Identification of copper‐induced genes in Pseudomonas fluorescens and use of a reporter strain to monitor bioavailable copper in soil
Identification of copper‐induced genes in Pseudomonas fluorescens and use of a reporter strain to monitor bioavailable copper in soil
复制标题
DOI：
10.1111/j.1574-6941.2001.tb00882.x
复制
发表时间：
2001-12
影响因子：
4.2
通讯作者：
Andreas Tom-Petersen;Carsten Hosbond;O. Nybroe
中科院分区：
文献类型：
--
作者：
Andreas Tom-Petersen;Carsten Hosbond;O. Nybroe
铜 (Cu) 是生物体所需的必需微量金属，但在较高浓度下具有潜在毒性。铜盐在农业中用作杀菌剂，在养猪业中用作生长促进剂。因此，它们被引入环境中，例如通过粪便，可能会对土壤细菌群落产生负面影响。在这项研究中，我们的目标是开发一种由本地土壤细菌携带的特定且稳定的铜报告基因结构，以测量土壤中铜的生物利用度。使用 Tn5::luxAB 启动子探针转座子对荧光假单胞菌菌株 DF57 进行诱变后，我们鉴定了四种响应 Cu 响应而 luxAB 表达升高的突变体。其中两个突变位点编码与 Cop 蛋白同源的蛋白，赋予丁香假单胞菌铜抗性，而第三个突变位点则与金属转运 ATP 酶同源。在第四个突变体 DF57-Cu15 中，被转座子打断的基因编码了一种带有 Cu 结合结构域的蛋白质，但与已知蛋白质的同源性较低。 DF57-Cu15 是最合适的 Cu 报告基因，因为它在纯培养物中具有高特异性响应和对 Cu 的耐受性。 DF57-Cu15 以浓度依赖性方式对来自 Cu 改良土壤微观世界的土壤溶液做出反应。螯合剂 EDTA 降低了土壤中荧光假单胞菌对铜的利用率。这表明细菌不一定能够利用复合物结合的铜。我们比较了可溶性铜的化学分析和对补充有粪肥或秸秆的含铜土壤微观世界的土壤溶液的报告分析。有机物增加了可溶性铜的量，但没有增加生物可利用的铜的量。铜可能与猪粪或大麦秸秆中的有机成分具有高亲和力。因此，通过化学分析测定可溶性铜并不总能预测铜对特定细菌的生物利用度。
Copper (Cu) is an essential trace metal required by living organisms but it is potentially toxic at higher concentrations. Cu salts are used as bactericides in agriculture and as growth promoters in pig production. They are consequently introduced to the environment, e.g. through manure, and might negatively affect the bacterial soil community. In this study, our goal was to develop a specific and stable Cu reporter construction harboured by an indigenous soil bacterium to measure bioavailability of Cu in soil. Following mutagenesis of Pseudomonas fluorescens strain DF57 with a Tn5::luxAB promoter probe transposon, we identified four mutants with elevated luxAB expression in response to Cu. Two of the mutated loci encoded proteins homologous to Cop proteins conferring Cu resistance to Pseudomonas syringae, while a third displayed homology to metal-transporting ATPases. In the fourth mutant, DF57-Cu15, the gene interrupted by the transposon encoded a protein carrying a Cu-binding domain but with low homology to known proteins. DF57-Cu15 was the most suitable Cu reporter due to its high specific response and tolerance to Cu in pure culture. DF57-Cu15 responded to soil solutions from Cu-amended soil microcosms in a concentration-dependent manner. The chelator EDTA reduced the availability of Cu to P. fluorescens in soil. This showed that complex-bound Cu is not necessarily available to bacteria. We compared chemical analysis of soluble Cu and the reporter assay on soil solutions from Cu-containing soil microcosms supplemented with either manure or straw. Organic matter increased the amount of soluble Cu but not the amount of bioavailable Cu. Probably, Cu binds with high affinity to organic constituents in pig manure or barley straw. Hence, determination of soluble Cu by chemical analysis cannot always predict the bioavailability of Cu to a specific bacterium.