INRODUCTION
This paper focuses on the general ways in which microbes
interact with metals. Some bacteria have evolved mecha-
nisms to detoxify heavy metals, and some even use them
for respiration. Microbial interactions with metals may have
several implications for the environment. Microbes may
play a large role in the biogeochemical cycling of toxic heavy
metals also in cleaning up or remediating metal-contami-
nated environments. There is also evidence of a correla-
tion between tolerance to heavy metals and antibiotic resis-
tance, a global problem currently threatening the treatment
of infections in plants, animals, and humans.
Metal Tolerance Mechanisms
In high concentrations, heavy metal ions react to form
toxic compounds in cells (Nies, 1999). To have a toxic ef-
fect, however, heavy metal ions must first enter the cell.
Because some heavy metals are necessary for enzymatic
functions and bacterial growth, uptake mechanisms exist
that allow for the entrance of metal ions into the cell. There
are two general uptake systems — one is quick and unspe-
cific, driven by a chemiosmotic gradient across the cell
membrane and thus requiring no ATP, and the other is slower
and more substrate-specific, driven by energy from ATP
hydrolysis. While the first mechanism is more energy effi-
cient, it results in an influx of a wider variety of heavy met-
als, and when these metals are present in high concentra-
tions, they are more likely to have toxic effects once inside
the cell (Nies and Silver, 1995).
To survive under metal-stressed conditions, bacteria have
evolved several types of mechanisms to tolerate the uptake
of heavy metal ions. These mechanisms include the efflux
of metal ions outside the cell, accumulation and complex-
ation of the metal ions inside the cell, and reduction of the
heavy metal ions to a less toxic state (Nies, 1999). Mergeay
et al. (1985) tested the minimal inhibitory concentrations
(MICs) of several different metal ions for
Escherichia coli
on agar medium, and the most toxic metal (with the lowest
MIC) was mercury, whereas the least toxic metal tested was
manganese (Table 1). Three examples of metal ions to
which bacteria have evolved well-studied resistance mecha-
Implications of Microbial Heavy Metal
Tolerance in the Environment
Reviews in Undergraduate Research, Vol. 2, 1-6 , 2003
Anne Spain1, Communicated by: Dr. Elizabeth Alm2
Although some heavy metals are essential trace elements, most can be, at high concentrations, toxic to all
branches of life, including microbes, by forming complex compounds within the cell. Because heavy metals are
increasingly found in microbial habitats due to natural and industrial processes, microbes have evolved several
mechanisms to tolerate the presence of heavy metals (by either efflux, complexation, or reduction of metal ions) or
to use them as terminal electron acceptors in anaerobic respiration. Thus far, tolerance mechanisms for metals
such as copper, zinc, arsenic, chromium, cadmium, and nickel have been identified and described in detail. Most
mechanisms studied involve the efflux of metal ions outside the cell, and genes for this general type of mechanism
have been found on both chromosomes and plasmids. Because the intake and subsequent efflux of heavy metal
ions by microbes usually includes a redox reaction involving the metal (that some bacteria can even use for energy
and growth), bacteria that are resistant to and grow on metals also play an important role in the biogeochemical
cycling of those metal ions. This is an important implication of microbial heavy metal tolerance because the
oxidation state of a heavy metal relates to the solubility and toxicity of the metal itself. When looking at the
microbial communities of metal-contaminated environments, it has been found that among the bacteria present,
there is more potential for unique forms of respiration. Also, since the oxidation state of a metal ion may determine
its solubility, many scientists have been trying to use microbes that are able to oxidize or reduce heavy metals in
order to remediate metal-contaminated sites.
Another implication of heavy metal tolerance in the environment is that it may contribute to the mainte-
nance of antibiotic resistance genes by increasing the selective pressure of the environment. Many have specu-
lated and have even shown that a correlation exists between metal tolerance and antibiotic resistance in bacteria
because of the likelihood that resistance genes to both (antibiotics and heavy metals) may be located closely to-
gether on the same plasmid in bacteria and are thus more likely to be transferred together in the environment.
Because of the prevalence of antibiotic resistant pathogenic bacteria, infectious diseases are becoming more diffi-
cult and more expensive to treat; thus we need to not only be more careful of the drastic overuse of antibiotics in
our society, but also more aware of other antimicrobials, such as heavy metals, that we put into the environment.
1University of Oklahoma; To whom correspondence
should be addressed: aspain@ou.edu
2Central Michigan University, Dept. of Biology
nisms – copper, zinc, and arsenic – are illustrated in this
review.
