Cadmium-regulated gene fusions in Pseudomonas fluorescens.
Cadmium-regulated gene fusions in Pseudomonas fluorescens.
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DOI：
10.1046/j.1462-2920.2000.00117.x
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发表时间：
2000-08
影响因子：
5.1
通讯作者：
S. Rossbach;M. L. Kukuk;T. L. Wilson;S. Feng;M. Pearson;M. Fisher
中科院分区：
文献类型：
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作者：
S. Rossbach;M. L. Kukuk;T. L. Wilson;S. Feng;M. Pearson;M. Fisher
为了研究土壤细菌应对环境中高浓度重金属污染的机制，采用基于lacZ的报告基因转座子Tn5B20进行诱变。普通土壤细菌荧光假单胞菌菌株ATCC 13525的基因组中的随机基因融合物用于创建5，000个荧光假单胞菌突变体的库。在有毒金属镉的存在下，筛选该突变体库的差异基因表达。14个突变体被确定为增加或减少基因表达的镉的存在。突变体的特征在于其金属依赖的基因表达和其金属耐受性。一半的突变体与差异基因表达特异性的金属镉反应，而其他一些突变体也响应浓度升高的铜和锌离子。突变体之一，菌株C8，也表现出增加的基因表达的溶剂乙醇的存在下，但在其他方面没有镉诱导的基因表达和一般的应激反应之间的重叠被检测到。使用任意聚合酶链反应（PCR），DNA测序和推导的蛋白质产物与遗传数据库中保存的序列的比较进行相应的遗传位点的分子分析。转座子靶向的一些遗传位点与任何已知基因没有任何相似性;因此，它们可能代表“新”位点。在重金属存在下差异表达的基因在金属耐受性中起作用的假设被验证的突变体之一。该突变株C11对镉和锌离子高度敏感。在突变体C11中，转座子插入到一个遗传区域，该区域显示与编码双组分系统的传感器/调节蛋白对的基因相似，该双组分系统调节金属抗性细菌中的基因表达，包括真养罗尔斯通氏菌的czcRS，铜绿假单胞菌的czrRS和铜绿假单胞菌的copRS。虽然本研究中使用的荧光假单胞菌菌株尚未从富含金属的环境中分离出来，但它至少含有一个遗传区域，使其能够应对浓度升高的重金属。
To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ-based reporter gene transposon Tn5B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5,000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals.