Download Lead resistance in micro-organisms

Microbiology (2014), 160, 12–25
Review
DOI 10.1099/mic.0.070284-0
Lead resistance in micro-organisms
Anna Jarosławiecka and Zofia Piotrowska-Seget
Correspondence
Department of Microbiology, University of Silesia, Jagiellońska Street 28, Katowice 40-032, Poland
Anna Jarosławiecka
[email protected]
Lead (Pb) is an element present in the environment that negatively affects all living organisms. To
diminish its high toxicity, micro-organisms have developed several mechanisms that allow them to
survive exposure to Pb(II). The main mechanisms of lead resistance involve adsorption by
extracellular polysaccharides, cell exclusion, sequestration as insoluble phosphates, and ion efflux
to the cell exterior. This review describes the various lead resistance mechanisms, and the
regulation of their expression by lead binding regulatory proteins. Special attention is given to the
Pbr system from Cupriavidus metallidurans CH34, which involves a unique mechanism
combining efflux and lead precipitation.
Introduction
Heavy metal resistance in bacteria results from their
primary contact with metals that appear naturally in the
environment. However, intensive human activity and
exploitation of natural deposits has led to the expansion
of metal-resistant micro-organisms (Bruins et al., 2000).
Apart from 13 other metals and metalloids, lead (Pb) is
considered to be one of the major pollutants (Sparks,
2005). Industrial activities, such as production of batteries
and pigments and metal smelting, as well as manufacture of
products such as lead arsenate insecticides or lead water
pipes are the main sources of Pb in the environment.
Natural processes including soil erosion, volcanic emission
and mobilization of Pb from minerals contribute only to a
minor degree to Pb pollution of the environment (Gadd,
2010; Gerba, 1996; Siegel, 2002; Spiro & Stigliani, 2002;
Yokel & Delistraty, 2003). The total Pb concentration in
industrial areas can reach up to 10 000 mg kg21, while the
average value in soils ranges from 10 to 100 mg kg21
(Akmal & Jianming, 2009; Schwab et al., 2005).
Additionally, the level of Pb(II) in industrial wastewaters
reaches 200–250 mg l21, whereas, according to accepted
quality standards, it should not exceed 0.05–0.10 mg l21
(Sag et al., 1995).
Pb(II) enters bacterial cells through the uptake pathways
for essential divalent metals such as Mn(II) (Laddaga et al.,
1985; Tynecka et al., 1981) and Zn(II) (Bruins et al., 2000;
Grass et al., 2002; Laddaga & Silver, 1985; Makui et al.,
2000). Pb is not known to be of any biological importance
and is toxic at very low concentrations (Bruins et al., 2000).
The toxicity of Pb depends upon its bioavailability. Since
most Pb(II) is bound to clay minerals and complex organic
molecules, the bioavailable fraction of Pb(II) to which
microbes are exposed may be rather low (Kotuby-Amacher
Abbreviations: CDF, cation-diffusion facilitator; EP, extracellular
polymer; MBD, metal binding domain; MFS, major facilitator superfamily;
MT, metallothionein; PPB, polyphosphate bodies.
12
et al., 1992; Singh et al., 1996). Thus, in laboratory
experiments it is necessary to choose the right medium
components to avoid the precipitation and complexation
of Pb(II), since this leads to an apparent decrease of Pb
toxicity (Roane, 1999; Sani et al., 2001).
Pb(II) toxicity occurs as a result of changes in the
conformation of nucleic acids and proteins, inhibition of
enzyme activity, disruption of membrane functions and
oxidative phosphorylation, as well as alterations of the
osmotic balance (Bruins et al., 2000; Vallee & Ulmer,
1972). Pb(II) also shows a stronger affinity for thiol and
oxygen groups than essential metals such as calcium and
zinc (Bruins et al., 2000). Despite the high toxicity of Pb,
many micro-organisms have evolved mechanisms that
enable them to survive Pb exposure (Fig. 1).
Micro-organisms resistant to Pb(II) have been isolated from
metal contaminated soils, industrial wastes, and from plants
growing on metal contaminated soil. Among these isolates the
following examples have been identified: the Gram-positive
bacteria Bacillus cereus, Arthrobacter sp. and Corynebacterium
sp.; the Gram-negative bacteria Pseudomonas marginalis,
Pseudomonas vesicularis and Enterobacter sp.; and fungi
Saccharomyces cerevisiae and Penicillium sp. Psf-2 (Chen &
Wang, 2007; Hasnain et al., 1993; Sun & Shao, 2007; Roane &
Kellogg, 1996; Trajanovska et al., 1997; Zanardini et al., 1997).
Some lead-resistant bacteria have been found to play a specific
role in the growth of lead-exposed plants. For example, the
endophyte Bacillus sp. MN3-4 increases Pb(II) accumulation
in Alnus firma, and Pseudomonas fluorescens G10 and
Mycobacterium sp. G16 promote plant growth and reduce
Pb toxicity in Brassica napus (Sheng et al., 2008; Shin et al.,
2012). Additionally, the activity of the lead-resistant bacteria
Streptomyces sp. and Ps. vesicularis has been noticed on the
marble facade of a cathedral, where it was manifested in the
form of red stains. These bacteria were capable of producing a
red and red–brown pigment, identified as the lead tetroxide,
minium (red lead, Pb3O4) (Zanardini et al., 1997).
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Lead resistance in micro-organisms
Pb2+
Pb2+
Pb2+
OM
IM
Pb2+
Pb2+
Pb2+
PbrA
Pb2+
Pb2+
Pb2+
Pb2+
ZntA
Pb2+
Pb2+
MT
CadA
Fig. 1. Selected mechanisms of cell protection against lead toxicity (based on Dopson et al., 2003, modified). Pb(II) could be
kept away from the micro-organisms through its precipitation as insoluble phosphates outside the cell (circles), adsorption on
extracellular polysaccharides (violet stars) or by polymers naturally occurring in the cell wall. After entering the cell through the
essential metal transporters, Pb(II) can be further inactivated by binding to the metallothioneins (MT), sequestered as insoluble
phosphates or removed from the cell via transporters such as CadA, ZntA or PbrA. OM, outer membrane; IM, inner membrane.
The purpose of this review is to summarize current
knowledge of the various lead resistance mechanisms in
micro-organisms.
