Copper resistance mechanisms in bacteria and fungi · lar copper storage/carrier proteins (CutE and CutF), also described by Lee et al. [351, may be responsible for protecting the

FEMS Microbiology Reviews 14 (1994) 121-138

Federation of European Microbiological Societies

Published by Elsevier

121

FEMSRE 00386

Copper resistance mechanisms in bacteria and fungi

Carlos Cervantes *+3 and Felix Gutierrez-Corona b

’ Institute de Investigaciones Quimico-Biologicas, Universidad Michoacana, 58240 Morelia, Mich., Mexico, and ’ Institute de Imestiga- cion en Biologia Experimental, Facultad de Quimica, Universidad de Guanajuato, Guanajuato, Gto., Mexico

(Received 5 July 1993; accepted 29 November 1993)

Abstract: Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high

concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for

copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and

the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are

related to reduced copper transport, enhanced efflux of cupric ions, or copper complexation by cell components. Copper tolerance

in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of

copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and

phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.

Key words: Copper resistance; Bacteria; Plasmid; Copper uptake and efflux; Fungi; Copper chelation

Introduction

Copper is an essential micronutrient for most, if not all, living organisms since it is the con- stituent of many metalloenzymes and proteins involved in electron transport, redox, and other important reactions [l]. The role of copper in redox reactions is due to its ability to undergo Cu(I) + Cu(I1) transitions which are dependent on the surrounding chemical environment [2]. Copper requirements by microorganisms are usually satisfied with very low concentrations of the metal (in the order of l-10 PM). In contrast, copper present at higher levels in its free ionic form (Cu”> is toxic to microbial cells [3,4]. Cop-

* Corresponding author. Tel. (Fax): (043) 14 21 52.

SSDI 0168-6445(93)E0112-W

per toxicity is mainly due to its interactions with nucleic acids [5], to the alteration of enzyme active sites and to the oxidation of membrane components, processes that can be related to the ability of copper to generate toxic hydroxyl free- radicals [6,7]. On the other hand, organically complexed copper is relatively nontoxic to microorganisms [8].

Given copper’s dual role of essentiality/ toxicity, microorganisms must possess delicate mechanisms to maintain intracellular copper within such a restricted level that neither inter- feres with normal metal homeostasis nor poses a risk of toxicity. Little information on copper transport in microorganisms has been reported, in part by lacking of a suitable radioisotope of the metal (the currently available 64Cu isotope has a half-life of only about 12 h). Copper properties as

122

an essential but toxic metal are shared by other micronutrients such as zinc, cobalt, and nickel, and are clearly distinguished from the unessential toxic cations of lead, mercury, and silver [9].

Copper and copper-containing compounds are widely used as bactericides [lo], algicides, fungicides, veterinary food additives, plant antibacte- rial agents, and for the preservation of natural and man-made materials 1111. Although copper is commonly encountered in the environment, toxic concentrations of the metal are only found associated with certain mineral ores and industrial or agricultural discharges [121. High levels of copper generally exert a selective pressure on microorganisms that may result in the appearing of resistant variants possessing copper resistance genetic determinants 113-151. Bacteria and fungi are ubiquitous organisms with fundamental properties as bioconverters, only recently considered as playing important roles in the biogeochemistry of heavy metals [16,17]. For this reason, understanding the responses of microorganisms to metals is of scientific interest and also may be relevant to devise biotechnological solutions for the recovery and environmental decontamination of valuable and/or toxic metals [18].

In this article we attempt to summarize the current knowledge on copper resistance in bacteria and fungi with emphasis on copper resistance mechanisms. Several aspects of resistance to copper in bacteria [15,18-301 and fungi [l&31-33,155] have been previously reviewed.

Copper resistance in bacteria

Chromosomal copper resistance

Genes related to normal copper metabolism in bacteria usually reside in the chromosome [34] whereas copper resistance genes are located in plasmids. However, copper resistance determined by chromosomal genes (or not clearly linked to plasmid DNA) has been found in naturally occurring as well as in laboratory-induced mutant Gram-negative bacteria (summarized in Table 1).

