The copA genes of the fives isolates encode multi-copper oxidases that oxidize Cu(I) to Cu(II) but not phenolic compounds or polymers as other multi-copper oxidases reported [41, 42]. Phylogenetic analyses of 16S rRNA gene sequences indicate that the isolates belong to Sphingomonas, Stenotrophomonas and Arthrobacter genera. The phylogenetic tree obtained from the sequence analysis of 16S rRNA gene was similar to those results predicted from the sequence analysis of CopA protein (Figure 3 and 4), showing a high concordance between structural and functional genes. Mobile genetic elements (MGE) could

be involved in the spreading of Cu resistance determinants, facilitating the adaptation of bacterial communities to copper [43]. Bacteria exposed to copper for a long period of time may compound screening assay acquire MGE such as plasmids carrying copper determinants and, therefore, they become copper-resistant bacteria [43–45]. In agreement with this hypothesis, this study showed the presence of the copA gene in metagenomic DNA from the three Cu-polluted soils and the absence of copA gene in metagenomic DNA from the non-polluted soil. This study demonstrates

that Gram-negative Cu-resistant strains isolated from long-term Cu-contaminated soils carried plasmid with Cu-resistance determinants. The presence of plasmids encoding copA genes in Sphingomonas sp. strain O12, Sphingomonas sp. strain A32, Sphingomonas sp. strain A55 and Stenotrophomonas sp. C21 (Figure 5) confirm that MGE are involved in copper resistance in these isolates. The copA (pcoA) genes encoding multi-copper oxidases have been characterized in plasmids such as pPT23D, Transferase inhibitor pRJ1004 and pMOL30

from Escherichia coli RJ92, Pseudomonas syringae pv. tomato PT23 and Cupriavidus metallidurans CH34, respectively [20, 21, 24]. The multi-copper oxidase copA gene was present in the genome of the Gram-positive bacterium Arthrobacter sp. O4, but plasmids were not detected in this strain. The CopA protein sequence Dimethyl sulfoxide from Arthrobacter sp. O4 possesses a high similarity (68%) with the multi-copper oxidase gene of Arthrobacter sp. FB24, which is located in a plasmid [46, 47]. As plasmid this website isolation in some bacterial strains is difficult, the presence of the copA gene from Arthrobacter sp. O4 in a plasmid could not be excluded. Conclusions This study have shown that the bacterial community diversity of agricultural soil of central Chile analyzed by DGGE was similar in Cu-polluted and non-polluted soils. The copA gene encoding multi-copper oxidase was detected only in metagenomic DNA of Cu-polluted soils suggesting that copA genes are widely spread in contaminated environments. Cu-resistant bacteria were isolated from these long-term polluted soils. The MIC studies on bacterial isolates indicated that Cu-resistant bacteria were also resistant to other heavy metal such as Ni2+, Hg2+ and CrO4 2-.