Novel polymerase chain reaction primers for the specific detection of bacterial copper P-type ATPases gene sequences in environmental isolates and metagenomic DNA

Aims: In the last decades, the worldwide increase in copper wastes release by industrial activities like mining has driven environmental metal contents to toxic levels. For this reason, the study of the biological copper-resistance mechanisms in natural environments is important. Therefore, an appropriate molecular tool for the detection and tracking of copper-resistance genes was developed. Methods and Results: In this work, we designed a PCR primer pair to specifically detect copper P-type ATPases gene sequences. These PCR primers were tested in bacterial isolates and metagenomic DNA from intertidal marine environments impacted by copper pollution. As well, T-RFLP fingerprinting of these gene sequences was used to compare the genetic composition of such genes in microbial communities, in normal and copper-polluted coastal environments. New copper P-type ATPases gene sequences were found, and a high degree of change in the genetic composition because of copper exposure was also determined. Conclusions: This PCR based method is useful to track bacterial copper-resistance gene sequences in the environment. Significance and Impact of the Study: This study is the first to report the design and use of a PCR primer pair as a molecular marker to track bacterial copper-resistance determinants, providing an excellent tool for long-term analysis of environmental communities exposed to metal pollution.