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Vol. 12 No. 2 - April 2006

Bacterial Resistance: A Tool For Remediation of Toxic Metal Pollutants

By: O.P. Shukla, U.N. Rai, Smita Dubey & Kumkum Mishra


Bioremediation is a process that utilizes inexpensive microbial biomass to sequester toxic heavy metal and is particularly useful for the removal of contaminants from industrial effluents. Bioremediator agents are prepared from the naturally abundant and waste biomass of algae, moss, fungi and bacteria that have been killed while the biomass is pre-treated by washing with acids and bases before final drying and granulation. Bacterial biomass is more useful and effective during the remediation of contaminated sites. This process offers the advantage of low operating cost, minimization of volume of chemical or biological sludge to be disposed and high efficiency in detoxifying very dilute effluent. These advantages have served as the primary incentives for developing full-scale bioremediation process to minimize heavy metal pollution.

Plasmid Mediated Toxic Metal Resistance

Many bacterial strains contain genetic determinants of resistance to heavy metals such as mercury, silver, arsenic, bismuth, cadmium, chromium, nickel, lead and undoubtedly others. These resistance determinants are often found on plasmids and transposons, which facilitate their analysis by molecular genetic technique. In the frequent absence of any obvious source of direct selection, these resistances occur with surprisingly high frequencies. It has been suggested that heavy metal resistances may have been selected in earlier times, and that they are merely carried along today for a free ride with selection for antibiotic resistances. We doubt that there is such a thing as a free ride as for as these determinants are concerned. For example, in Tokyo in the late 1970s both heavy metal resistances and antibiotic resistances were found with high frequencies in Escherichia coli isolated from hospital patients ,where as heavy metal resistance plasmids without antibiotic resistance determinants were found in E. coli from an industrially polluted river. Selection occurs for resistances to both types of agents in the hospital, but only for resistance to toxic heavy metals in the river environment. Redfard et al. found mercury resistance microbes in agricultural soil with no known mercurial input. In such settings, resistance microbes may be very rare, but they may come into much greater quantitative prominence after industrial or agricultural pollution. These major recent progresses have consisted of the cloning and DNA sequence analysis of determinants for mercury, arsenic, cadmium and tellurium resistances and initial reports of still additional resistances

Resistance to Arsenic

The mechanism of arsenate resistance is reduced accumulation of arsenate by induced resistant cell. Arsenate ions enter bacterial cells via the phosphate transport systems. Arsenate is toxic to bacteria because it is an analog of phosphate and can inhibit enzymes such as kinases. Also, arsenylated sugar hydrolyze spontaneously, resulting in a loss of free energy in glycolysis. Resistance to arsenate is determined by plasmid in S. aureus and enteric bacteria. The arsenate resistance determinants of both S. aureus and E. coli specify an efflux system, which has recently been studied in some detail by measuring the loss of arsenic from preloaded resistance cells.

Arsenic resistances are governed by plasmids that also code for antibiotic and other heavy metal resistances. The presence of the resistance plasmid does not alter the kinetic parameters of the cellular phosphate transport system; even the Ki for arsenate as a competitive inhibitor of phosphate transport is unchanged. Direct evidence for plasmid governed enzyme dependent efflux of arsenate indicated that the reduced net uptake of arsenic resulted from rapid efflux. Molecular genetic studies, cloning, Southern blotting and minicell polypeptide synthesis followed the physiological and biochemical studies of the arsenic resistance determinants. The DNA sequence of a Staphylococcus arsenic resistance determinants was recently completed.

The plasmid determined arsenic resistance system has always had the same biochemical mechanism, reduced uptake due to an ATPase efflux system in both gram-negative and gram-positive bacteria.

Resistance to Mercury

Mercuric ions are toxic to bacteria be cause they bind avidly to sulpha hydryl groups and inhibit macromolecule synthesis and enzyme action. Many enzymes have critical thiol group and are sensitive to mercury in vitro. Transcription and translation are particularly sensitive. This may be due to the inhibition of precursor synthesis or mercury binding to polynucleotide. Resistance to mercurial is a common plasmid determined property of both gram positive and gram negative bacteria. A decreasing incidence of mercurial resistance in hospital strains has coincided with the discontinuation of mercurial disinfectant usage. Also, mercury is frequently specified by drug resistance plasmids and is also common in soil Pseudomonas and Bacilli. It has recently been found in Thiobacillus ferrooxidanns. Bacterial resistance to mercury is determined by enzymatic reduction of the ion to mercury, which is much toxic. The enzyme that catalyses the reduction of mercury is the intracellular, cytoplasmic, FAD-containing mercuric reductase. The reductase mechanism also involves a plasmid specified mercury specific transport system. It seems to be required to direct mercury through cytoplasmic membrane, when it would otherwise encounter sensitive enzymes. It seems that the reductase and transport function might interact physically. Indeed, some reductase protein appears to be membrane-associated in E. coli minicells.

