Characterization of copper-resistant bacteria and bacterial communities from copper-polluted agricultural soils of central Chile

Abstract

Background: Copper mining has led to Cu pollution in agricultural soils. In this report, the effects of Cu pollution on bacterial communities of agricultural soils from Valparaiso region, central Chile, were studied. Denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes was used for the characterization of bacterial communities from Cu-polluted and non-polluted soils. Cu-resistant bacterial strains were isolated from Cu-polluted soils and characterized.

Results: DGGE showed a similar high number of bands and banding pattern of the bacterial communities from Cu-polluted and non-polluted soils. The presence of copA genes encoding the multi-copper oxidase that confers Cu-resistance in bacteria was detected by PCR in metagenomic DNA from the three Cu-polluted soils, but not in the non-polluted soil. The number of Cu-tolerant heterotrophic cultivable bacteria was significantly higher in Cu-polluted soils than in the non-polluted soil. Ninety two Cu-resistant bacterial strains were isolated from three Cu-polluted agricultural soils. Five isolated strains showed high resistance to copper (MIC ranged from 3.1 to 4.7 mM) and also resistance to other heavy metals. 16S rRNA gene sequence analyses indicate that these isolates belong to the genera Sphingomonas, Stenotrophomonas and Arthrobacter. The Sphingomonas sp. strains O12, A32 and A55 and Stenotrophomonas sp. C21 possess plasmids containing the Cu-resistance copA genes. Arthrobacter sp. O4 possesses the copA gene, but plasmids were not detected in this strain. The amino acid sequences of CopA from Sphingomonas isolates (O12, A32 and A55), Stenotrophomonas strain (C21) and Arthrobacter strain (O4) are closely related to CopA from Sphingomonas, Stenotrophomonas and Arthrobacter strains, respectively.

Conclusions: This study suggests that bacterial communities of agricultural soils from central Chile exposed to long-term Cu-pollution have been adapted by acquiring Cu genetic determinants. Five bacterial isolates showed high copper resistance and additional resistance to other heavy metals. Detection of copA gene in plasmids of four Cu-resistant isolates indicates that mobile genetic elements are involved in the spreading of Cu genetic determinants in polluted environments.

Figure 1

Location of sampling sites of agricultural soils in Valparaíso region, central Chile. North Chagres, South Chagres and Ñilhue are Cu-polluted sites. La Vinilla is a non-polluted site.

Figure 2

DGGE of 16S rRNA genes of bacterial communities from agricultural soils.A. DGGE of bacterial communities from North Chagres (lanes N1-N3), South Chagres (lanes S1-S3), Ñilhue (lanes Ñ1-Ñ3) and La Vinilla (V1-V3). B. Dendrogram showing cluster analysis of 16S rRNA gene profiles of bacterial communities from the four agricultural soils. Gel image analysis was performed by using Phoretix 1D software package. Bands were automatically detected and manually corrected. A binary matrix was generated by presence or absence bands. The sample similarities were analyzed by MVSP.

Figure 3

Identification of bacterial isolates by 16S rRNA gene sequence analysis. The phylogenetic tree was constructed using neighbor-joining method. Values of 1000 bootstrap replicates above 60% are given at the branching point. Sequences of the bacterial isolates Sphingomonas sp. strain O12, Sphingomonas sp. strain A32, Sphingomonas sp. strain A55, Stenotrophomonas sp. strain C21 and Arthrobacter sp. strain O4 are highlighted (black circles). Vertical bar represents 0.02 units of evolutionary distance.

Figure 4

Phylogenetic tree showing the relatedness of multi-copper oxidase CopA of the bacterial isolates. The phylogenetic tree was constructed using neighbor-joining method. Values of 1000 bootstrap replicates above 50% are given at the branching point. Sequences of CopA proteins of the bacterial isolates Sphingomonas sp. strain O12, Sphingomonas sp. strain A32, Sphingomonas sp. strain A55, Stenotrophomonas sp. strain C21 and Arthrobacter sp. strain O4 are highlighted (black circles).

Figure 5

Detection of plasmids encoding copA genes in copper-resistant bacterial isolates.A. Agarose gel electrophoresis of plasmids isolated from Sphingomonas sp. strain O12 (lane 2) Sphingomonas sp. strain A32 (lane 3), Sphingomonas sp. strain A55 (lane 4) and Stenotrophomonas sp. strain C21 (lane 5). No plasmid was observed in Arthrobacter sp. strain O4 (lane 6). C. metallidurans strain MSR33 that carries the pMOL30, pMOL28 and pTP6 plasmids was used as a positive control (lane 1). B. Detection of copA gene in plasmids of bacterial isolates. The copA gene was detected in Sphingomonas sp. strain O12 (lane 2), Sphingomonas sp. strain A32 (lane 3), Sphingomonas sp. strain A55 (lane 4) and Stenotrophomonas sp. strain C21 (lane 5). C. metallidurans strain MSR33 (lanes1) was used as a positive control.