Copper
Copper is used by cells in small quantities in cellular en-
zymes (e.g., cytochrome c oxidase). However, because
copper is so widely used in mining, industry, and agricul-
ture, high levels of copper may exist in some environments.
As such, bacteria have evolved several types of mechanisms
to resist toxicity due to high copper concentrations. With
respect to the prevalence of copper resistance in the envi-
ronment, Lin and Olson (1995) studied bacteria isolated from
a water distribution system experiencing copper corrosion,
and 62% were found to be copper resistant. Of these resis-
tant bacteria, 49% had
cop or
cop-like gene systems, in-
cluding both compartmentalization and efflux systems
(Cooksey, 1993).
In the plant pathogen
Pseudomonas syringae, resistance
to copper via accumulation and compartmentalization in the
cell’s periplasm and outer membrane is due to four proteins
encoded on the plasmid-borne
cop operon (Cooksey, 1994).
The proteins are found in the periplasm (CopA and CopC),
the outer membrane (CopB), and the inner membrane
(CopD) and work together to compartmentalize copper away
from sensitive cellular functions. In
E. coli, resistance to
copper is based on an efflux mechanism by which copper is
removed from the cell. The efflux proteins are expressed
by plasmid-bound
pco genes, which are in turn are depen-
dent on the expression of chromosomal
cut genes (Cooksey,
1993). Two
cut genes (
cutC and
cutF) were identified by
Gupta et al. (1995) and were shown to encode a copper-
binding protein and an outer membrane lipoprotein.
Cooksey (1993) also states that most bacterial species in
the environment have acquired at least one of the afore-
mentioned copper management systems, and that the evo-
lution of copper resistance may have come about through
the modification of copper uptake genes found on chromo-
somes.
Zinc
Zinc is another essential trace element. It is not biologi-
cally redox reactive and is thus not used in respiration. It
is, however, important in forming complexes (such as zinc
fingers in DNA) and as a component in cellular enzymes
(Nies, 1999). Bacterial cells accumulate zinc by a fast, un-
specific uptake mechanism and it is normally found in higher
concentrations (but is less toxic) than other heavy metals
(Nies, 1999). Uptake of zinc ions is generally coupled to
that of magnesium, and the two ions may be transported by
similar mechanisms in bacteria (Nies and Silver, 1995).
Two general efflux mechanisms are responsible for bac-
terial resistance to zinc. One is a P-type ATPase efflux1
system that transports zinc ions across the cytoplasmic mem-
brane by energy from ATP hydrolysis. A chromosomal gene,
zntA, was isolated from
E. coli K-12 and was found to be
responsible for the ATPase that transports zinc and other
cations across cell membranes (Beard et al., 1997). The
other mechanism involved in zinc efflux is an RND-driven2
transporter system that transports zinc across the cell wall
(not just the membrane) of gram-negative bacteria and is
powered by a proton gradient and not ATP (Nies, 1999).
Reviews in Undergraduate Research
2
Table 1. Minimal inhibitory concentrations (MICs) of
several heavy metals in Escherichia coli. The MICs were
determined on an agar medium at different acidities,
allowing for the dissolution of the metal ions. Minimal
inhibitory concentrations refer to the smallest concentration
necessary to inhibit growth; thus, lower MIC values indicate
more toxic metals and higher MICs indicate less toxicity.
1 A P-type ATPase is defined as an ATPase that forms a phos-
phorylated intermediate while catalyzing a reaction (Nies
and Silver, 1995).
2 RND refers to a family of proteins that are involved in the
transport of heavy metals (Nies, 1999).
Heavy Metal
MIC (mM)
Mercury
0.01
Silver
0.02
Gold
0.02
Chromium (Cr(VI))
0.2
Palladium
0.2
Platinum
0.5
Cadmium
0.5
Cobalt
1
Nickel
1
Copper
1
Zinc
1
Thallium
2
Uranium
2
Lanthanum
2
Yttrium
2
Scandium
2
Ruthenium
2
Aluminum
2
Lead
5
Iridium
5
Osmium
5
Antimony
5
Indium
5
Rhodium
5
Gallium
5
Chromium (Cr(II))
5
Vanadium
5
Titanium
5
Beryllium
5
Chromium (Cr(III))
10
Manganese
20
Arsenic
Arsenic, which is not considered a heavy metal but rather
a semi-metal with metallic and non-metallic properties, is
toxic to bacteria, as well as other domains of life. For ar-
senic to have toxic effects, though, it first needs to be in a
bioavailable form. Arsenic uptake by bacteria is mediated
by phosphate transporters and is generally pumped back
out of the cell by an efflux pump (Nies and Silver, 1995).