Cell wall and exopolysaccharides
One of the mechanisms that micro-organisms use to avoid
the toxicity of metals of no biological function is to limit
their movement across the cell envelope (Bruins et al.,
2000). Early studies initiated on the interactions between
Pb(II) and the cell envelope in Micrococcus luteus and
Azotobacter sp. revealed that these ions are mostly present
in the cell wall and cell membrane, with a minor portion
present in the cytoplasmic fraction (Tornabene & Edwards,
1972; Tornabene & Peterson, 1975). The cell wall is a
natural barrier for Pb(II), since the functional groups of
several macromolecules are involved in binding this metal.
In Gram-negative bacteria, this role is played mainly by
lipopolysaccharide, a significant component of the outer
membrane. In Gram-positive bacteria, peptidoglycan
together with teichoic and teichuronic acids are responsible for Pb binding (Fig. 1) (Beveridge & Fyfe, 1985). This
protection mechanism is an uncontrolled process, and
specific resistance mechanisms become operative when the
cell envelope reaches its saturation point (Beveridge & Fyfe,
1985).
Çabuk et al. (2006) reported that hydroxyl and carboxyl
groups, as well as nitrogen based bio-ligands including
amide and sulfonamide, were involved in the binding of
Pb(II) by Bacillus sp. ATS- 2. In consequence, this strain was
able to bind 91.7 % of the Pb(II) added to growth medium at
50 mg l21 (Çabuk et al., 2006). In S. cerevisiae, amide and
phosphate groups were recognized to participate in the
http://mic.sgmjournals.org
immobilization of Pb(II) in the cell wall, whereas carbonyl,
phosphate, hydroxyl and amino groups had this role in
Pseudomonas aeruginosa ASU6a (Çabuk et al., 2007; Gabr
et al., 2008). In Synechococcus sp., molecules with amide,
amino, hydroxyl or carboxyl groups participate in the
binding of metal ions, including Pb(II) (Shen et al., 2008).
The strains mentioned above differed in their efficiency
when removing Pb(II) from metal solutions. Lead was
accumulated to 30, 64.5 and 123 mg Pb g21 of microbial
biomass for S. cerevisiae, Ps. aeruginosa and Synechococcus
sp., respectively (Çabuk et al., 2007; Gabr et al., 2008; Shen
et al., 2008). In E. coli, almost 97 % of Pb(II) was bound in
the cell membrane, while almost no Pb(II) was detected in
peptidoglycan (Kumar & Upreti, 2000).
Many micro-organisms synthesize extracellular polymers
(EPs) that bind cations of toxic metals, thus protecting
metal-sensitive and essential cellular components (Fig. 1)
(Bruins et al., 2000). The composition of EPs is very
complex, including proteins, humic acids, polysaccharides
and nucleic acids, which chelate metals with different
specificity and affinity (Pal & Paul, 2008; Guibaud et al.,
2003; Roane, 1999). Pb(II) binding by EPs has been
reported for Bacillus firmus, Pseudomonas sp. (Salehizadeh
& Shojaosadati, 2003), Cyanobacteria (Freire-Nordi et al.,
2005; Paperi et al., 2006), Halomonas sp. (Amoozegar et al.,
2012) and Paenibacillus jamilae (Perez et al., 2008). EP
produced by the latter strain is highly Pb(II) specific, and
adsorbs ten times more Pb(II) than other metals such as
Cd(II), Co(II), Ni(II), Zn(II) and Cu(II) (Aguilera et al.
2008; Morillo et al., 2006). This EP exhibits a characteristic
feature, namely a high content of uronic acids (28.29 %),
which are considered to play an important role in its Pb(II)
binding specificity. Immobilization of Pb(II) in the EP
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13
A. Jarosławiecka and Z. Piotrowska-Seget
from Pa. jamilae was not influenced by the presence of
other metals in solution, and was strictly pH dependent
(optimal absorption was at pH 6), with the maximum
binding capacity being 303 mg (g EP)21 (Perez et al.,
2008). In the case of Ps. marginalis, isolated from metal
contaminated soil, EPs had the capacity to bind Pb(II) up
to 2.5 mM (0.3 mM of soluble lead in minimal medium,
pH 6). EPs were produced by this strain independently of
its exposure to the metals, and the bound Pb(II) was visible
as dark granules outside the cell, when observed through
transmission electron microscopy (Roane, 1999). EP with a
similar capacity to the EPs from Pa. jamilae [316 mg (g
EP)21] and a higher content of uronic acids (38 % of EP)
was found in Alteromonas macleodii subsp. Fijiensis (Loaëc
et al., 1997). Adsorption of Pb(II) to EPs as well as
accumulation of lead inside the cell has been also noted in
the cyanobacterium Gloeothece PC6909. After crossing the
membrane, Pb(II) was found to be bound to intracellular
inclusions, identified as polyphosphate bodies (PPB). The
primary role of PPB is to store energy and phosphorus;
however, they also contribute to the precipitation of metal
ions and to neutralization of their toxic effects. In addition,
accumulation of Pb(II) in the vacuolar structures of this
bacterium was also observed (Pereira et al., 2011).
Extra- and intracellular precipitation of Pb(II)
Pb(II) is known to react with several anions such as
chlorides, phosphates and hydroxyl ions to form insoluble
precipitates. Additionally, Pb(II) forms strong complexes
with organic compounds such as tryptone, cysteine, yeast
extract or succinic acid (Babich & Stotzky, 1979; Mayer &
Godwin, 2006). The precipitation of Pb(II) is used by
several micro-organisms to lower the concentration of free
Pb(II) by sequestering it in the form of phosphate salts
outside and inside the cell (Fig. 1). The chemical
composition of these precipitates varies between strains.
For example, Citrobacter freundii precipitates Pb(II) as an
extracellular phosphate (PbHPO4). Pb(II) precipitation in
this strain is presumably a consequence of the hydrolysis of
organic phosphorus by an acid phosphatase and the
concomitant precipitation of Pb(II) on the cell surface
(Aickin & Dean, 1977, 1979; Aickin et al.,1979).
