Identification of copper-induced genes in Pseudomonas fluorescens and use of a reporter strain to monitor bioavailable copper in soil

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.