Lee et al. [351 reported the isolation of Es- cherichia coli mutants altered in the transport of

Table I

Chromosome-encoded resistance to copper in bacteria

Species Mechanism of resistance Ref

No.

Desulfolihrio spp.

Klehsiella tmeumoniae

Escherichia co/i

K. aerogenes

Thiobaciilus ferrooxidans Suifolobus Cyanobacteria

Leptospirillum E. coli

T. cuprinus Vibrio alginolyticus

Xanthomonas campestris

Complexing copper by

produced sulfide

Transient physiological

adaptation

Lacking of a major outer

membrane protein

Copper-binding by

capsular polysaccharides

Unknown

Unknown

Copper complexing by

metallothionein-like

protein

Unknown

Binding and efflux of

copper

Unknown

Chelation by secreted

copper-binding proteins

Unknown

1401

[411

[421

WI

[341 [481 Ml

1471 [%I

[4hl

[611

[791

copper. From the analysis of copper susceptibility and copper transport properties of the mutants, these authors tentatively identified two copper uptake systems, designated CutA and CutB. CutA was also involved in zinc uptake as well as in copper transport [351. Cytoplasmic copper is believed to be reduced by glutathione to Cu(I)

which can cause oxidative damage to the cell by producing hydroxyl radicals [29]. Two intracellular copper storage/carrier proteins (CutE and CutF), also described by Lee et al. [351, may be responsible for protecting the bacterial cell from Cu(I) toxicity and for delivering copper to the sites of synthesis of copper proteins ([291; Fig. 1). Structural genes cutC and cutD have been assigned a role in copper efflux (Fig. 1). The proteins CutC and CutD are probably copper efflux ATPases [29]. The c&E gene has been mapped, cloned and sequenced and was shown to code for a 512-amino acid protein with a presumptive copper-binding region [361. The copper-binding motif of the CutE protein is related to a copper-binding region identified in Pseudomonas syringae cop

plasmid gene products ([371; see below).

123

2,1. Cu

l

CutA ~ Curb ¢ -

+ ~ Toxic effects

CutEo~I~&utF Idetallo- proteins

I

ATP I AD P+ Pi |

~_ Cute ~(:~CutD

C)

Cu 2+ Fig. 1. Model for the functioning of chromosomal (Cut) genc products involved in copper metabolism in Excherichia colt. Arrows show the fate of copper until cxccss is extruded from the cell by the membrane ATPasc. Adapted from Brown et al.

[2q] with permission.

0 i

J

Bacterial copper transport and resistance have been related with P-type ATPases [38] by recent developments [24]. These enzymes constitute a family of membrane cation translocation pumps found in both prokaryotes and eukaryotcs where they all show significant sequence conservation [24]. Examples of P-type ATPases are the bacterial Kdp K- uptake enzyme, the muscle sar- coplasmic reticulum Ca 2- effiux ATPase, the ani- mal cell N a + / K + exchange pump, and the CadA Cd 2- efflux enzymc from Staphylococcus attreus plasmids [24]. Odermatt ct al. [39,156] recently idcntified two P-type ATPase genes, copA and copB, in Enterococcus hirae: disruption of either copA or copB leads to copper sensitivity, suggesting that they code for copper cfflux ATPascs. Thc gcne for a human lcthal diseasc of copper deficiency, the Menkcs syndrome, also appears to dctcrmine a P-type ATPasc [24]. Intcrcstingly, this ATPase is more similar to the bacterial Cd 2" and Cu 2' ATPases than to eukaryotic ATPases [20,24].

An additional gene, cutR, is responsible for regulating the cut operon [35]. Regulation of the cut system has been hypothesized to be carried

out by a two-component system C u t R / C u t S [24], in a similar way as it is proposed for the plasmid- conferred systems (see below). The gcne products of the cut operon have been proposed to interact with a plasmid-mediated copper resistance system (see below) to regulate copper homeostasis [35]. The possibility exists that a coordinated control may function between the chromosomal and plasmid systems [24].

