Biochemical Society Transactions (2002) Volume 30, part 4
12 Van Veen, H. W., Abee, T., Kortstee, G. J. J., Konings, W. N.
and Zehnder, A. J. B. (1993) J. Bacteriol. 175, 200–216
13 Dunn, T., Gable, K. and Beeler, T. (1994) J. Biol. Chem.
269, 7273–7278
14 Keasling, J. D. and Hupf, G. A. (1996) Appl. Environ.
Microbiol. 62, 743–746
Mechanisms of mercury bioremediation
A. M. M. Essa1, L. E. Macaskie and N. L. Brown
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Abstract
Mercury is one of the most toxic heavy metals, and
has significant industrial and agricultural uses.
These uses have led to severe localized mercury
pollution. Mercury volatilization after its reduc-
tion to the metallic form by mercury-resistant
bacteria has been reported as a mechanism for
mercury bioremediation [Brunke, Deckwer,
Frischmuth, Horn, Lunsdorf, Rhode, Rohricht,
Timmis and Weppen (1993) FEMS Microbiol.
Rev. 11, 145–152; von Canstein, Timmis,
Deckwer and Wagner-Dobler (1999) Appl. En-
viron. Microbiol. 65, 5279–5284]. The reduction\
volatilization system requires to be studied
further, in order to eliminate the escape of the
metallic mercury into the environment. Recently
we have demonstrated three different mechanisms
for mercury detoxification in one organism, Kleb-
siella pneumoniae M426, which may increase the
capture efficiency of mercury.
Introduction
The discharge of heavy metals into the environ-
ment as a result of agricultural, industrial and
military operations, and the effects of this pol-
lution on ecosystems and human health, have been
of concern for some years [1]. Mercury is one of
the most toxic heavy metals, and has significant
industrial use due to its ability to form amalgams
with other metals (e.g. in gold extraction or dental
amalgams). It has also been used in the chloralkali
process to produce NaOH and chlorine, and in
batteries and other electrical apparatus. Mercurial
compounds have been also used in agriculture
as insecticides, fungicides, herbicides and bac-
tericides [1]. These various uses in industry and
Key words : heavy metal, mercuric salts, mercuric sulphide,
volatilization, waste water.
1
To whom correspondence should be addressed (e-mail
AME846!bham.ac.uk).
# 2002 Biochemical Society
15 Tuovinen, O. H. and Kelly, D. P. (1974) Arch. Microbiol.
95, 153–164
Received 12 March 2002
agriculture have led to severe localized mercury
pollution in the aquatic systems and in soils [2,3].
Mechanisms of mercury resistance
Micro-organisms can survive in the presence of
high concentrations of mercuric salts due to
several different mechanisms, as detailed below.
Enzymic reduction to Hg0 and volatilization
The most common mechanism of mercury re-
sistance in bacteria is the enzymic reduction of
bivalent mercuric ions (Hg#+) to the elemental
form (Hg!) by the cytoplasmic flavoenzyme
mercuric reductase. The mechanism of reductive
mercuric ion resistance and the genes responsible
for encoding this resistance have been reviewed
recently [4,5]. Narrow-spectrum mercury resist-
ance determinants confer resistance only against
Hg#+ salts and a small number of organomercurial
derivatives, whereas broad-spectrum determi-
nants also encode resistance to a wide range of
organomercurials. The mechanism of Hg#+ detox-
ification common to all narrow-spectrum mercury
resistance determinants in Gram-negative bacteria
is the transport of Hg#+ ions into the cytoplasm,
followed by their reduction to elemental mercury,
Hg!. Transport (uptake) is due to the specific
transporter, MerT (and in some cases also to the
auxiliary transporters MerC and\or MerF [6]).
Hg! appears to be eliminated by passive diffusion
from the cell under normal physiological con-
ditions [5]. Subsequent volatilization of the el-
emental mercury removes this material from the
immediate environment of the cell before ap-
preciable re-oxidation to Hg#+ is likely to occur.