Several mechanisms for resistance to arsenic have been
identified. Chen et al. (1986) proposed a model for the plas-
mid-mediated mechanism of the efflux of arsenate and ars-
enite in gram-negative bacteria. The nucleotide sequence
of a fragment of DNA containing the
ars operon3 was stud-
ied, and three genes,
arsA,
arsB, and
arsC, were found to
encode for the proteins ArsA, ArsB, and ArsC, respectively.
ArsA is a protein with ATPase activity and thus is involved
in translocation of the metal ions across the cell membrane.
ArsB interacts with ArsA on the inner membrane of the cell,
and the two proteins form the arsenite pump. ArsC is a
smaller protein that alters the specificity of the arsenite pump
to allow for the efflux of arsenate. Thus, ArsC is only re-
quired for tolerance to arsenate, and ArsA and ArsB are re-
quired for tolerance to both species of arsenic.
Gladysheva et al. (1994) isolated and studied the protein
ArsC encoded by the
arsC gene on plasmid R773 and found
that the protein actually catalyzes the reduction of arsenate
to arsenite in
E. coli, using NADPH as the reducing power;
this suggests that the arsenite pump is not altered by the
ArsC protein, but that it is rather the substrate (arsenate) is
altered (or reduced) to fit the arsenite pump. Ji et al. (1994)
isolated and purified another arsenate reductase protein; this
one, however, was encoded by a gene on plasmid p1258 of
Staphylococcus aureus, a gram-positive bacterium. This
arsenate reductase (ArsR) was found to be active in the pres-
ence of thioredoxin and NADPH.
A chromosomal operon homologous to the
ars operon
found on plasmid R773 was identified in
E. coli (Dioro et
al., 1995), and was also responsible for encoding resistance
to arsenic. Dioro suggests that this chromosomal operon
might have been a precursor to plasmid-mediated arsenic
resistance mechanisms involving the reduction of arsenate.
Arsenic Biogeochemistry – An Example of Microbial
Interactions with Metals in the Environment
Because arsenic is also toxic to humans and is a known
carcinogen, the United States Environmental Protection
Agency (US EPA) has established a maximum contaminant
concentration level of 50 µg/L of arsenic in drinking water,
which is proposed to go down to 10 µg/L within several
years (http://www.epa.gov/ogwdw000/ars/ars9.html, visited
1/08/01). Despite these mandates, however, arsenic con-
tamination remains a worldwide threat. Arsenic concentra-
tions are higher in groundwater than in surface water where
the presence of arsenic is mainly due to dissolved minerals
from weathered rocks and soils. The United States Geo-
logical Survey (USGS) found that 10% or more of ground-
water in several counties in the Midwest and Northeast U.S.
exceeded arsenic concentrations of 50 µg/L (http://
co.water.usgs.gov/tace/arsenic, visited 1/08/01). Addition-
ally, in groundwater from the area surrounding and includ-
ing Hanoi, Vietnam, arsenic concentrations have been found
to range from 1-3050 µg/L with an average concentration
of 159 µg/L. In highly affected areas, arsenic concentra-
tions averaged over 400 µg/L. Water analyzed after treat-
ment processes had concentrations ranging from 25-91µg/
L, but with 50% of wells tested still being over the 50 µg/L
arsenic concentration standard (Berg et al., 2001). High
arsenic concentrations pose a significant chronic health
threat to millions drinking contaminated water, and in some
groundwater, concentrations of arsenic are indeed high
enough to allow for arsenic resistance mechanisms in mi-
crobes to remain ecologically favorable.
Many studies have been done on microbial metabolism
of arsenic in aquatic environments and the effects microbes
have on the speciation and mobilization of arsenic. Since
aquatic sediments can be anaerobic, and because arsenic
concentrations in sediments can range from 100-300 µg/L,
microbe-mediated arsenic reduction may be common.
Brannon and Patrick (1987) found that the addition of ar-
senate to an anaerobic sediment resulted in the accumula-
tion of arsenite, indicating the reduction of arsenate to ars-
enite by microbes. Ahmann et al. (1997) further showed
that native microorganisms from the Aberjona watershed
were, in fact, responsible for the arsenic flux in the anoxic
contaminated sediments. Harrington et al. (1998) also dem-
onstrated the ability of microbes in sediments from Coeur
d’Alene Lake to reduce arsenate. In reducing conditions, it
was found that arsenite was the dominant form of arsenic.