Levinson et al. (1996) divided the process of intracellular
lead precipitation into three stages: (1) binding of metal to
the cell surface; (2) metabolism-dependent intracellular
uptake, and (3) final precipitation of Pb(II) as crystals of
lead phosphates. For instance, Staphylococcus aureus
precipitates Pb(II) inside the cell as lead phosphate
Pb3(PO4)2. This kind of protection allows this strain to
withstand a 600-fold higher dose of Pb(II) compared to a
sensitive strain (Levinson et al., 1996; Levinson & Mahler,
1998). The marine bacterium Vibrio harveyi is capable of
precipitating Pb(II) inside the cell in the form of an
unusual phosphate compound Pb9(PO4)6. This process is
regulated partially by quorum sensing. The exact mechanisms of this regulation are not known, but it has been
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suggested that quorum sensing controls the availability of
inorganic phosphates (Mire et al., 2004). Precipitation of
Pb(II) in the form of Pb9(PO4)6 was also observed in
Providencia alcalifaciens 2EA as a result of phosphatase
activity that releases inorganic phosphates (Naik et al.,
2012). In Klebsiella aerogenes NCTC418 grown in phosphate limited medium, Pb(II) was precipitated inside the
cell as PbS (Aiking et al., 1985). Karnachuk et al. (2002)
showed that sulfate reducing bacteria used anglesite
(PbSO4) as the electron acceptor and reduced this
compound to poorly soluble galena (PbS). No toxic effect
of Pb(II) on these bacteria during the transformation
process was observed (Karnachuk et al., 2002). The
phosphate solubilizing Enterobacter cloacae (Park et al.,
2011) and Burkholderia cepacia immobilize Pb(II) in the
form of pyromorphite Pb5(PO4)3Cl (Templeton et al.,
2003). Lead precipitation has also been found in other
strains, such as Shewanella putrefaciens CN32, Ps. fluorescens and the endophyte Bacillus sp. MN 3-4; however, the
chemical compositions of these precipitates have not
been determined (al-Aoukaty et al., 1991; Shin et al.,
2012; Smeaton et al., 2009). Intracellular sequestration of
Pb(II) by micro-organisms was also demonstrated by in
situ experiments. Precipitates containing lead were present
in different soil bacterial strains and identified as PPB
(Perdrial et al., 2008). In the cyanobacterium Synechocystis
PCC 6803 exposed to Pb(II), an increase of PPB in
number and size was reported (Arunakumara & Xuecheng,
2009).
Binding of Pb(II) by specific proteins
Metallothioneins (MTs) are frequently referred to as
proteins involved in protecting cells against toxic metals;
however, their primary role is in zinc homeostasis (Fig. 1)
(Blindauer, 2011). MTs, first discovered in Synechococcus
PCC 7942, are encoded by the smt locus, which consists of
two divergently transcribed genes smtA and smtB. smtA
encodes a class II MT, and the product of smtB represses the
transcription of smtA. SmtA is involved in zinc homeostasis,
as the deletion of smtA in Synechococcus PCC 7942 led to its
hypersensitivity to Zn(II) (Blindauer, 2011). In addition to
Zn(II), Pb(II) is one of the few metalions that is capable of
switching on the expression of smtA. Apart from
Synechococcus PCC 7942, the induction of smt genes and
biosynthesis of MT in the presence of Pb(II) has been
detected in B. cereus, Streptomyces sp., Salmonella choleraesuis 4A and Proteus penneri GM-10 (Huckle et al., 1993;
Murthy et al., 2011; Naik et al., 2012; Rifaat et al., 2009).
Another gene (bmtA) encoding a Pb(II) binding metallothionein has been identified in Ps. aeruginosa WI-1 (Naik
et al., 2012). Additionally, it has been suggested that a
metallothionein-like protein binds Pb(II) in Bacillus megaterium (Roane, 1999).
Extracellular enzymes, such as superoxide dismutase from
Streptomyces subrutilis, can also be engaged in Pb(II)
binding (So et al., 2001).
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Microbiology 160
Lead resistance in micro-organisms
Biotransformation of lead compounds
A number of micro-organisms inhabiting soil and water can
transform inorganic and organic lead compounds into
volatile forms, which diminishes their toxic effect (Thayer,
2002). This effect has been observed in lake sediments
(Silverberg et al., 1977; Wong et al., 1975). Nevertheless, the
contribution of microbes to this process requires further
investigation, since methyl lead compounds are also present
in the environment as a result of pollution (Gadd, 2010;
Thayer, 2002). The biomethylation of Pb(II) has been
reported to be catalysed by Pseudomonas sp. (Walton et al.,
1988; Wong et al., 1975), Alcaligenes sp. (Wong et al., 1975),
Acinetobacter sp. (Wong et al., 1975), Flavobacterium sp.
(Wong et al., 1975), Aeromonas sp. (Wong et al., 1975) and
Bacillus sp. (Walton et al., 1988). In general, two forms of
volatile methylated lead have been identified as products of
microbial activity: tetramethyl (Me4Pb) and trimethyl
(Me3Pb) lead. For example, Aeromonas sp., isolated from
Lake Ontario, transformed lead acetate into tetramethyl lead
(Me4Pb). This reaction was observed both in the lake water as
well as in laboratory cultures (Silverberg et al., 1976). Arctic
marine bacteria cultured under polar conditions were capable
of converting Pb(II) into trimethyl lead (Me3Pb) (Pongratz &
Heumann, 1999) while Pseudomonas sp., Acinetobacter sp.,
Flavobacterium sp. and Aeromonas sp. transformed lead
nitrate or trimethyl lead acetate into tetramethyl lead (Me4Pb)
(Hughes & Poole, 1989; Reisinger et al., 1981; Thayer &
Brinckman, 1982; Wong et al., 1975).
Binding of Pb(II) by siderophores
Siderophores are small, high-affinity iron chelating compounds secreted by various micro-organisms. Apart from
iron, siderophores may interact with other metals outside
of the cell (Saha et al., 2012; Schalk et al., 2011). They have
been implicated in the complexation of Pb(II) by Ps.
aeruginosa 4EA and by endophytic Pseudomonas putida
KNP9, isolated from mung bean (Phaseolus vulgaris) (Naik
& Dubey, 2011; Tripathi et al., 2005). Siderophores
produced by these two strains have been identified as
yellow–green, fluorescent pyoverdines, which are characteristic for Pseudomonas. The pyoverdines produced by
the plant growth promoting strain Ps. putida KNP9
reduced the concentration of Pb(II) in mung bean roots
by 93 % and in shoots by 56 % (Tripathi et al., 2005).
Studies by Naik & Dubey (2011) revealed that production
of siderophores by Ps. aeruginosa 4EA may double in
response to Pb(II) exposure. In Ps. aeruginosa PAO1,
pyochelin also contributed to Pb(II) binding, apparently
with a higher affinity than pyoverdine (Braud et al., 2010).
Efflux systems
Ions that escape extra- and intracellular binding can be
excluded from the cell through the activity of efflux
systems, the most effective mechanisms providing resistance to toxic metals (Bruins et al., 2000). In this
mechanism, proteins from the superfamily of P-type
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ATPases transport metal cations across the cytoplasmic
membrane using ATP as the energy source (Apell, 2004).