Sulfate-reducing Desulfocibrio spp. tolerate high concentrations of copper by complexing the metal with produced sulfide, thus reducing copper toxicity [4(I]. Unstable 'training' of Klebsiella pneurnoniae to tolerate high copper concentrations has been reported [41]. Copper tolerance, lost after growing the cells in metal-free culture medium, was considered as the result of a transient physiological adaptation [41]. Luktenhaus [42] selected copper-resistant Escherichia colt mutants and found that they lacked a major outer membrane protein, presumably involved in copper transport.

Some bacterial strains used for ore leaching procedures [43], notably Thiobacillus [44-46], Leptospirillum [47], and Sulfolobus [48], were found to be resistant to copper, as well as to other heavy, metals, but no studies on the mechanisms of copper resistance have been reported.

Many bacterial types possess a capsule as their outermost envelope which may function as a bar- rier preventing toxic compounds from reaching the cell surface. It has been reported that, in bacteria forming biofi[ms, capsular cxopolymers interact with surrounding metal ions mainly through polysaccharide carboxy'l groups [49]. Binding of copper by bacterial exopolymcrs has been shown [50,51]. By analyzing the interactions between copper and purified polyanion exopoly- mers from the marine bacterium Alteromonas atlantica, Gcesey et al. [50] suggested that high- affinity copper-binding sites present in bacterial exopolymcrs influence the partitioning of copper ions in the environments where bacteria thrive. Capsulated strains of Klehsiella aerogenes have been shown to tolerate higher copper levels as compared to noncapsulated variants [52]; it is believed that K. aerogenes capsular polysaccharides sequester toxic copper ions. Copper may

J24

also be unspecifically bound by negatively charged groups on thc bacterial cell surface [53,54]. This property has been exploited as a means for detox- ifying copper-polluted waste streams by using bactcria [54-56]. Bacterial iron-binding sidcro- phorcs arc also known to bind copper [57].

Intraccllular accumulation of copper by a metallothionein-likc protcin has bccn reported in cyanobactcria [58]; a cadmium-binding mctallothionein analogue protein was previously found in a Pseudomonas strain [59]. The amino acid sequence of a metaIlothionein (M'I') purified from the cyanobacterium Synechococcus sp. has been reported [58]. Huckle ct al. [60] recently cloned the structural gene for this MT from the Syne- chococcus chromosome and found that several ions (notably zinc, copper, and cadmium) specifi- cally stimulated transcription at the MT locus. Although the precise function of the prokaryotic MT is not known, by analogy to eukaryotic Ml's, it is believed to play roles in essential metal ion homeostasis and in resistance to non-essential rectal ions [611].

Harwood-Scars and Gordon [61] showed that thc marine bacterium Vibrio alginolyticus synthesizes extraeellular copper-binding proteins in rc- sponse to copper challenge; metal chelation was proposed as a mechanism of copper detoxification.

A copper resistance determinant from the chromosome of Xanthomonas campestris was recently cloned and will be discussed below with the plasmid systems.

Plasrnid-mediated copper resistance

Most adaptivc dispensable bacterial genes (e.g. those coding for antibiotic resistance, virulence properties and degradation of rare organic sub- strates) arc not usually located on the chromosome but in extrachromosomal genetic elements called plasmids. Plasmid-eonferred copper rcsistancc determinants arc often capable of transfer- ring in viw) to new bacterial hosts by conjugation, or to othcr genetic elements by transposition [21]. These transfer properties represent a natural way of dispersing new adaptivc genetic abilities in bacterial populations inhabiting heavy-metal pol-

"1 able 2

Plasmid-mcdiatcd cuppcr rcsi:,tancc m bacteria

Spccics Plasmid Mcchani'ml ~I Rcl. resistance No.

