Heavy-metal resistant actinomycetes

Abstract

Water and soil pollution has become a major concern in the world, as much of the population relies on groundwater as its major source of drinking water as well as on soil as cultivable land. Heavy-metal contamination brings a potential health hazard that can cause metal toxicoses in animals and humans (Volesky and Holan 1995). Microorganisms play an important role in the environmental fate of toxic metals and radionuclides because various biological mechanisms transform soluble and insoluble forms of xenobiotics. These mechanisms are part of natural biogeochemical cycles. They are potentially useful for both in-situ and ex-situ bioremedial treatment processes for solid and liquid wastes (Gadd 2000). The use of microorganisms for recovering of metals from waste streams, as well as the employment of plants for landfill application (Watanabe 1997), has raised growing attention. A wide variety of fungi, algae, and bacteria are now under study or are already in use as biosorbents for heavy-metal remediation (Gadd 1992; Diels et al. 1993; Kotrba et al. 1999). Bacteria have evolved strategies to cope with toxic metals in the environment, and bacterial operons that confer resistance to cadmium, mercury, copper and arsenic have been described (Silver and Phung 1996; Nies and Brown 1998; Xu et al. 1998). Recently some chromosomal genes conferring metal tolerance have been detected in several bacterial species as well (Cai et al. 1998; Xiong and Jayaswal 1998; Hassan et al. 1999). Actinomycetes are Gram-positive bacteria with high content of guanine and cytosine (G + C) (55-75 mol%) that generally exhibit a filament growth with ramifications. They are metabolically versatile being a significant component of soil microbial population and they were proved to habit in marine and aquatic sediments (Peczynska-Czoch and Mordarski 1984). Another characteristic of actinomycetes is the great variation of its secondary metabolism with active compounds as final products. Actinomycetes stand out within the prokaryotes microorganisms by their wide morphologic diversity, presenting a continuous transition from round cells, rod-shaped cells, to hyphae that are fragmented with ramifications or stable with ramifications (Goodfellow et al. 1988; Larpent and Sanglier 1989). Copper is a heavy metal that has been used for years as an ingredient of bactericides and fungicides and as a growth enhancer of pigs (Trajanovska et al. 1997). Copper is found in high concentrations (0.1 to 20 mM) in contaminated soils thus being a threat to soil bacteria population. There is a copper filter plant near an agricultural area of Tucum'n, Argentina that flows its wastewater to a natural channel. It has been determined that during the process copper contamination is produced in the environment (Amoroso et al. 1996). Copper is needed at low concentration (<0.1 μM) to cell normal functions but it is very toxic at high concentrations because its production of free radicals causes a serious damage to the cell (Gutteridge and Wilkins 1983; Simpson et al. 1988). Nevertheless, bacteria exposure to toxic levels of copper has led to the development and evolution of various genetics mechanisms that regulate the uptake and resistance to copper (Trevors 1987). Plasmid pRJ1004, that confers copper resistance to E. coli, is one example of these mechanisms (Tetaz and Luke 1983). pRJ1004 is a conjugate 78-megadalton plasmid found in an E. coli strain isolated from the effluent of a piggery where pigs were fed a supplemented diet with copper sulphate. It was shown to carry a copper-resistance determinant, pco (plasmid-borne copper resistance) (Tetaz and Luke 1983). Brown et al. (1995) characterised and described the nucleotide sequence of the pco resistance determinant, together with copper transport studies. There is a lot of information available on copper resistance genetic mechanisms in Gram-negatives bacteria (Williams et al. 1993; Harwood and Gordon 1994; Munson et al. 2000) but little has been done in Gram-positives. In spite of this, Trajanovska et al. (1997) reported the detection of copper and other heavy metals resistance genes in Gram-positives and Gram-negatives isolated from a lead contaminated area. There is still less information about the genes that codifies heavy metal resistance in actinomycetes. Recent experiences indicate that mercury resistance in Streptomyces is related to the presence of giant linear plasmids (Ravel et al. 1998, 2000a,b). Cadmium is a very toxic metal and has been found in the environment at increased concentrations producing different pathologies in humans and animals (Friberg et al. 1979).The accelerated growing of industrial activities producing this contamination has increased the cadmium liberation at a higher rate than the one of the natural geochemical processes (Nriagu and Pacyma 1988). The monitoring of the viability of cadmium resistant actinomycetes in culture medium and in soil samples is of considerable importance because of the potential capacity of these strains in the bioremediation of cadmium (Amoroso et al. 1998). One of the easier ways is to follow the viability by fluorescence. The green fluorescent protein (GFP) has proved to be a particularly useful and sensitive gene reporter. Recently a red-shifted variant of GFP was developed, which gives brighter fluorescence and higher levels of expression in mammalian cells. Enhanced Green Fluorescent protein (EGFP) shows a 35-fold enhancement of fluorescence over wild-type GFP when excited at 488 nm, and possesses excitation and maximal emission of 488 nm and 507 nm, respectively (Sun et al. 1999). Using actinomycetes strains isolated from heavy metals contaminated soils we studied their resistance to copper at different concentrations and then assayed an hybridisation experience with probes constructed using two copper resistance genes (pcoR and pcoA) from pRJ1004 of E. coli. With the aim to study the survival of the cadmium resistance strain Streptomyces R25 (Amoroso et al. 1998) in cadmium contaminated soil we carried out transformations experiments using pIJ8660 that contain the EGFP gene to transform this strain and analyze their survivals.