Efflux of Pb(II) is mediated by P-type ATPases from the
PIB family, which are also involved in the transport of
Zn(II) and Cd(II). However, other PIB ATPases also exist
that are specific for other metals such as Cu(I) (Argüello,
2003). PIB pumps have been widely recognized in most
bacterial and archaeal species (Coombs & Barkay, 2005).
Among them, CadA from S. aureus, ZntA from E. coli,
CadA2 from Ps. putida KT2440 and PbrA from Cupriavidus
metallidurans CH34 are known to transport Pb(II), as well
as Cd(II) and Zn(II), to the periplasm (Fig. 1) (Argüello,
2003; Hynninen et al., 2009; Leedjärv et al., 2008, Rensing
et al., 1998). Other transmembrane metal transporters
belong to the CBA family of transporters, and the cationdiffusion facilitator (CDF) family. The former are present
in Gram-negative bacteria, and are chemiosmotic antiporters transferring metals from the cytoplasm to the
periplasm. CDF transporters are chemiosmotic ion–proton
antiporters and also transport ions from the cytoplasm to
the periplasm (Nies, 2003). So far, only CzcCBA1 from Ps.
putida KT2440, a CBA transporter, has been recognized to
participate in the export of Pb(II) from the cell, while none
of the transporters from the CDF group has yet been
shown to have this activity (Hynninen, 2010).
Metalloregulatory proteins and Pb(II) sensing
MerR family regulators. Bacteria can control the intra-
cellular concentration of Cd(II), Zn(II), Co(II), Cu(I), Ag(I),
Au(I), Hg(II) and Pb(II) through metalloregulatory proteins
belonging to the MerR family (Brown et al., 2003). These
proteins regulate the expression of genes encoding defence
systems active against toxic metals or elevated levels of
microelements. Differences within the C-terminal ligand
binding domains of MerR family regulators allow them to
recognize a specific metal (Chen & He, 2008). One of the
MerR-like proteins is PbrR from C. metallidurans CH34
(CmPbrR). This protein is encoded by pbrR, located on
pMOL30, and it regulates expression of the pbr lead
resistance genes and, consequently, synthesis of the lead
efflux pump (Borremans et al., 2001; Taghavi et al., 2009). It
has been shown that CmPbrR is highly specific towards
Pb(II), with an almost 1000-fold specificity over other metals
such as Hg(II), Cd(II), Zn(II), Co(II), Ni(II), Co(II) and
Ag(I) (Chen et al., 2005). CmPbrR and its two homologues
from the same micro-organism (chromosomally encoded
CmPbrR2, previously PbrR691, and CmPbrR3) are the only
known lead specific regulatory proteins so far identified
(Chen & He, 2008; Taghavi et al., 2009). The molecular
mechanism of selectivity and recognition of Pb(II) by
CmPbrR has been studied for CmPbrR2. CmPbrR2 has
three highly conserved cysteine residues: Cys78, Cys113 and
Cys122 (Fig. 2a), which have been proved to be involved in
Pb(II) binding; however, the existence of a fourth ligand
cannot be excluded (Chen & He, 2008). The coordination of
Pb(II) is specific, as it possesses a stereochemical lone pair of
electrons on one side, not engaged in binding to ligands. As a
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15
A. Jarosławiecka and Z. Piotrowska-Seget
(a)
DNA binding domain
α1
β1
CmMerR
CmPbrR
CmPbrR2
CmMerR
CmPbrR
CmPbrR2
α2
β2
β3
α3
14
MENNLENLTIGVFAKAAGVNVETIRFYQRKGLLLEPDKPYGSIRRYGEADVTRVRFVKSA
-----MNIQIGELAKRTACPVVTIRFYEQEGLLPPPGRSRGNFRLYGEEHVERLQFIRHC
------MMRIGELGKKADCLVQTVRFYESEGLLPEPARSEGNFRLYDEVHLQRLLFIRRC
: ** : * :
* *: ** *: : *** * : * . : * * * . : * : *: : .
α4
α5
78, 79
113
QRLGFSLDEIAELLRLEDG--THCEEASSLAEHKLKDVREKMADLARMEAVLSELVCACH
RSLDMPLSDVRTLLSYRKRPDQDCGEVNMLLDEHIRQVESRIGALLELKHHLVELREACS
RAKDMTLDEIRQLLNLRDRPELGCGEVNALVDAHIAQVRTKMKELRALERELMDLRRSCD
* * .. * : :: :*. :: * :: * :* :*
** ..
:
: * ::
2-turn α-helix
122
132
CmMerR ARRGNVS------------CPLIASL---------------CmPbrR GARPAQS------------CGILQGLSDCVCDTRGTTAHPSD
CmPbrR2 SARTSRQGGASLAGSAMPECGILNSLAEPA-----------* :: .*
. *
.
(b)
CadC
CmtRMtb
CmtRSc
SmtB
AztR
α1
N
7
11
---MK-----------KKDTCEIFCYDEEKVNRI-QGDLQTVDISGVSQILKAIADENRA
------------------------------------MLTCEMRESALARLGRALADPTRC
------------------------------------MLTLAADIDVLARFGRALADPIRC
-------MTKPVLQDGETVVCQGTHA----AIASELQAIAPEVAQSLAEFFAVLADPNRL
MNKHKKKQDLDLIQSSDTPTCDTHLVHLDNVRSSQAQILPTDKAQQMAEIFGVLADTNRI
. :: .:
. : ** *
DNA binding domain
α3
CadC
CmtRMtb
CmtRSc
SmtB
AztR
α2
αR
β1
β2
58 60
57 61
KITYALCQDEELCVCDIANILG-VTIANASHHLRTLYKQGVVNFRKEGKLALYSLGDEHI
RILVALL-DGVCYPGQLAAHLG-LTRSNVSNHLSCLRGCGLVVATYEGRQVRYALADSHL
RILLALR-QAPAYPADLADSLG-ISRTRLSNHLACLRDCGLVVTVPDGRRTRYELADERL
RLLSLLA-RSELCVGDLAQAIG-VSESAVSHQLRSLRNLRLVSYRKQGRHVYYQLQDHHI
RLLSALA-SSELCVCDLAALTKMNSESAVCHQLRLLKAMRLVSYRREGRNVYYSLADSHV
::
*
::*
: :
. .:* *
:*
:*: . * * * ::
α5
CadC
CmtRMtb
CmtRSc
SmtB
AztR
102
RQIMMIALAHKKEVKVNV-----------------ARALGELVQVVLAVDTDQPCVAERAASGEAVEMTGS
GHALDDLRAAVVAVEADRTCADADEKGCC------VALYQNALD------HLQECR--------------INLYRSLVE------NNTYATGTG-----------α6
C
Fig. 2. Amino acid sequence alignment of metalloregulators involved in Pb(II) sensing (generated using CLUSTAL Omega 1.2.0).