I:M'herichia coh R 124

.¥unthonumus pXv( 'up camp(ts'tri3

h'. coil Rtsl

My<oha('tenum pVTl s( rof~daceum

IM'udomonas syringae pPT23

E. <oh pRJ 11){14

P. ~yrmgae pPS 11 X <ampestn.~ pXV 10A Ah'aligetles el~lrol~hU.~ pMOI.30

Decreased OmpF {St~[ protein s,,nthcsJs

t.'nkno~,n JSnl

Reduced copper [85] HCCII nl tl [~ll[ioI1

SLllfatc- is-l] dcpcndcnl copper precipitation

("hclation by 16S] copper-binding pr~Icins

Enhanced 178] copper el'flux

('oppcr-binding [~'~6] Unknown [Sl] Unknown [27]

luted environments [62]. The use of culture mcdia with different abilities to complex cupric ions [63,64] has resulted in reported MIC copper val- ues ranging from 1 to 20 mM for plasmid-containing resistant strains, depending on the testing conditions.

Plasmid-determined resistance to copper has been found in diverse Gram-negative bacterial species ([15]: Tablc 2), but only threc copper resistance determinants have been analyzed to the molecular level [20,22,24,28-30].

The Pseudomonas system Thc wide use of copper solutions to control

microbial plant diseases is considered as the selective factor for acquisition of copper resistance by plant-associated bacteria [65-68]. The first well-studied bacterial copper resistance determinant comes from a plasmid identified in a strain of the tomato pathogen l{s'eudomonas syrmgae pv. tomato [68]. This plasmid, called pPT23, con- fers inducible copper resistance [69] and is highly conserved among I: .wringae strains [66,70,71]. The copper resistance determinant from pPT23 has been chined [72] and its nucleotide sequence determined [37].

Four structural genes, arranged in the cop operon, were initially identified; two of them, copA and copB, are necessary for expression of full copper resistance whereas plasmids with deletions in copC or copD still confer partial resistance [37]. From sequence analysis, a peptide of eight amino acids containing aspartic acid, histidine and mcthionine residues is repeated several times in both CopA and CopB, and is proposed to function as a copper-binding domain [37]. CopA also showed limited, but significant, sequence homology with the copper proteins azurin and plastocyanin [37]. The predicted polypeptides CopA and CopB generally lacked cysteine and thus do not pertain to the mctallothionein class.

Cha and Cooksey [73] expressed the cop operon and found that CopA and CopC are periplasmic proteins able to bind 11 and 1 copper atoms per polypeptide, respectively (Fig. 2). CopB is an outer membrane protein which binds additional copper ([73]; Fig. 2) and that may be involved in cross-membrane copper transport [24]. By DNA-hybridization studies, Cooksey et al. [71] found that copB gone from p[asmid pPT23 shows homology with plasmid or chromosomal DNA from copper-resistant pathogenic and saprophytic plant-associated bacteria. The finding that homology was significant even with DNA from copper-sensitive strains, led these workers to propose that cop genes may have a function other than copper resistance and that plasmid cop genes might have evolved in pseudomonads from chromosomal genes [71]. The fourth gene product, CopD, is associated with the inner membrane, where participates in copper transport [74]. The involvement of copC and copD in copper transport was recently suggested by the finding that expression of both these genes in P. syringae, lacking copA and copB, caused copper hypersen- sitivity [74].

Regulation of the cop operon was initially reported to be carried out by a trans-acting product, CopR, the genc of which is located distal to copD and which may also be encoded by the chromosome of some plant-pathogenic pseudomonads [71]. From DNA sequence analysis, Cooksey [30] recently identified two additional

125

Cu 2÷

cu2+ ?

2

Fig. 2. Cellular location of the polypeptides encoded by the plasmid copper resistance determinant from Pseu(h~monas wringae. Outer membrane (OM) and inner membrane (IM) proteins CopB and Copl), respectively, participate in copper transport and/or efflux. ('opA and ('opC are periplasmic proteins capable of binding cupric ions. Redrawn from Silver

ctal. [24] with permission.

regulatory components of the cop operon. A chromosomally determined DNA-binding protein, CopM, and protein CopS encoded by a gene distal to copD in the plasmid determinant [24,30]. The couple CopR plus CopS seem to be members of a two-component regulatory system of the type which are known to control the expression ot" many physiologically important genes and oper- ons [75].

