Biology

Microbiological tolerance of metals

Briefly describe the methods used by bacteria and fungi to tolerate environmental heavy metals. Include references and your views on the potential commercial applications of bacteria and fungi.

Mechanisms used by Bacteria and Fungi to Tolerate Heavy Metals.

A good indication of the health of an ecosystem is the diversity of its microflora. An ecosystem that displays a rich array of bacteria, fungi, and other microorganisms, can be described as a healthy environment. Stresses applied to an organism, can cause a change in that microbe's growth rate. When a microorganism, such as a bacterium or fungus, is exposed to various stresses, a typical response is a decrease in the rate of growth of the organism. An important stress like low temperature results in a reduction of the molecular motion in biological systems (Taylor, 1987). With a decrease in molecular motion comes a subsequent reduction in growth rate. In the natural environment the microflora must overcome countless obstacles. Such stresses as temperature, water activity, and nutrient availability are impediments that fungi and bacteria face constantly. On top of the naturally occurring stresses, artificial stresses influence the activity of fungi and bacteria. These include changes in soil pH, man-made drought and flooding and the dumping of heavy metals.
Heavy metal stress is caused by excessive amounts of the class B/borderline heavy metals that includes copper, nickel, zinc, lead, silver, cadmium and mercury (Nieboer and Richardson, 1990). To tolerate heavy metals, organisms have two basic mechanisms at their disposal: avoidance and sequestration (Baker and Walker, 1990). This paper is concerned with the techniques used by bacteria and fungi to tolerate excessive heavy metals present in the ecosystem. Table 1 describes the general methods bacteria and fungi use to tolerate metal stresses discussed in this paper. There are many different methods used apart from the ones below. Bacteria and fungi can employ a range of mechanisms and are not just limited to a particular mechanism.

Table 1. Mechanisms of heavy metal tolerance discussed.
Cellular Process Tolerance Mechanism
Cell Wall Avoidance
Chemical transformation Avoidance
Metal-binding proteins Sequestration
Chelating agents Sequestration

The tolerance displayed by bacteria and fungi may be thought of as the result of man-made pollution. However, lots of these mechanisms of tolerance are genetic and probably arose from the pre-human environments plagued by volcanic pollution. Since the earth contains these heavy metals in varying amounts, we can assume that all organisms on the planet have adapted ways of coping with these metal ions. Some of the metal ions mentioned above are required in trace amounts for growth of some organisms. Cooper, zinc and lead are essential micronutrients, but are toxic when present in excess (Tomsett, 1993). This poses a difficulty in maintaining homeostasis. Fungi and bacteria have to evolve to accept certain metal ions, but reject excessive amounts.

Avoidance
The mechanisms of avoidance in fungi and bacteria are similar. The principal component that helps these organisms and that sets it apart from other cells is the cell wall that exists outside the more fluid plasma membrane. Both fungi and bacteria have a cellular wall that helps in keeping unwanted materials extracellular. This method of avoidance is common to bacteria and fungi and is a basic mechanism of heavy metal tolerance.
A soil fungus can be seen by its filamentous mycelial strands that aggregate to form a visible shape. These strands comprise the vegetative portion of the fungus (Hawker, 1966). The mycelia of a fungus can act occasionally to increase heavy metal tolerance. Ramo Rao et al. (1997) have shown that certain strains of the Curvularia sp. have cadmium tolerance due to its ability to bind cadmium to surface mycelia. They used an isolate of Curvularia lunata that showed a high tolerance to cadmium when grown in a cadmium rich medium. Floating the mycelia in an EDTA solution leached off the cadmium that was bound to the fungus. Ramo Rao et al. (1997) found that 90% of the cadmium taken up by the fungus was extractable by EDTA and therefore bound to the surface of the fungus. They also showed that the cadmium present was in ionic form and not bound to any proteins. They did not account for the other 10% of cadmium uptake by the fungus suggesting that other methods of tolerance may exist.
Cadmium resistance in fungi seems to be the result of avoidance techniques. Zinc has similar properties to cadmium and as a result the avoidance techniques are similar to those due to increased amounts of cadmium. The fungi seem to bind cadmium and zinc in its structural components including its cell wall (Tohoyama et al., 1990). The tolerance to toxic amounts of cadmium was shown by Tohoyoma et al. (1990) to be under the genetic control of the CAD2 gene. Understanding the genetics behind resistance helps in deriving the molecular mechanisms of heavy metal tolerance.
Another type of avoidance technique is by transforming the chemical to an easily volatilized form. Certain strains of Bacillus spp are resistant to mercury by means of chemical transformation. The mercury resistant strains of Bacillus spp. will produce mercury reductase. The reductase allows the volatilization of elemental mercury from mercury (Bharathi et al., 1990). The production of a secondary molecule allows the bacteria to release the toxic mercury, avoiding metal stress. Apart from the case of mercury reductase, the mechanisms of heavy metal tolerance in bacteria have not been fully defined (Silver and Phung, 1996).

