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首页> 外文期刊>Applied Microbiology >Development of a Markerless Gene Replacement System for Acidithiobacillus ferrooxidans and Construction of a pfkB Mutant
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Development of a Markerless Gene Replacement System for Acidithiobacillus ferrooxidans and Construction of a pfkB Mutant

机译：酸性氧化硫硫杆菌无标记基因替换系统的开发和pfkB突变体的构建

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The extremely acidophilic, chemolithoautotrophic Acidithiobacillus ferrooxidans is an important bioleaching bacterium of great value in the metallurgical industry and environmental protection. In this report, a mutagenesis system based on the homing endonuclease I-SceI was developed to produce targeted, unmarked gene deletions in the strain A. ferrooxidans ATCC 23270. A targeted phosphofructokinase (PFK) gene ( pfkB ) mutant of A. ferrooxidans ATCC 23270 was constructed by homologous recombination and identified by PCR with specific primers as well as Southern blot analysis. This potential pfkB gene (AFE_1807) was also characterized by expression in PFK-deficient Escherichia coli cells, and heteroexpression of the PFKB protein demonstrated that it had functional PFK activity, though it was significantly lower (about 800-fold) than that of phosphofructokinase-2 (PFK-B) expressed by the pfkB gene from E. coli K-12. The function of the potential PFKB protein in A. ferrooxidans was demonstrated by comparing the properties of the pfkB mutant with those of the wild type. The pfkB mutant strain displayed a relatively reduced growth capacity in S~(0) medium (0.5% [wt/vol] elemental sulfur in 9K basal salts solution adjusted to pH 3.0 with H_(2)SO_(4)), but the mutation did not completely prevent A. ferrooxidans from assimilating exogenous glucose. The transcriptional analysis of some related genes in central carbohydrate metabolism in the wild-type and mutant strains with or without supplementation of glucose was carried out by quantitative reverse transcription-PCR. This report suggests that the markerless mutagenesis strategy could serve as a model for functional studies of other genes of interest from A. ferrooxidans and multiple mutations could be made in a single A. ferrooxidans strain.
机译：嗜酸性，嗜自生自养的酸性氧化硫硫杆菌铁氧化物是重要的生物浸出细菌，在冶金工业和环境保护中具有重要价值。在本报告中，开发了一种基于归巢内切核酸酶I-SceI的诱变系统，可在A. ferrooxidans ATCC 23270菌株中产生靶向的，未标记的基因缺失。A。ferrooxidans ATCC 23270的靶向磷酸果糖激酶（PFK）基因（pfkB）突变体通过同源重组构建了H2O3，并通过PCR用特异引物和Southern印迹分析进行鉴定。该潜在的pfkB基因（AFE_1807）的特征还在于在缺乏PFK的大肠杆菌细胞中表达，并且PFKB蛋白的异源表达表明它具有功能性PFK活性，尽管它明显低于磷酸果糖激酶-（约800倍）。由大肠杆菌K-12的pfkB基因表达的图2所示的蛋白（PFK-B）。通过比较pfkB突变体与野生型的特性，证明了潜在的PFKB蛋白在氧化铁曲霉中的功能。 pfkB突变菌株在S〜（0）培养基中表现出相对降低的生长能力（用H_（2）SO_（4）调节至pH 3.0的9K基础盐溶液中的0.5％[wt / vol]元素硫），但是该突变不能完全阻止A. ferrooxidans吸收外源葡萄糖。通过定量逆转录-PCR对野生型和突变型菌株中补充或不补充葡萄糖的中央碳水化合物代谢中的一些相关基因进行了转录分析。该报告表明，无标记诱变策略可以用作功能研究氧化铁农杆菌其他目的基因的模型，并且可以在单个铁氧化农杆菌菌株中进行多个突变。

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