(a) Metalloregulators from the MerR family: CmMerR (Tn501), CmPbrR2 and CmPbrR from C. metallidurans CH34. (b)
Metalloregulators from SmtB/ArsR family: SmtB from Synechococcus sp. strain PCC 7942, CadC from S. aureus pI158, CmtR
from M. tuberculosis, CmtR from St. coelicolor, AztR from Anabaena PCC 7120. The cysteine residues involved in the Pb(II)
sensing in metalloregulators and corresponding residues are marked in yellow. Amino- and carboxyl-terminal domain, secondary
structures and DNA binding domain are marked in the figures. Description in the text (Cavet et al., 2003; Hobman et al., 2012;
Pennella & Giedroc, 2005).
consequence, Pb(II) forms a hemidirected geometry with all
the ligands on one side of the metal and with the pair of
electrons on the other. CmPbrR2 most likely uses
hemidirected geometry to recognize Pb(II) over other ions
16
(Chen et al., 2007; Chen & He, 2008). This unique feature has
been utilized in the construction of lead specific indicators
for monitoring the intracellular presence of Pb(II) in human
cells, and biosensors sensing Pb(II) contamination in the
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Microbiology 160
Lead resistance in micro-organisms
environment (Chakraborty et al., 2008; Chen et al., 2005;
Chiu & Yang, 2012; Corbisier et al., 1999).
Acidithiobacillus ferrooxidans ATCC 23270 (Zheng et al.,
2012).
In recent studies, residues Cys14, Cys79 and Cys134 have
been identified in CmPbrR to be involved in the sensing of
Pb(II), and in promoter (PpbraA) activation (Fig. 2a)
(Hobman et al., 2012). Cys14 is situated in the helix–turn–
helix DNA binding domain of CmPbrR and may play an
essential role in the PbrR–DNA interaction. In addition,
Cys79, which is present in all the divalent metal ion
responsive MerR regulators examined so far, is necessary
for the detection of +2 charged ions over +1 ones. In
contrast, Cys134 is unique and is not found in other
characterized MerR regulators (Hobman et al., 2012).
Upon lead binding, the PbrR transcriptional regulator
induces the expression of genes encoding Pb(II) specific
transporters to pump out metal ions out of the cell (Chen
et al., 2007).
AztA from the cyanobacterium Anabaena PCC 7120 is
a Zn(II)/Pb(II)/Cd(II) translocating PIB-type ATPase,
expression of which is regulated by an SmtB/ArsR family
repressor, AztR (Liu et al., 2005). AztR, which has high
sequence similarity to SmtB, differs slightly in its structure
since it lacks the a5 zinc specific binding site, which is
essential for the allosteric regulation of DNA binding by
SmtB and ZiaR (VanZile et al., 2002; Turner et al., 1996;
Thelwell et al., 1998). AztR possesses only one metal
binding site, a3N, for Zn(II), Cd(II) and Pb(II), which is
similar to the S3(N9O) metal site from CadC in S. aureus
(Fig. 2) (Liu et al., 2005).
Another transcriptional regulator from the MerR family is
ZntR, which regulates the expression of the zntA gene
encoding ZntA. This PIB ATPase transports Pb(II) as well
as Zn(II) and Cd(II) ions out of the cell in E. coli (Binet &
Poole, 2000; Brocklehurst et al., 1999). Binet & Poole
(2000) demonstrated that all these metal ions can be
inducers for zntAR, and the Cd(II)- and Pb(II)-dependent
regulation of zntA is also mediated by ZntR. The
interactions between Zn(II) and ZntR are well known,
but ZntR interactions with Pb(II) and Cd(II) remain
poorly studied (Binet & Poole, 2000).
SmtB/ArsR family regulators. CadC from S. aureus is a
regulatory protein from the SmtB/ArsR family, which
regulates the expression of cadAC, encoding a membrane
bound Cd(II)/Zn(II)/Pb(II) specific PIB-type ATPase,
CadA (Busenlehner et al., 2003; Yoon et al., 1991). CadC
responds to the metal ions in the following order: Pb(II) .
Cd(II) . Zn(II) (Rensing et al., 1998). In the CadC
structure, an a3N metal binding site containing three
cysteine residues, Cys7, Cys58 and Cys60, ensures selection
of the correct ion (Fig. 2b) (Sun et al., 2001). CadC binds
Pb(II) through a trigonal coordination complex, with
cysteine thiolate ligands derived from the N-terminal
domain (Cys7/11) and a pair of cysteines in the a4 helix
(Cys58/60) (Fig. 2) (Busenlehner & Giedroc, 2006).
Another protein that belongs to the SmtB/ArsR family is
CmtR from Mycobacterium tuberculosis (CmtRMtb). CmtR
has also been found to sense Pb(II) in Mycobacterium
smegmatis (Cavet et al., 2003). In contrast to other SmtB/
ArsR Cd(II)/Pb(II) sensing proteins, CmtRMtb possesses a
unique pair of a4C metal binding sites, formed from the a4
helix and a C-terminal tail (Banci et al., 2007). Pb(II) is
predicted to be bound to Cys57 and Cys61 in the helix a4,
and to Cys102 in the C-terminal region (Fig. 2). A CmtR
sensor with an a4N site sensing Pb(II) and Cd(II) has been
found in Streptomyces coelicolor (Wang et al., 2010).
Recently, another CmtR transcriptional regulator responsive to Cd(II) and Pb(II) has also been reported in
http://mic.sgmjournals.org
Other regulators. ZraSR (formerly HydH/G) is a two-
component regulatory system, which regulates the
expression of zraP, encoding ZraP, a periplasmic Zn(II)
binding protein in E. coli involved in zinc homeostasis
(Noll et al., 1998). ZraS activates ZraR (a cytoplasmic
response regulator) by phosphorylation. Although ZraP
was not reported to bind Pb(II), its regulators ZraSR are
induced by high concentrations of Zn(II) and Pb(II) (Lee
et al., 2005; Leonhartsberger et al., 2001).
