25 U), MgCl2 (3 mM). Genomic DNA was prepared from single colonies re-suspended in 100 μl of Tris-EDTA buffer (TE, pH 7.5), heated at 95°C for 5 min and centrifuged briefly. The supernatant (2 μl) was used for PCR reactions. The universal primers

forward 27f and reverse 1492r were used for 16S rRNA gene amplification. The primers forward Coprun F2 and reverse Coprun R1 were used for the amplification of the copA gene. The forward primer 5’-GTCGTTAGCTTGCCAACATC-3’ and the reverse primer 5’-CGGAAAGCAAGATGTCGAATCG-3’ [31] were used for chrB gene (chromate resistance) amplification. The forward primer 5’-ACCATCGGCGGCACCTGCGT-3’ and the reverse primer 5’-ACCATCGTCAGGTAGGGGAACAA-3’ were used for merA gene (inorganic mercury resistance) amplification [32]. The forward primers 5’-TCGCCCATATATTTTAGAAC-3’ and the reverse primer 5’-GTCGGGACAGATGCAAAGAAA-3’ were used for merB gene (organic mercury resistance) amplification [32]. DNA amplification learn more of chrB, merA and merB was carried out using the following conditions: 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, plus a final extension at 72°C for 7 min. C. metallidurans MSR33 was used as positive control for copA, chrB, merA and merB genes [31]. PCR products were visualized by agarose gel selleck electrophoresis

followed by staining with GelRed (1:10,000 v/v). 16S rRNA and copA genes sequence analyses The PCR products were visualized by agarose gel electrophoresis. Bands were cut from the gel with a scalpel and DNA was recovery using Zimoclean Gel

DNA Recovery Kit (Irvine, CA, USA). The purified DNA was sequenced directly by an Applied Biosystem 3730XL DNA sequence (Carlsbad, CA, USA), using the primers 27f and 1492r for 16S rRNA gene and Coprun F2 and Coprun R1 for copA gene sequencing, respectively. The nucleotide sequences of 16S rRNA genes were aligned with sequences available in the GenBank (http://​www.​ncbi.​nlm.​nih.​gov/​). The nucleotide sequence of copA gene was translated into a protein sequence using blastx. Then, partial sequences of CopA were aligned with other CopA sequences 3-mercaptopyruvate sulfurtransferase from Cu-resistant bacteria [18]. A phylogenetic analysis was performed to study the evolutionary relationships of the sequences based on the alignments calculated by CLUSTAL W using the default options. The evolutionary history was inferred using the Neighbor-Joining method. Evolutionary analyses were conducted in MEGA 5.05 software [33]. The 16S rRNA gene sequence of strains O12, A32, A55, C21 and O4 were submitted to the EMBL Nucleotide Sequence Database under accession number EMBL:HE608567, EMBL:HE608568, EMBL:HE608569, EMBL:HE608570 and EMBL:HE608571, respectively. The copA gene sequence of strains O12, A32, A55, C21 and O4 were submitted to the EMBL Nucleotide Sequence Database under accession number EMBL:HE716432, EMBL:HE716433, EMBL:HE16434, EMBL:HE16435, EMBL:HE16436, respectively.