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 bioremediationA. M. M. Essa1, L. E. Macaskie and N. L. Brown

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.

AbstractMercury 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.

IntroductionThe 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.1To whom correspondence should be addressed (e-mailAME846!bham.ac.uk).

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 resistanceMicro-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 volatilizationThe 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 inGram-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 HgSMercuric 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

# 2002 Biochemical Society 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 wastewaterMercury 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

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 asHgS due to the production of H2S ; and (3) mercury precipitation as mercury–sulphur com-pounds due to the production of volatile thiol(s).

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

# 2002 Biochemical Society673

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.

References1 Goldwater, L. (1972) Mercury : A History of Quicksilver,

York Press, Baltimore2 Nriagu, J. O. (1979) Biogeochemistry of Mercury in the

Environment, Elsevier/North-Holland Biomedical Press,New York

Metal Insertion into ProteinsMetal 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

AbstractThe 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.

IntroductionThe 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.1To whom correspondence should be addressed (e-mailaugust.boeck!lrz.uni-muenchen.de).

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) inEnvironmental 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 inBiological Systems : Mercury and its Effects onEnvironmental 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. andBrown, 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. andKeasling, 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. andWeppen, P. (1993) FEMS Microbiol. Rev. 11, 145–152

12 von Canstein, H., Timmis, K. N., Deckwer, W. D. andWagner-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 functionsmay 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]).

# 2002 Biochemical Society 674