The relationship between chromosomal and plasmid genes in the P. syringae determinant very much resembles the E. coli C u t / P c o system described above. Recent DNA sequence analyses have also revealed a clear-cut relationship between the predicted polypeptide sequences of the cop and the E. coli pco determinants [24]. The amino acid sequence identities of the apparently homologous protein pairs are: P c o A / C o p A , 76c7~,; P c o B / C o p B , 54%; P c o C / C o p C , 66c~; and P c o D / C o p D , 38% [24]. Sequence similarity has also been fl)und betwecn copR/pcoR and copS /pcoS [30].

In contrast to the copper-effiux strategy of the

12~

E. coil determinants (scc below), howcvcr, metal sequestration has been dcmonstratcd as the basis for copper resistance in plant-associatcd bacteria ([73]; Fig. 2). Increased copper accumulation was shown by Cooksey and Azad [76] in P. syringae with a cloncd cop operon as well as in related copper-resistant pscudomonads; some strains accumulated as much as 12(1 mg of copper per gram (dry weight) of cells. The copper in resistant cells appears to bc in the cupric (Cu 2-) state since bacterial colonies still maintain the bluc color of these ions [66]. Copper complexing was highly spccific as copper-induced cells did not accumu- late other mctals [76]; a halt" of the ceil-bound coppcr was released by EI)TA, suggcsting that the metal was bound to the cell envelope, probably through the outcr membrane protein CopB.

This is the first plasmid-dctermincd heavy metal resistance system that involves scquestra- tion of the toxic ion. Othcr heavy-metal resistance mechanisms result from reduced accumulation, cffiux, or chemical modification of the ions involved [21]. Coppcr complcxing, however, has bcen rcported for several chromosome-encoded resistancc systems (Table 1) and is commonly found in copper-rcsistant fungi (see below).

"Fhe E. coli system An E. coli copper resistance plasmid, pRJ 1004,

was found in a strain isolated from the effluent of a piggery [77] where copper sulfate, used as a food supplement, possibly acted as a selective agent. Inducible resistance to copper conferred by pRJl004 was found to be due to decreased metal uptake [78]. Rouch et al. [157] cloned the copper resistance determinant from pRJ1004 and four pco (for plasmid copper) genes, pcoARBC, were initially identified, and their corresponding gene products were assigned roles for the resistance phenotype. Further studies led to the finding of additional putative pco genes now arranged as pcoABCDRSE [24,28]. As mentioned above, the pco genes show significant sequence homology to the cop genes, but the precise fun- cion of the Pco proteins is still unknown. The PcoA protein conserves the potential copper- binding domain of the CopA protein and thus appears to function as a copper-binding polypcp-

tide. The CopB protein, however, does not retain the potential copper-binding sites present in CopB and is proposed to have a function unre- lated to copper binding [29]. The PcoC protein shares a putative copper recognition motif with the CopB protein suggesting a role of PcoC in copper binding [29]. Sequence analysis showed that PcoD have potential t ransmembrane seg- ments and methionine and histidinc groups located on the outer face of the inner membrane. which are also conserved in CopD [29].

PooR is a repressor responsible for regulation of the pco operon; from sequence analysis, PooR was found to show homology with DNA-binding regulatory proteins of the two-component class, as it was mcntioncd above for thc pairs C u t R / CutS and C o p R / C o p S from the E. coli chromosome and the Pseudomonas plasmid copper resistance determinants [24]. Further nucleotidc sequence analysis of the pco operon showed the presence of another regulatory gene, named pcoS. From its deduced amino acid sequence, PcoS was predicted to be a membrane polypeptide with the attributes of an auto-phosphorylating protein which qualifies it as the sensor portion of the two-component P c o R / P c o S regulatory system [24]. Williams et al. [158] recently found pco homologs among copper-resistant enteric bacteria from pig farms in thc UK and Australia.