The C. metallidurans CH34 Pbr efflux system
A Pbr efflux system which removes Pb(II) from the cell was
first reported by Borremans et al. (2001) in C. metallidurans
CH34 (formerly known as Alcaligenes eutrophus, Ralstonia
metallidurans and Wautersia metallidurans). This bacterium
is resistant to 20 heavy metals and is used as a model microorganism in studies of metal resistance and metal tolerance
(Borremans et al., 2001; Janssen et al., 2010). The Pbr efflux
is encoded by pbr, located within a mer-pbr-czc island on
pMOL30 (Monchy et al. 2007). Seven genes of the pbr locus
(pbrUTRABCD) are organized into two divergently oriented
transcription units, pbrUTR and pbrABCD (Hynninen et al.,
2009). The two pbr promoters are both regulated by the
transcriptional activator PbrR (Fig. 3) (Permina et al., 2006;
Hynninen et al., 2009). So far, pbr has only been found on
plasmids in several lead-resistant bacteria, frequently with
pbrU*, pbrT, pbrR
p/o
pbrA, pbrB, pbrC, pbrD
PbrR
Pb2+
Fig. 3. Regulation of pbr by PbrR. In the presence of Pb(II), PbrR binds
to the pbr promoter (p/o) and initiates its transcription in two opposite
directions. pbrU is inactivated by the insertion with transposone
TnCme2 (marked with asterisk). (Based on Hynninen, 2010).
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17
A. Jarosławiecka and Z. Piotrowska-Seget
(a) C. metallidurans CH34
pbrT
pbrR
pbrA
pbrBC
pbrD
(b) A. faecalis NCIB8687, Sh. frigidimarina NCIMB400, K. pneumoniae NTUH-K2044, K. pneumoniae CG43
pbrR
pbrA
pbrBC
pbrR
pbrA
pbrB
(c) R. picketti 12D and 12J
pbrT
(d) H. arsenicoxydans
pbrB
pbrR
pbrC
pbrT
pbrA
Fig. 4. The pbr genes from various bacteria (based on Hynninen et al., 2009).
some modifications (Hynninen et al., 2009; Permina et al.,
2006). In comparison to C. metallidurans CH34, in all other
known lead-resistant bacteria the pbrD gene is missing,
whereas some of them lack pbrT (Alcaligenes faecalis
NCIB8687, Shewanella frigidimarina NCIMB400, Klebsiella
pneumoniae NTUH-K2044, K. pneumoniae CG43) or
pbrC (Ralstonia picketti 12D and 12J). Additionally, in
Herminiimonas arsenicoxydans, pbrC and pbrB are separated,
and pbrT is next to pbrC (Fig. 4) (Chen et al., 2004;
Hyninnen, 2009). The pbrU gene, located downstream of
pbrT in C. metallidurans CH34, is inactivated by the
insertion of the TnCme2 transposon (Monchy et al., 2007).
Proteins encoded by pbr and the mechanism of Pb(II)
resistance
The group of proteins encoded by pbr in C. metallidurans
CH34 includes: (1) PbrT, the membrane protein; (2) PbrA, a
PIB-type ATPase, the main efflux transporter; (3) PbrB,
undecaprenyl pyrophosphate phosphatase, an integral
membrane protein; (4) a putative signal peptidase PbrC;
(5) PbrD, a putative intracellular lead binding protein, and
(6) PbrR, a positive regulator (Fig. 5) (Borremans et al.,
2001; Hynninen et al., 2009; Monchy et al., 2007). PbrT is a
lead uptake permease belonging to the iron/lead transporter
superfamily (Debut et al., 2006), which participates (along
with other transporters for essential metals) in the influx of
Pb(II). In the cytoplasm, Pb(II) is most likely bound by the
PbrD protein, reducing its toxic effect, and the expression of
pbrA is activated. PbrD might play the role of a chaperone
for Pb(II) inside the cell. It possesses a potential metal
binding motif, rich in cysteine residues (Cys-7X-Cys-Cys7X-Cys-7X-His-14X-Cys) with a large number of proline
18
and serine residues. Although PbrD is not required for full
lead resistance, cells deficient in this protein show a
decreased accumulation of Pb(II) (Borremans et al., 2001).
The PbrA protein, supplemented by other PIB ATPases,
CadA and ZntA, exports Pb(II), Cd(II) and Zn(II) ions to
the periplasm. Then, inorganic phosphate groups released
by PbrB precipitate lead, preventing the re-entry of Pb(II)
into the cell (Fig. 5). This mechanism is the only system
known so far which is capable of combining efflux and ion
precipitation (Hynninen et al., 2009). Although the Pbr
efflux system was first predicted to be Pb(II) specific,
Hynninen et al. (2009) proved that it also participates in the
protection of the cell against Cd(II) and Zn(II), whereas the
specific mechanism of Pb(II) resistance requires the
cooperation of PbrA and PbrB (Borremans et al., 2001;
Hynninen et al., 2009). The roles of PbrT, PbrC and PbrD
are being challenged, since their absence does not impair the
overall ability of this mechanism to neutralize toxic ions
(Borremans et al., 2001; Hynninen et al. 2009).
The mechanism of Pb(II) removal from the periplasm to
the cell exterior in C. metallidurans CH34 is not known,
but may involve the CzcCBA efflux system (Hynninen et al.,
2009). CzcP (a P1B4-type ATPase, first assigned to the PIB
family of Pb(II)/Cd(II)/Zn(II) efflux ATPases) was also
suggested to be a transporter involved in the further efflux
of Pb(II) from the cell. However, additional research
showed that it does not participate in this activity (Monchy
et al., 2007; Scherer & Nies, 2009).
In C. metallidurans CH34, the pbrR2cadApbrC2 gene
cluster located on chromosome 1, and zntA located on
chromosome 2, cooperate with pbr in providing Pb(II)
resistance (Monsieurs et al., 2011; Taghavi et al., 2009).
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Microbiology 160
Lead resistance in micro-organisms
Pb2+
OM
Pb2+--P
Pb2+--P
Pb2+
Pb2+
PbrA
ZntA
Pb2+
CadA
P
Pb2+--P
Periplasm
PP PP
PbrB
ATP
Pb2+
ADP
+Pi
Pb2+
PbrD
PbrA, a member of the PIB ATPase family, is not Pb(II)
specific, and also transports Zn(II) and Cd(II) to the
periplasm (Hynninen et al., 2009). Efflux through PbrA can
compensate for the transport of Pb(II) into the cell via the
PbrT protein (Borremans et al., 2001). Detailed information about the PbrA protein is included in the next section
concerning PIB ATPases.
PbrB is a membrane protein, which has been identified
as a C55 lipid phosphatase, with six predicted transmembrane domains and a periplasmically located active site.
Presumably, this protein does not participate in the cell
wall synthesis as other C55 lipid phosphatases do, being
only a part of the Pbr efflux system. The other
phosphatases may contribute to the release of phosphate
groups. However, it is rather unlikely that the expression of
genes responsible for their synthesis is induced by Pb(II)
(Hynninen et al., 2009). Inactivation of pbrB and the
absence of PbrB results in lead sensitivity in C. metallidurans CH34 (Borremans et al., 2001; Taghavi et al., 2009).