Resistance to Tellurium

Tellurite and tellurate resistances have been detected from plasmidmediated determinants. The resistance mechanism is not known. Early work on tellurite resistance in bacteria showed that although reduction of TeO3-2 to black Te0 can be seen, it is apparently not the resistance mechanism because it occurs with both sensitive and resistant cells. Alkylation of tellurium is apparently also not the mechanism of resistance. Recently, Jobling and Ritchie cloned the tellurium resistance determinants from a large alcaligenes plasmid in to E. coli, where it was expressed from a 3.55 kb DNA fragment. However, there is a low frequency mutation on the plasmid that results in a tellurite resistance determinants on RP4.

Resistance to Chromium

Bacterial resistance to chromate has been found in several Pseudomonas strains and also with a plasmid in Streptococcus lactis . Horitsu et al showed that a CrO42 a sensitive Pseudomonas ambigua strain accumulated six times more chromate than a resistant strain. Ontake et al. recently concluded that the basis of plasmidmediated chromateresistance in pseudomonas is the reduction in chromate uptake by the plasmid bearing strain. With P. fluorescens containing a chromate resistance plasmid the V max for chromate uptake was reduced, the K for chromate uptake was unchanged, and Ki for chromate as a competitive inhibitor of sulfate transport was unchanged. There was no difference in chromate efflux between the sensitive and resistant P. fluorescens strain,which suggests that the block may be at the level of uptake rather than efflux. Thus, there is an interplay between plasmids governed fucntions and chromosomal gene determined properties. The sulfur source used for cell growth greatly affects chromate resistance level. The rate of chromate uptake by P. aeruginosa also was regulated by the sulfur source.

Resistance to Cadmium

There are six or more systems for bacterial cadmium resistance known today. However, little physiological and biochemical work has been done. Only one of these systems has been cloned, and DNA sequencing has just been completed in our laboratory. Therefore, our understanding of bacterial cadmium resistance is preliminary and tentative.

Cadmium ions are taken into sensitive bacterial cells by the energydependent manganese transport system, where they cause rapid cessation of respiration by binding to sulfahydryl group in protein. Resistance to cadmium is a common plasmid specified function in S. aureus. In other bacterial genera cadmium resistance is frequently associated with the large “penicillinase” plasmids. Some plasmid specified both Cad A and Cad B resistance determinants, whereas other carry Cad A determinants. A small multicopy Cad A plasmid has also recently been described. It has been known for some time that high level resistance to cadmium involves decreased accumulation. It is now clear from the detailed studies of Tynecka et al. that cadmium resistance is caused by a plasmid encoded efflux system.

Resistance to Copper

Plasmid determined copper resistance has been reported on an antibiotic resistance plasmid, in E. coli isolated from pig fed copper supplements as growth stimulants, and in P. syringae from plants treated with copper as an antibiotic reagent. Although the genes determining copper resistance from P. syringae have been cloned and transferred into E. coli, there have been no detail studies of the mechanism of copper resistance. Thus the proposed cellular copper sequestration might be the basic mechanism of copper resistance.

Resistance to Silver

Microbial silver toxicity is found in situation of industrial pollution, especially those associated with mining and use of photographic film. In hospitals, silver salts are the preferred antimicrobial agent for tropical use on patients with large burns. It is thus not surprising that silver resistant bacteria and silver resistant plasmids have been described and found in polluted industrial and mining sites. Plasmid determined silver resistance is very strong. The ratio of minimal inhibitory silver concentration for resistant and sensitive strain can be greater than 100:1, and under some condition resistance cell can be grown in concentration of up to 0.5 M added silver salts. The level of resistance depends on silver complexing components. However, it is unclear how this type of resistance mechanism can explain the accumulation of amounts of silver equivalent to up to 30% of cell mass by resistant cells. Apparently, silver is reduced to metallic silver equally by sensitive and by resistant cells, so that reduction of silver does not appear to be a primary resistance mechanism.


Analysis of DNA sequence of some heavy metal resistance system have advanced our understanding of mercury, cadmium and arsenic resistance mechanisms enormously. Understanding of tellurium resistance is less complete because the biochemical mechanism is not known. There remain resistance system for about ten additional toxic heavy metal. Heavy metal resistant strains isolated from environmental or clinical sources generally have these resistances on plasmid. Chromosomal mutation to heavy metal resistance can be produced in the laboratory but does not generally occur in nature. The chromosomally determined mutation leading to arsenate, cadmium, chromate and cobalt resistances are due to changes in the membrane transport system responsible for uptake of the beneficial materials such as phasphate, manganese, sulfate and magnesium along with these toxic materials.

O.P. Shukla, U.N. Rai & Smita Dubey are at Ecotoxicology & Bioremediation Division, National Botanical Research Institute (NBRI), Lucknow and Kumkum Mishra is at Department of Botany, Lucknow University, Lucknow (India).

This article has been reproduced from the archives of EnviroNews - Newsletter of ISEB India.

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