l,ee et al. [35] proposed a unique physical interaction between chromosomal (metabolic) and plasmid (resistance) genes in copper-resistant E. coil cells. Chromosomal genes cutA (uptake) and cutD (efflux) are required for the expression of copper resistance in E. coli, whereas plasmid gent products of pcoA and pcoB were proposed to interact with proteins CutC and CutD by forming a tbur-component efflux system [35]. More- over. chromosomal regulatory gene cutR re- pressed pco plasmid genes in the absence of a functional regulatory pcoR [35]. This genetic in- terplay would allow bacterial cells to regulate changing environmental copper concentrations according to cellular needs. As proposed by this model, chromosomal genes control normal low- copper situations, whereas plasmid genes will function when high (toxic) copper is present [35]. An additional, low-level copper resistance deter-

127

minant, called cdr, was found in the E. coil plasmid pRJl004 and a role in copper-damage repair was tentatively assigned [157]. This would be a distinct resistance mechanism since it does not deal with copper itself but with its toxic effects.

Other plasmid-mediated systems The chromosome of Xanthomonas campestris

pv. juglandis, a pathogen of walnuts, was the source of the third, still unpublished, sequenced copper resistance determinant [79]. Although of chromosomal origin, the four structural genes, copABCD, of the Xanthomonas determinant showed significant homology with both cop and pco gcncs [20]: surprisingly, the sequences were more similar for the Xanthomonas/E. coli dctcr- minants than wcrc for the Xanthomonas (a pscu- domonad)/Pseudomonas genes.

Copper resistance has also been encountered in strains of Xanthomonas campestris pv. cesica- toria, a pathogen of peppers and tomatoes, bear- ing large plasmids pXvCu [8(I] or pXVIOA [81]. The cloned copper resistance determinant from pXVIOA, which did not express in E. coli, showed strong nucleotide sequence homology with pXvCu and only slight similarity to P. syringae copper resistance genes [81]. Voloudakis et al. [82], however, recently found significant homology between copper resistance plasmid DNA from diverse Xanthomonas strains and the copA gene from P. syringae, suggesting a possible common origin for the copper resistance determinants from these plant-associated bacteria. Garde and Ben- der [83] used a probe from pXV10A to detect copper resistance determinants from a variety of soil bacteria: genomic DNA from copper-sensitive strains failed to hybridize to the pXVIOA probe, thus allowing the authors to propose a non-endogenous origin of the Xanthomonas copper resistance genes. Since the t'. ~yringae resistance determinant is postulated of chromosomal origin ([83]; see above), exchange of plasmid DNA between pseudomonads and xanthomonads is suggested as responsible for the similarities between the copper resistance determimmts of these bacteria [82]).

A distinct plasmid-cncoded copper resistance

mcchanism has bccn found in a strain of My- cobacterium scrofulaceum isolated from a polluted river [84]. Sulfate-dependent precipitation of the metal (as copper sulfide) was shown to bc rcsponsiblc for coppcr rcsistancc [84]. This mcchanism resembles the one mentioned above for Desulfocibrio strains [40] that was probably not plasmid-linkcd.

In E. coli, R-factors Rtsl [85] and R124 [86] also confcr coppcr resistance. Cotter ct al. [87] reported that Rtsl-containing E. coli cells accumulated less coppcr than a plasmid-frec derivative, although their copper uptake results wcrc somewhat masked by a rapid unspecific copper- binding. Copper resistance determined by plasmid R124 was associated with a lower level of major outer mcmbranc protein OmpF [86]. To our knowledge, no further studies about copper resistance have bccn reported with these plasmids.

Copper resistance in multiply heavy-metal-resistant Alcaligenes eutrophus isolatcs [88] is dctcr- mincd by large plasmids pMOI,85 [89] and pMOL30 [27]. To emphasize thcir rcsistancc properties, Mcrgcay ct al. [62] have proposed these strains to bc classified as Alcaligenes eutrophus var. metallotolerans. Drcsslcr ct al. [90] reported the isolation of Alcaligenes and Pseu- domonas strains showing resistance to copper of probablc plasmid origin. Thcsc strains also contain genetic determinants for resistance to other toxic metals [90].

