Identification of copper resistant microorganisms by PCR.

ALBARRACÍN, V.H.,; BENITO, J.M.,; SIÑERIZ, M,; AMOROSO, M.J.,; ABATE, C.M.

Environmental Microbiology Methods and Protocols, Series “Methods in Biotechnology”,

Humana Press. Inc.,

Lugar: New Jersey, USA; Año: 2004; p. 243 - 248

Copper is an essential trace element required as enzyme cofactor and it is involved in diverse biological processes including respiration, destruction of free radicals, and iron homeostasis. It is also used in industrial applications such as production of steel and other alloys, galvanization of iron, electroplating, manufacture of batteries, TV tubes and pigments. However, mining and leaching from natural deposits contribute to environmental contamination. This kind of pollution posses a great risk to the survival of natural microorganism populations in soil and sediments where industries discharge their effluents. Exposition to this metal, leads microorganisms to develop resistance mechanisms such as reduced influx, facilitated efflux, sequestration, and modification of copper among others . Investigation assays are performed in many unrelated microorganisms such as Synechococcus, Saccharomyces cerevisiae , Candida   albicans , Enterococcus hirae, Helicobacter spp., Escherichia coli , Pseudomonas spp. , Methanobacterium bryantii  Vibrio alginolyticus , Salmonella typhimurium , Xanthomonas campestris , Alcaligenes eutrophus , and gram positives such as actinomycetes . Trajanovska et al, investigated the nature of the genetic systems encoding copper resistance, using primers constructed on the base of the nucleotide sequence of the pco (plasmid-borne copper resistant) resistant determinant that is carried by the plasmid pRJ1004, as a growth promoter. Nevertheless, DNA isolation and purification of both genomic and plasmid DNA is the crucial step for the success development of this molecular method. Among the protocols used to the isolation of genomic and plasmid DNA in Streptomyces strains, the Kirby mix, the lysozyme treatment, the salting out and the CTAB procedures are used for Streptomyces genomic DNA isolation. For Streptomyces plasmid isolation, an adaptation of the alkaline lysis procedure is most commonly used. The hot alkaline lyses-acid phenol method produces CCC Streptomyces and E.coli plasmids that become suitable for restriction digests . The aim of this chapter is to describe a genomic DNA isolation technique for different actinomycete strains and a PCR amplification assay using pcoA and pcoR primers useful for copper resistant actinomycetes genes identification.