They also found that dissimilatory iron-reducing bacteria
(DIRB) and sulfate-reducing bacteria (SRB) are capable of
both arsenic reduction and oxidation and thus may contrib-
ute to the cycling of arsenic in sediments.
Microbial reduction of arsenate in aquatic sediments is
important because arsenite (the reduced form) is more toxic
and more soluble (and thus, more mobile) than arsenate,
which forms relatively insoluble, non-bioavailable com-
pounds with ferrous oxides and manganese oxides. Spe-
ciation of arsenic is affected or controlled by not only oxi-
dation and reduction processes by microbes, but also by
methylation by microbes, and adsorption to other particles
(Aurilio et al., 1994). It was found that DIRB responsible
for the dissolution of iron oxides bound to arsenic can also
free soluble arsenic into the sediment (Cummings et al.,
1999). Another study done on arsenic biogeochemistry in
Lake Biwa Japan showed that arsenic concentration and
speciation may also depend on eutrophication4 (Sohrin et
al, 1997).
Reviews in Undergraduate Research
3
3
ars operon refers to the operon found on plasmid R773 in
gram-negative bacteria that encodes for the efflux of arsen-
ate and arsenite
Many scientists have sought microbial community mem-
bers responsible for arsenate reduction. Hoeft et al. (2002)
found that, in the anoxic water of Mono Lake (California),
two subgroups (Sulfurospirillium and Desulfovibrio) of the
Proteobacteria lineage were present and most likely using
arsenate as an electron acceptor for growth. They also found
an interesting cycling of arsenic occurring; the presence of
nitrate rapidly re-oxidized any arsenate that had been pro-
duced. Thus, in some environments, both oxidation and
reduction of arsenic may occur. In another study of aerobic
contaminated mine tailings, it was found that members of
the
Caulobacter,
Sphingomonas, and
Rhizobium families
may be responsible for the reduction and mobilization of
arsenic (Macur et al., 2001).
While it has been shown that microbes are capable of
arsenic reduction, the question remains whether microbes
reduce arsenate for detoxification purposes (as described in
the section on arsenic tolerance mechanisms) or for growth
during anaerobic respiration. In a sample of agricultural
soil, it was determined that the reduction of arsenate was
not involved in respiration because rates of arsenate reduc-
tion did not contribute to microbial growth (Jones et al.,
2000). Thus, arsenate reduction in this case is probably
due to intracellular detoxification by mechanisms, similar
to those described in
E. coli and
S. aureus. Conversely,
Laverman et al. (1995) showed that the bacterial strain SES-
3 could grow using a diversity of electron acceptors, in-
cluding Fe(III), thiosulfate, and arsenate coupled to the oxi-
dation of lactate to acetate. Another study reported the
growth of strain MIT-13 (isolated from the Aberjona water-
shed) by using arsenate as an electron acceptor, and the in-
hibition of arsenate reduction by molybdate (Ahmann et al.,
1994). Additionally, an organism from the genus
Desulfitobacterium isolated from lake Coeur d’Alene was
shown to reduce arsenate, but it was not determined whether
this reduction supported growth (Niggemeyer et al., 2001).
From arsenic-contaminated mud from Australia, however,
a
Bacillus strain was isolated and characterized as being
able to respire with arsenate (Santini et al., 2002).
Uranium Reduction – An Example of Microbial Metal
Bioremediation
Because of radionuclides present in soils and groundwa-
ter due to nuclear waste during the Cold War Era, much
effort has been put forth to see whether microbes can con-
tribute to remediation by reducing and immobilizing toxic
metals, such as uranium (Francis and Dodge, 1998). It has
been shown that DIRB in the family
Geobacteraceae are
involved in uranium reduction in contaminated aquifer sedi-
ments (Holmes et al., 2002) and also in technetium reduc-
tion (Lloyd et al., 2000). Certain sulfate reducers have also
been shown to be capable of reducing uranium; in
Desulfovibrio vulgaris, it was shown that cytochrome
c3 was
responsible and necessary for uranium reduction activity
(Lovley et al., 1993). Additionally, members of
Clostridium
species have been shown to reduce uranium under anaero-
bic conditions (Francis et al., 1994). This has implications
in the bioremediation of uranium contaminated aquifers and
sediments, as soluble and mobile uranium poses much more
of a threat to public health and the environment than an
insoluble precipitate.