Copper resistance in fungi

Copper binding in fungal cells

It is known that the binding of copper in fungi, as well as that of other metals, comprises two phases. The first is a metabolism-independent surface binding pathway, whereas the second phase is an energy-dependent metal influx [31]. Binding o1" significant amounts of metals to the fungal cell wall is a very. rapid process, often taking place in a few minutes [91,92]. When cultured in the presence of copper, the cell walls of Penicillium italicum [93], Neurospora crassa

12b~

[93,94], P. ochro-chloron [95], Aureobasidium pullulans [96] and Mucor rouaii (Ramirez-Salgado el al., unpublished results) accumulated 5 -40% of the metal added to the culture medium, which, in some cases, gave rise to a blue-colored mycelium. Copper-tolerant strains of Rhizopus stolonifer, CunninghameUa blakesleeana [97] and M. rowcii (Ramirez-Salgado el at., unpublished) bound sig- nificantly more copper into the mycelial biomass than the correspondent copper-sensitive parental strains.

Germann and Lerch [98] reported that 'slime', a wall-less mutant of N. crassa [99], showed a reduced accumulation of copper when cultured in the presence of the metal as comparcd to the walled strain. This finding adds evidence about cell wall involvement in metal binding properties of fungal celts.

A copper-resistant strain of M. rouxii, cultured in thc presence of a low concentration (1.6 /,~M) of the metal, accumulated a ten-fold higher amount of copper in the cell wall fraction and in a mixture of cytosol and membranes, as compared to the metal-sensitive parental strain (Ta- ble 3). In the presence of a much higher copper concentration (3.2 mM), however, the differencc in metal accumulation was reduced to a 50% highcr amount of copper in the cell wall fraction, but not in the cy toso l /membrane fraction, of the resistant strain (Table 3). As previously suggested for other fungi [96,97], these findings point to the involvement of the cell wall in the development of copper resistance. However, it seems that, at

"Fable 3

Suhcellular distribution of copper in strains of Mucor nmxti

Copper (mM) Strain " Copper bound in growth (nmol /mg dry weight) medium

('ell walls Mixed traction h

(}.(X)I 6 Cup" 0.30 0.20 0.0016 ( 'up ' 2.80 2. I{) 3.2 ( 'up" 42.6 38.6 3.2 ( 'up ' 64.9 35.3

" ( 'up ' , copper-sensitive: ( 'up ~. copper-resislant. ~' Mixed membrane l'r:lction plus cytosolic fraction. l)ata from Ram~rez-Salgado el al., unpublished results.

- - ® . . .

Fig. 3. Ultraslructural appearance of cell walls (CW) from a ( 'up ' strain of Trichoderma t'iride grown at low (A, 0.0016 raM) and high (B. 3.2 mM) concentration of copper. Bars: A. 100 nm: B. 2(~) nm. From CortcSs-Penagos et al.. unpublished.

least in M. rouxii, cytosolic a n d / o r membrane- associated components may also participate in metal tolerance processes.

The biopolymers of fungal cell walls, such as the polysaccharides chitosan and chitin, have been reported to be useful in the removal of valuable metals, particularly uranium [17,100]. The Phy- comycetes, such as R. stolonifer, C blakesleeana and M. rouxii, have a significant proportion of chitosan in the outer laycr of their vegetative cell walls [101]. It has been proposed that the copper-binding properties of mycelial cells of some fungi developed in the presence of the metal may be due to qualitative or quantitative changes in cell wall components, leading to an improved copper-binding ability and, thereby, to metal detoxification [94,102]. When cultured in the presence of toxic concentrations of copper, the cell wails of C. blakesleeana contain reduced amounts of chitosan and chitin and a higher proportion of a hydroxypro[ine-rich protein [103,104], which was absent in the cell walls of control cells. The changes in fungal cell walls by culturing in the presence of copper may be re- flected at the structural level [103,104]. Fig. 3 shows that a copper-resistant strain of Tricho- derma citqde, when cultured with toxic conccntra- fions of copper, produced about five-lk)ld thicker

129

cell walls (Cort6s-Penagos et al., unpublished re- suits).

