Studies of soil bacterial community by DGGE revealed that heavy metal contamination in agricultural soils close to copper and zinc smelters may provoke changes in the composition of soil bacterial community and a decrease of the bacterial diversity [11, 16]. However, changes in the soil bacterial community exposed to heavy metal may vary depending of soil properties, heavy metal bioavailability and the indigenous microbial
groups in soil . The genes conferring copper resistance in bacteria are often present in plasmids and organized in an operon [17–19]. The copper resistance is encoded by the cop genes (copA, copB, copC and copD) in Cupriavidus metallidurans CH34, Pseudomonas syringae pv. tomato PT23, Xanthomonas axonopodis pv. vesicatoria selleck kinase inhibitor E3C5 and Pseudomonas aeruginosa PAO1 and by the pco genes (pcoA, pcoB, pcoC and pcoD) in Escherichia coli strain RJ92 [20–24]. The copA gene encoding a multi-copper oxidase (pcoA gene in E. coli) is one of the main genetic determinants involved in Cu-resistance in Gram-negative bacteria. It encodes the multi-copper oxidase that oxidase Cu(I) to the less toxic chemical form of Cu(II) [1, 25, 26]. A different copA gene that encodes a Cu-transporting P-type ATPase
involved in Cu homeostasis has been described in E. coli and other bacteria . The copA gene encoding a multi-copper oxidase is widely present in Cu-resistant bacterial strains and may represent a relevant marker to study the Cu-resistance in bacteria . The aims of this study were to investigate the effect of this website long-term Cu pollution selleck inhibitor on the bacterial community and the characterization of Cu-resistant bacteria from agricultural sites located close to copper smelters from the Aconcagua valley, central Chile. Methods Chemicals The metal salts CuSO4·5H2O, ZnCl2, K2CrO4, NiCl2·H2O, HgCl2, CoCl2·6H2O, CdCl2·2H2O (analytical grade) were purchased from Merck (Darmstadt, Germany) and used to prepare stock solutions of Cu2+, Zn2+, CrO4 2-, Ni2+, Co2+, Cd2+ (800 mM), and Hg2+ (150 mM). HNO3, HClO4 and H2SO4 (Suprapur) and standard Titrisol solution were obtained from Merck (Darmstadt, Germany). Taq DNA polymerase and bovine serum albumin for PCR were
heptaminol obtained from Invitrogen (Carlsbad, CA, USA). Taq DNA polymerase Stoffel fragment was obtained from Applied Biosystems (Darmstadt, Germany). Tryptic soy broth (TSB) and R2A medium were purchased from BD Diagnostic Systems (Heidelberg, Germany). Formamide and ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and urea from Bio Rad (Hercules, CA, USA). Acrylamide was obtained from Winkler (Santiago, Chile). Soil sampling Three composite soil samples were collected from four different agricultural sites in Valparaiso region (central Chile). Each composite sample contained 12 bulk soil cores from the surface stratum (0–10 cm depth) taken from three sampling points located in an area of 250 m2 per site.