Correlation of Metal Tolerance and Antibiotic Resistance
Bacterial resistance to antibiotics and other antimicro-
bial agents is an increasing problem in today’s society. Re-
sistance to antibiotics is acquired by a change in the genetic
makeup of a bacterium, which can occur by either a genetic
mutation or by transfer of antibiotic resistance genes be-
tween bacteria in the environment (American Academy of
Microbiology, 2000).
Because our current antibiotic are becoming less useful
but used more heavily against antibiotic resistant pathogenic
bacteria, infectious diseases are becoming more difficult and
more expensive to treat. The increased use of antibiotics in
health care, as well as in agriculture and animal husbandry,
is in turn contributing to the growing problem of antibiotic-
resistant bacteria. Products such as disinfectants, sterilants,
and heavy metals used in industry and in household prod-
ucts are, along with antibiotics, creating a selective pres-
sure in the environment that leads to the mutations in mi-
croorganisms that will allow them better to survive and
multiply (Baquero et al., 1998).
According to Jeffrey J. Lawrence’s (2000) discussion of
the Selfish Operon Theory, clustering of genes on a plas-
mid, if both or all genes clustered are useful to the organ-
ism, is beneficial to the survival of that organism and its
species because those genes are more likely to be trans-
ferred together in the event of conjugation. Thus, in an
environment with multiple stresses, for example antibiotics
and heavy metals, it would be more ecologically favorable,
in terms of survival, for a bacterium to acquire resistance to
both stresses. If the resistance is plasmid mediated, those
bacteria with clustered resistance genes are more likely to
simultaneously pass on those genes to other bacteria, and
those bacteria would then have a better chance at survival.
In such a situation, one may suggest an association with
antibiotic resistance and metal tolerance. For example,
Calomiris et al. (1984) studied bacteria isolated from drink-
ing water and found that a high percent of bacteria that were
tolerant to metals were also antibiotic resistant.
CONCLUSIONS
Although some heavy metals are important and essential
trace elements, at high concentrations, such as those found
in many environments today, most can be toxic to microbes.
Microbes have adapted to tolerate the presence of metals or
can even use them to grow. Thus, a number of interactions
Reviews in Undergraduate Research
4
4 Eutrophication is a condition in which the presence of large
amounts of biomass/organic matter in surface waters due to
high nutrient loads results in low oxygen concentrations,
poor water quality, and fish kills.
between microbes and metals have important environmen-
tal and health implications. Some implications are useful,
such as the use of bacteria to clean up metal-contaminated
sites. Other implications are not as beneficial, as the pres-
ence of metal tolerance mechanisms may contribute to the
increase in antibiotic resistance. Overall, it is most impor-
tant to remember that what we put into the environment can
have many effects, not just on humans, but also on the envi-
ronment and on the microbial community on which all other
life depends.
ABOUT THE AUTHOR
Anne Spain carried out research related to the material in
this article at Central Michigan University in Mt. Pleasant,
Michigan from the fall of 2000 until the spring of 2002.
Her work focused on finding a correlation between heavy
metal tolerance and antibiotic resistance in
Escherichia coli
that had been isolated from central Michigan recreational
waters. She also worked on trying to characterize the mi-
crobial community of arsenic-contaminated aquifers from
several counties in Southeastern Michigan by amplification
of community 16S rDNA and analysis by polymerase chain
reaction (PCR) and denaturing gradient gel electrophore-
sis. Due to complications with PCR, however, and limited
time at the university, the project was not completed. Anne
is currently enrolled as a graduate student at the University
of Oklahoma where she is studying environmental micro-
biology and microbial ecology. She hopes to obtain her
PhD after several years and continue in academia, conduct-
ing research and eventually teaching.
ACKNOWLEDGEMENTS
Research was funded by an Undergraduate Student Research
and Creative Endeavors Grant awarded by Central Michi-
gan University and an Undergraduate Summer Research
Fellowship awarded by the American Society for Microbi-
ology. Help and support were given by Dr. Elizabeth Alm,
Janice Burke, Erica Francis from the Biology Department
at Central Michigan University and by Dr. Sheridan Haack
from the USGS.
FURTHER READING
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51: 730-750.
Nies, D.H., and Silver, S. (1995). Ion efflux systems in-
volved in bacterial metal resistances. Journal of Industrial
Microbiology
14: 186-199.
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