Sequestration
Techniques employed by fungi and bacteria in dealing with heavy metal stress also include the sequestration of metal ions. These techniques involve forming complexes outside the cell or transporting the ions into the cell and sequestering the metal ions inside organelles.
Melanin is a dark pigmented polymer suggested to have an involvement in heavy metal tolerance. In cultures of the fungus Aspergillus pullulans and Cladosporium resinae the production of extracellular melanin has been shown to bind copper (Gadd and de Rome, 1988). Another soil fungus, Gaeumannomyces graminis var. graminis produced melanin in its cell wall upon exposure to copper (Caesar-Tonthat et al., 1995). The melanin produced would accumulate in the cell walls of the mycelia. Using electron microscopy, Caesar-Tonthat et al. (1995) looked at silver stained fungal cells exposed to CuS and saw that the CuS was localized in the melanin layer in fungal cell walls. This enabled the fungus to tolerate a toxic copper stress.
Bacteria employ a similar tactic to fungi with respect to sequestration tolerance mechanisms. A sulphate reducing bacteria amended with lead was seen to tolerate the toxic metal ions. Bharathi et al. (1990) showed that detoxification occurred by formation of lead-sulphur complexes. The formation of metal ion complexes is a common way of dealing with heavy metal stress.
In fungi a group of proteins known as the metallothioneins have been studied which form complexes and aid in metal tolerance. A metallothionein is a small protein that can form metal complexes due to the numerous cysteine residues present. An example of this effect is the copper specific metallothionein found in the fungus Agaricus bisporus (Munger and Lerch, 1985). Recently metallothioneins have been found in the Cyanobacteria (Silver and Phung, 1996). This metallothionein was the first to be characterized in bacteria.
The use of vacuoles to sequester toxic metal ions has not been demonstrated in many bacterial and fungal isolates. A type of metallothionein has been shown to transport cadmium in the tobacco plant (Vogeli-Lange and Wagner, 1990). The transported cadmium is sequestered in vacuoles in the tobacco leaf cell. Although, not bacteria or fungal sequestration, there is the possibility of this mechanism of tolerance application. Both bacteria and fungi have metallothioneins and may use them for the sequestration in vacuoles of toxic heavy metals. Further research in this area would be needed to see if this is true.

Conclusion
Heavy metal tolerant bacteria and fungi have many different possibilities in commercial applications. Fungi and bacteria have an important role in nutrient cycling in the environment. Zibilske and Wagner (1982) showed that toxic amounts of metal ions in soil affected the diversity of the microflora. The change in the microflora was both a qualitative and quantitative decrease in bacteria and fungi. The resultant loss could affect the soil's nutrient cycling capability. Without bacteria and fungi, the health of an ecosystem is placed in jeopardy.
In excessively polluted areas, these bacteria and fungi can be used as a chelating agent. Understanding the mechanisms of metal tolerance will help to select for isolates suitable for each microcosm's needs. An ecosystem could be "cleaned up" by using metal tolerant bacteria and fungi that sequester heavy metal ions. In order for this possible use of metal tolerant bacteria and fungi there needs to be further research into the mechanism of sequestration and the isolation of suitable tolerant strains.
Bacteria and fungi use many techniques to tolerate toxic levels of heavy metals. It is important to note that sequestration and avoidance are not mutually exclusive. As previously mentioned in the study by Ramo Rao et al. (1997), only 90% of cadmium tolerance could be accounted for by avoidance techniques. The other 10% could be tolerated by another mechanism. Research in this field is important due to the increased dumping of heavy metals into the environment. Most research is centered on isolating bacteria and fungi that are metal tolerant. Little research, however, has been done on the molecular mechanisms of heavy metal tolerance by bacteria and fungi. To understand the nature of metal tolerance better we need to recognize how the mechanisms through which a fungus or bacterium tolerates heavy metal stress.

References

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Baker, A. J. M. and P. L. Walker. (1990). Ecophysiology of metal uptake by tolerant plants, In: Heavy Metal Tolerance in Plants: Evolutionary Aspects, ed: A. J. Shaw. pp 155-173.

Bharathi, P. A. L., V. Sathe, and D. Chandramohan. (1990). Effect of lead, mercury, and cadmium on a sulphate-reducing bacterium. Environ. Pollut. 67: 361-374.

Gadd, G. M. and L. de Rome. (1988). Biosorption of copper by fungal melanin. Appl. Microbiol. Biotechnol. 29: 610-617.

Hawker, L. E. (1966). Fungi: an introduction. Hutchinson: London, UK. Pp 9-17.

Munger, K. and K. Lerch. (1985). Copper-metallothionein from the fungus Agaricus bisporus - chemical and spectroscopic properties. Biochem. 24: 6751-6756.

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Silver, S. and L. T. Phung. (1996). Bacterial heavy metal resistance: new surprises. Annu. Rev. Microbiol. 50: 753-789.

Taylor, M. J. (1987). Physio-chemical principles in low-temperature biology, In: The effects if low temperature on biological systems, eds: B. W. W. Grant and G. J. Morris. Edward Arnold, UK, pp 3-71.

Tohoyama, H., M. Inohoue, M. Joho, and T. Murayama. (1990). Resistance to cadmium is under the control of the CAD2 gene in the yeast Saccharomyces cerevisiae. Curr. Genet. 18: 181-185.

Tomsett, A. B. (1993). Genetics and molecular biology of metal tolerance in fungi, In: Stress Tolerance of Fungi, ed: D. H. Jennings. Marcel dekker Inc. New York, NY. pp 69-95.

Vogeli-Lange, R., and G. J. Wagner. (1990). Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves. Plant Physiol. 92: 1086-1093.

Zibilske, L. M. and G. H. Wagner. (1982). Bacterial growth and fungal genera distribution in soil amended with sewage sludge containing cadmium, chromium, and copper. Soil. Sci. 134(6): 364-370.