The mmfB gene, located on the Tn6048 transposon, shows
significant similarity to pbrB and hence might compensate
for the loss of this gene (Monsieurs et al., 2011). PbrB is
fused with the PbrC protein, a putative signal peptidase;
however, PbrC is not essential for the functioning of the Pbr
efflux system. It is assumed that its role can be substituted by
other peptidases, or that peptidase activity is not essential for
the correct functioning of PbrB. PbrC might be important
for the maturation or activity of PbrB in the periplasm
(Hobman et al., 2012; Hynninen et al., 2009).
Although pbrU is not expressed in C. metallidurans CH34, it
has been suggested that this gene encodes a permease,
located in the inner membrane and belonging to the major
facilitator superfamily (MFS) (Monchy et al., 2007; Taghavi
et al., 2009). Apart from pbrU, eight other genes encoding
MFS proteins have been found to be upregulated by Pb(II).
Interestingly, the most overexpressed gene encoding an
MFS is located inside the cluster responsible for the
biosynthesis of a siderophore. This fact suggests that there
is cross-talk between the responses to lead and iron
(Taghavi et al., 2009).
http://mic.sgmjournals.org
IM
Fig. 5. The lead transport system in C.
metallidurans CH34 (based on Hynninen et al.,
2010, modified). Pb(II), entering the cell through
the Zn(II) and Mn(II) transporters, is actively
pumped out by PbrA to the periplasmic space
and then precipitated with phosphates (P)
released by the PbrB protein. PbrA is supported
in this role by CadA and ZntA proteins. PbrD
is a putative intracellular Pb(II) binding protein
(Borremans et al., 2001; Hynninen et al., 2009).
OM, outer membrane; IM, inner membrane.
In C. metallidurans CH34, Pb(II) also induces general stress
mechanisms via alternative sigma factors such as s24/rpoK,
s32/rpoH and s28/fliA (Grosse et al., 2007).
PIB ATPases PbrA, CadA, CadA2 and ZntA
As mentioned above, the PIB ATPase PbrA participates in
Pb(II) efflux in C. metallidurans CH34 (Hynninen et al.,
2009). This transporter possesses two metal binding
domains (MBD): the N-terminal cytoplasmic domain and
a domain located in the transmembrane region (Argüello,
2003; Borremans et al., 2001; Dutta et al., 2006, 2007).
Primarily, the N-terminal domain was suggested to be
involved in Pb(II) binding, as its amino acid sequence differs
from the equivalent one in ZntA from E. coli and CadA from
S. aureus (Borremans et al., 2001; Hynninen et al., 2009).
However, mutations in the N-terminal MBD in ZntA from
E. coli proved that this part of the transporter is not
necessary for metal recognition; hence a similar conclusion
was drawn for PbrA from C. metallidurans CH34 (Mitra &
Sharma, 2001; Hou et al., 2001; Hynninen et al., 2009).
Instead, the transmembrane region has been proposed as the
area responsible for the recognition of a specific metal,
which has been confirmed experimentally (Argüello, 2003;
Dutta et al., 2006, 2007; Liu et al., 2006). The sequence of
conserved amino acids in the transmembrane MBD from
PbrA in C. metallidurans CH34 is the same as in ZntA from
E. coli, CadA from S. aureus, CadA2 from Ps. putida and
ZntA from C. metallidurans CH34. The PbrA transporter is
not Pb(II) specific and is also capable of transporting Cd(II)
and Zn(II) to the periplasm (Argüello, 2003; Borremans
et al., 2001; Hynninen et al., 2009; Leedjärv et al., 2008;
Rensing et al., 1998).
Efflux of Pb(II) has also been confirmed for CadA from S.
aureus (a PIB-type ATPase) and its homologue ZntA from
E. coli (Rensing et al., 1997, 1999). The CadA protein is
encoded by the cadAC genes, located on plasmid pI258.
This transporter, driven by ATP hydrolysis, functions as an
efflux pump, transferring ions from the cytoplasm to the
periplasm. Low level resistance to the metal is provided by
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19
A. Jarosławiecka and Z. Piotrowska-Seget
the pump itself; however, the full efflux function is
achieved in cooperation with CadC, which negatively
regulates expression of the cadA gene (Nies, 1992; Rensing
et al., 1999; Silver & Phung, 2005).
CadA2 is the main efflux transporter for Cd(II) and Pb(II)
in Ps. putida KT2440. Surprisingly, it might be supported
in this role by CzcCBA1, which so far has only been known
for transporting Zn(II), Cd(II) and Co(II) from the
periplasm to the cell exterior in C. metallidurans CH34
(Leedjärv et al., 2008). It has also been postulated that Ps.
putida KT2440 might possess an additional mechanism for
lead resistance, as mutations in genes encoding lead
resistance transporters only slightly impaired the ability
of this bacterium to resist the toxicity of Pb(II) (Hynninen,
2010). In Ps. putida KT2440, PAP2 phosphatase encoded
by pp4813 is present, and can substitute for the activity of
PbrB in C. metallidurans CH34. It is hypothesized that the
novel family of transporters exemplified by MrdH in this
strain may also participate in lead resistance (Haritha et al.,
2009; Hynninen, 2010).
ZntA is a Zn(II), Pb(II) and Cd(II) specific PIB ATPase that
transports these ions in E. coli. The expression of the zntA
gene is regulated by ZntR, a transcriptional regulator from
the MerR family (Brocklehurst et al., 1999). The N-terminal
domain of the ZntA ATPase possesses a highly conserved
GXXCXXC motif, characteristic for P1-type ATPases, as well
as an additional CCCDGAC sequence (Liu et al., 2005). Liu
et al. (2005) proved that the first 46 residues of the Nterminal domain together with the CCCDGAC sequence
play a significant role in the binding of Pb(II), while having
no influence on either Zn(II) or Cd(II) binding (Liu et al.,
2005). The transport of specific metal ions by this pump is
further determined by structural features in the transmembrane domain (Mitra & Sharma, 2001).
Summing up, it has to be pointed out that although ZntA,
CadA and their close homologues function as transporters
involved in bacterial resistance to Pb(II) and Cd(II), their
primary physiological role is participation in zinc homeostasis (Nies, 1992; Rensing et al., 1998; Raimunda et al.,
2012).
