TTCACCACCTGGGAGTTCAG-3′
PST_4066-R	5′-TCATCACCGACAAGGAAGCT-3′
katE-F	5′-GCTGGACCCGACCAAAAT-3′
katE-R	5′-CGGACGGTTGATCGGAAT-3′
PST_1360-F	5′-CGCTGGTTGTGGTCAATACG-3′
PST_1360-R	5′-ACTGCCTGGGTCTGGGTCAT-3′
PST_2535-F	5′-TTCCACTTGATCGCCTTGCT-3′
PST_2535-R	5′-GTGGTTTCACGCCCCAGTAC-3′
sodC-F	5′-CACGGCTTTCATATCCACG-3′
sodC-R	5′-CACGGCTTTCATATCCACG-3′
sodB-F	5′-AAGGAAGAGTTCACCAAGACCG-3′
sodB-R	5′-ACGAAGTCCCAGTTCACCAG-3′

图1 不同细菌中fur基因的染色体位置以及Fur蛋白的二级结构比较A：不同细菌中fur基因及其侧翼基因的比较；B：施氏假单胞菌A1501 Fur蛋白与其他菌体Fur蛋白氨基酸序列比对

Fig.1 Comparative analysis of the chromosomal location of fur gene and primary structure of Fur protein in different bacteria

图2 A1501 fur基因及Fur蛋白在不同铁离子浓度培养条件下的表达水平A：LB培养基不同铁离子浓度下fur基因的相对表达分析；B：LB培养基不同铁离子浓度下Fur蛋白的表达水平

Fig.2 Expression of fur gene and Fur protein in wild type A1501 under different iron conditions

图3 野生型A1501和fur突变株的碳源代谢谱比较

Fig.3 Changes in the carbon metabolite profiling between wild type A1501 and fur mutant

图4 野生型A1501、fur突变株和回补株在铁过量和铁限制条件下的生长情况A：菌株在LB铁过量培养基中的生长情况；B：菌株在LB铁限制培养基中的生长情况。

Fig.4 Growth curves of wild type A1501, fur mutant and C-fur under iron-excessive and iron-limited conditions

图5 野生型A1501和fur突变株胞内铁元素水平A：野生型A1501及fur突变株菌株在LB培养条件下胞内铁含量测定；B：铁转运相关的基因在野生型和fur突变株中的表达分析

Fig.5 Intracellular iron level between wild type A1501 and fur mutant

图6 H2O2冲击条件后野生型A1501、 fur突变株及回补株存活率的比较

Fig.6 Comparision of survival rates among wild type A1501, fur mutant and complementary strain after treatment with H2O2

图7 qRT-PCR检测H2O2冲击条件下野生型A1501、 fur突变株氧化胁迫相关基因表达

Fig.7 Expression of oxidative-response genes in wild type A1501, fur mutant after treated with H2O2 by qRT-PCR

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施氏假单胞菌A1501铁吸收调节蛋白Fur的表达特性与功能鉴定

Expressional and Functional Characterization of the Ferric Uptake Regulator Fur in Pseudomonas stutzeri A1501

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