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Bacterial Resistance: A Tool For Remediation of Toxic Metal
Pollutants
By: O.P. Shukla, U.N. Rai, Smita Dubey & Kumkum Mishra
Introduction
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.
Conclusion
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). |