Resistance of fungi to Pb(II)
Little is known about lead resistance mechanisms in fungi,
but even low concentrations of this metal can affect their
growth and reduce biomass. The growth of S. cerevisiae is
disturbed at 0.05 mM Pb(II). This concentration of metal
reduces the biomass by 60 % (Donmez & Aksu, 1999).
Pb(II) induces a variety of changes in S. cerevisiae such as
the lengthening the lag phase and slowing growth,
decreasing the DNA/RNA ratio and damaging DNA. It
inhibits the assimilation of ammonium ions, as well as
reducing viability and metabolic activity (Bussche &
Soares, 2011; Chen & Wang, 2007; Van der Heggen et al.,
2010; Sakamoto et al., 2010; Suh et al., 1999a; Soares et al.,
2002, 2003; Yuan & Tang, 1999). Pb(II) leads to the death
20
of yeast cells, caused by reactive oxygen species which
trigger apoptosis (Bussche & Soares, 2011).
Fungi have developed different strategies to ameliorate the
toxicity of lead. In Aureobasidium pullulans, EPs bind more
than 90 % of the Pb(II) that it was exposed to. Removal of
EPs enables Pb(II) to penetrate the cell wall and the cell
membrane and so enter the cytoplasm (Suh et al., 1999b,
2001). In the marine fungus Corollospora lacera, 93 % of all
lead was located extracellularly in the mycelia, while 1.7–
5.5 % was found inside the cell. Penicillium sp. Psf-2,
isolated from the Pacific sediment, was able to withstand
24 mM Pb(NO3)2 in a liquid culture. Lead deposits have
been detected in the cytoplasm and vacuole, as well as on
the outer layer of the cell wall of this fungus. Intriguingly,
these precipitates seem to have been composed of pure lead
only (Sun & Shao, 2007; Taboski et al., 2005).
Interactions between S. cerevisiae and Pb(II) are more
complex and three phases can be distinguished in the
reaction of the cell (Chen & Wang, 2007). In the first
phase, Pb(II) is quickly adsorbed on the surface of the cell
and then passively transported through the cell wall. This
phase is rapid, lasts about 3–5 min and is independent of
metabolism. Accumulation of Pb(II) causes changes on the
cell wall’s surface, which becomes rough (Suh et al., 1999a).
In the next step, Pb(II) crosses the cell membrane into the
cytoplasm, though the transport mechanism has not been
characterized. Pb(II) penetrates into the cells easily. This
suggests that the yeast cell wall is not a significant barrier to
Pb(II) and lead binding by functional groups within the
cell wall is poor. The yeast plasma membrane may control
to a certain degree the transport of Pb(II) to the cytoplasm,
since it takes close to an hour for these ions to cross the cell
membrane. However, it is still unknown how the
membrane regulates the flow of Pb(II). Finally, Pb(II) is
accumulated in the cytoplasm. This phase is independent
of metabolism (Suh et al., 1998, 1999a). In the cytoplasm,
glutathione (GSH) has been reported to be involved in the
neutralization of the toxicity of other heavy metals, such as
Cd(II) and Se(II) (Gharieb & Gadd, 2004). Van der Heggen
et al. (2010) revealed that, in the cytoplasm of S. cerevisiae,
Pb(II) is conjugated with glutathione, and the lead–
glutathione complex is transported to the vacuole, where
it is isolated from the rest of the cell. This complex is
transported through the vacuole membrane by yeast
cadmium factor 1, which is an ATP binding cassette
transporter. CAD2, a putative copper transporting ATPase
from S. cerevisiae, seems to have a role in the efflux of
Pb(II) (Li et al., 1997; Prévéral et al., 2006; Shiraishi et al.,
2000; Song et al., 2003; Szczypka et al., 1994). A protective
role of GSH against lead toxicity has also been observed in
Schizosaccharomyces pombe. Mutants of this strain deficient
in glutathione lost the ability to bind Cd(II) and became
more sensitive to Pb(II), Cu(II) and Zn(II) (Coblenz &
Wolf, 1994).
Fungi can also transform Pb(II) into pyromorphite, the
most stable lead mineral, indicating their significant role in
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Microbiology 160
Lead resistance in micro-organisms
the lead cycle in the environment (Clipson & Gleeson,
2012; Rhee et al., 2012). In turn, Aspergillus niger is capable
of solubilizing this mineral and subsequently immobilizing
Pb(II) as lead oxalates (Sayer et al., 1999).
Potential of lead-resistant micro-organisms in
environmental biotechnology
The increasing concentration of such hazardous heavy
metals as Pb(II) in the environment has stimulated scientists
to search for new possible ways of its removal or
neutralization. As mentioned above, micro-organisms show
a wide range of mechanisms for ameliorating the toxicity of
Pb(II) via its extra- and intracellular precipitation, adsorption on polysaccharides, binding to elements of the cell wall,
or by export through the different transporters. The
capability of micro-organisms to neutralize the toxic effect
of heavy metals or to detect heavy metals could be effectively
used in environmental biotechnology. Traditional physicochemical methods applied to the remediation of heavy metal
contaminated soil often damage its structure and quality, as
well as existing ecosystems. Additionally, these techniques
often require soil excavation and transport to a reclamation
site, and this in turn generates high costs and the need for
specialized equipment (Park et al., 2010). Moreover, other
techniques such as phytoremediation often depend on the
climate, water and soil conditions.
Thus, microbial methods in this context may offer an
attractive alternative. So far, lead-resistant micro-organisms have been successfully used for the immobilization
and biosorption of lead (Park et al., 2010; Wu et al., 2006;
Wilson et al., 2006), the enhancement of phytoremediation
as described in the case of Solanum nigrum L. (Gao et al.,
2012), the construction of lead detecting biosensors
(Chakraborty et al., 2008; Chen et al., 2005; Chiu &
Yang, 2012; Corbisier et al., 1999), bioaugmentation of lead
contaminated mine tailings followed by lead biomineralization (Govarthanan et al., 2013) and bioremediation of
lead contaminated soil (Chatterjee et al., 2012; Chen et al.,
2011; Guo et al., 2010). It seems that the combination of
the traditional and microbial approaches in environmental
biotechnology may yield promising results.
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Acknowledgements
Braud, A., Geoffroy, V., Hoegy, F., Mislin, G. L. A. & Schalk, I. J.
(2010). Presence of the siderophores pyoverdine and pyochelin in
The review was prepared during research on the project financed by
an individual grant from Ministry of Polish Science and Higher
Education N N305 185937. A. J. is a PhD student supported by a PhD
scholarship from the UPGOW project task 55 at the University of
Silesia within the European Social Fund.
the extracellular medium reduces toxic metal accumulation in
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