Formation of insoluble HgS
Mercuric sulphide (HgS) can be formed by the
direct reaction of Hg#+ with H S produced anaero-
#
bically by Clostridium cochlearium [7]. Aiking et al.
[8] reported that Klebsiella aerogenes NCTC418
produced HgS when grown in continuous aerobic
672
 Biometals 2002 : Third International Biometals Symposium

culture with the addition of 2 µg\ml HgCl . No
#
mercury reduction and volatilization was detected,
and the authors detected mercuric ion sensitivity
under sulphate-limited conditions. Elevated levels
of total cellular sulphide were detected in cells
grown in the presence of mercuric ions, probably
due to the formation of HgS.
Wang et al. [9] metabolically engineered a
novel aerobic sulphate reducing pathway for in-
creased secretion of sulphides. The assimilatory
sulphate reduction pathway was redirected to
overproduce cysteine, and excess cysteine was
converted into sulphide by cysteine desulphy-
drase. The engineered bacterium was used for the
aerobic precipitation of cadmium as cadmium
sulphide on the cell surface.
Removal of mercury from waste
water
Mercury removal processes utilize mainly physical
and chemical approaches that involve either trap-
ping and collecting mercury from contaminated
sites or the chemical precipitation of mercuric
compounds. Such processes are costly and may
leave hazardous by-products. Removal of mercury
by mercury-resistant bacteria in a laboratory test
reactor was first reported in 1984 [10], but no
details were given. The biological removal of
mercury from waste water by a mercury-reducing
biofilm was convincingly demonstrated by Brunke
et al. [11], who used both natural and engineered
mercuric reductase-containing bacteria. Subse-
quently, von Canstein et al. [12] recorded the
enzymic reduction of Hg#+ to water-insoluble Hg!
by mercury-resistant Pseudomonas putida, and
used this system for the removal of mercury from
waste water on an industrial scale. Pure cultures of
seven mercury-resistant strains of Pseudomonas
were immobilized inside a bioreactor. Neutralized
chloroalkali electrolysis waste water, with a mer-
cury concentration of 3–10 mg\l, was fed con-
tinuously into the bioreactor. A mercury retention
efficiency of 97 % was obtained within 10 h of the
inoculation of the bioreactor. Mercury reduction
by mercury-resistant bacteria is a good mechanism
for mercury bioremediation, but the recovery of
the metallic Hg! needs to be addressed, in order to
avoid its escape into the atmosphere.
We recently demonstrated three different
mechanisms of mercury detoxification of waste
water in one organism, Klebsiella pneumoniae
M426 (Figure 1). The first is the enzymic

Figure 1
Three different mechanisms of mercury resistance in Klebsiella pneu-
moniae M426
(1) Mercury volatilization after the reduction of Hg2+ to Hg0 ; (2) mercury precipitation as
HgS due to the production of H2S ; and (3) mercury precipitation as mercury–sulphur com-
pounds due to the production of volatile thiol(s).

673 # 2002 Biochemical Society

Biochemical Society Transactions (2002) Volume 30, part 4
reduction and volatilization of mercury, due to
the presence of the mercury-resistance determi-
nant Tn5073 (A. M. M. Essa, D. J. Julian, S. P.
Kidd, N. L. Brown and J. L. Hobman, un-
published work). The second mechanism is the
aerobic precipitation of ionic Hg#+ as insoluble
HgS, as a result of H S production. The third is
#
the biomineralization of Hg#+ as an insoluble
mercury–sulphur complex other than HgS. We
believe that this is due to the aerobic production of
a volatile thiol compound. This process showed
high efficiency of mercury removal in the presence
of high concentrations of mercury and at different
pH and salinity levels (A. M. M. Essa, L. E.
Macaskie and N. L. Brown, unpublished work),
and therefore may be applicable in an industrial
process with minimal prior treatment of the waste
water.
