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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). Downloaded from www.microbiologyresearch.org by 070284 G 2014 SGM IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 Printed in Great Britain 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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 14 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). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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 http://mic.sgmjournals.org 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 04:30:35 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. 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