References
1 Goldwater, L. (1972) Mercury : A History of Quicksilver,
York Press, Baltimore
2 Nriagu, J. O. (1979) Biogeochemistry of Mercury in the
Environment, Elsevier/North-Holland Biomedical Press,
New York
Metal Insertion into Proteins
Metal insertion into NiFe-hydrogenases
M. Blokesch, A. Paschos, E. Theodoratou, A. Bauer, M. Hube, S. Huth and A. Bo$ ck1
Department Biologie I, Mikrobiologie, Universita$ t Mu$ nchen, Maria-Ward-Strasse 1a, D-80638 Mu$ nchen, Germany
Abstract
The synthesis and the insertion of the metallo-
centre of NiFe-hydrogenases is a complex pro-
cess, in which seven maturation enzymes plus
ATP, GTP and carbamoyl phosphate are in-
volved. The review summarizes what is known
about the properties and activities of these aux-
iliary proteins, and postulates a pathway along
which maturation may take place.
Introduction
The synthesis and assembly of enzymes containing
metal centres is a relatively new field in bio-
Key words : auxiliary proteins, carbamoyl phosphate, cyano/
carbonyl ligands, endopeptidase, maturation.
Abbreviation used : CP, carbamoyl phosphate.
1
To whom correspondence should be addressed (e-mail
august.boeck!lrz.uni-muenchen.de).
# 2002 Biochemical Society
3 Bryan, G. W. and Langston, W. J. (1992) Environ. Pollut.
76, 84–131
4 Hobman, J. L., Wilson, J. R. and Brown, N. L. (2000) in
Environmental Metal-Microbe Interactions (Lovely, D. R.,
ed.), pp. 177–197, ASM Press, Washington
5 Hobman, J. L. and Brown, N. L. (1997) in Metal Ions in
Biological Systems : Mercury and its Effects on
Environmental Biology (Sigel, A. and Sigel, H., eds),
pp. 503–568, Marcel Dekker, New York
6 Wilson, J. R., Leang, C., Morby, A. P., Hobman, J. L. and
Brown, N. L. (2000) FEBS Lett. 472, 78–82
7 Pan-Hou, H. S. and Imura, N. (1981) Arch. Microbiol.
129, 49–52
8 Aiking, H., Govers, H. and Riet, J. T. (1985) Appl. Environ.
Microbiol. 50, 1262–1267
9 Wang, C. L., Lum, A. M., Ozuna, S. C., Clark, D. S. and
Keasling, J. D. (2001) Appl. Microbiol. Biotechnol. 56,
425–430
10 Williams, J. W. and Silver, S. (1984) Enzyme Microb.
Technol. 6, 530–537
11 Brunke, M., Deckwer, W. D., Frischmuth, A., Horn, J. M.,
Lunsdorf, H., Rhode, M., Rohricht, M., Timmis, K. N. and
Weppen, P. (1993) FEMS Microbiol. Rev. 11, 145–152
12 von Canstein, H., Timmis, K. N., Deckwer, W. D. and
Wagner-Dobler, I. (1999) Appl. Environ. Microbiol. 65,
5279–5284
Received 8 March 2002
chemistry. It emerged during mutational analyses
that, in addition to mutations in the structural
genes of metalloenzymes, there are additional
genetic lesions that prevent the generation of active
enzymes. These so-called auxiliary proteins may
possess a variety of functions, some of them
hitherto unprecedented. These include specific
cellular uptake of the metal, binding to an in-
tracellular metallochaperone or shuttle system,
donation of the metal to the apoprotein and
release of the metal donor thereafter, keeping the
folding of the apoprotein in a competent form.
Other functions may reside in changing the folding
state to internalize the metal centre after com-
pletion of the incorporation, or involve the
synthesis of organic moieties to which the metal is
attached prior to incorporation into the apo-
protein, as in the case of the molybdopterine
cofactors (for reviews see [1–4]).
674