It has been shown that, in addition to its important nutritional role, the presence of mycorrhizal fungi in plants plays a protective role against the toxic effects of copper and other heavy metals [105,106]. The protective action of mycorrhizal fungi may be based on the adsortive properties of fungal surfaces [106] or on the sequestration of copper by cytoplasmic components such as polyphosphate granules [107,108].

Copper uptake in fungi

Energy-dependent copper uptake usually pro- ceeds at a slower rate than metal surface-binding and can be inhibited by low temperatures, glucose analogues, acidic pH, and metabolic in- hibitors and uncouplers (for a review see [31]). Energy-dependent uptake of copper was demonstrated in yeasts [109-112]. Measuring copper transport in filamentous fungi is complicated by the high level of metal binding to the cell walls: however, energy-depcndcnt uptake of copper has been demonstrated in protoplasts of the copper- tolerant fungus P. ochro-chloron [113] and in the polymorphic fungus A. pullulans. This latter fungus and the yeast Saccharomyces cerec&iae are similar in several aspects of copper transport, but they differ from the yeast Candida utilis [111]. In both A. pullulans [159] and S. cerecisiae [112], copper uptake is relatively less specific than in C. utilis since in this yeast the process is not inhibited by other cations [111]. In addition, a stoichio- metric exchange of 2 mol of potassium for 1 mol of copper was reported in S. cererisiae [112] and A. pullulans [159], whereas that exchange was not detected in C. utilis [111]. On the other hand, copper uptake in S. cererisiae is inhibited by CCCP [112], but this uncoupler had relatively little effect in C. utilis [111].

Several studies have suggested a correlation between copper tolerance and an altered metal uptake (reviewed in [31]). Metal-resistant yeast strains showed a decreased influx of copper, cadmium and lithium [114-117]. It has also been reported that in a copper-tolerant strain of A. pullulans copper uptake was reduced as com-

pared to that of a sensitive strain [118]. By using protoplasts, Gadd et al. [117] demonstrated that decreased copper uptake by a copper-tolerant strain of S. ceret'isiae was due to changes in membrane transport properties rather than to alterations in cell wall permeability. Two separate mutant loci in Aspergillus nidulans, cupA and cupB, confer specific resistance to copper which was not due to an increased synthesis of copper- binding proteins [119]. A restricted uptake of copper was proposed as a mechanism responsible of copper resistance in A. nidulans [119].

Intracellular chelation of copper

A distinct mechanism of copper tolerance is the production of mctallothioneins. Thcsc are low-molecular-mass (< 10 kDa), cystcine-rich, metal-binding proteins inducibly synthesized in the presence of metals [120,121]. Metalloth- ioneins (MTs) are widely distributed throughout living organisms such as mammals (including hu- mans), plants, fungi [122,160], and cyanobacteria [58,123]. MTs have been proposed to be involved in a number of cellular processes including metal storage and detoxification, development, differ- entiation, control of metabolism, protection from free-radical toxicity, and UV response (reviewed in [124]).

Genetic studies in S. cere~'isiae showed that high levels of copper resistance could be at- tributed to a locus called CUP1 [125-127]. Cloning and molecular characterization of DNA sequences conferring copper tolerance to S. cerecisiae-sensitive strains showed that the resistance phenotype was due to a tandem amplification of a 2-kb genomic DNA segment of the CUP locus [126]. Further studies indicated that there exists a correlation between the levels of copper resistance and the extent of DNA amplification [32,121,128]. Copper-resistant strains of S. cere- t'isiae contain 2-14 copies of the 2-kb repeat unit in a tandem array (denoted CUPId), whereas copper-sensitive strains contain only one copy of the sequence (cupl ~) [129]. Transcription of CUP1 is 10-20-fold induced by the addition of copper but not of cadmium or zinc [130]. Copper-induced expression of the CUPI genc also requires

130

the participation of the ACEI (also called CUP2) transcription factor which is encoded by the CUP2 gene [131,132]. Sequence analysis of the A C E I gene revealed a predicted polypeptide possessing an N-terminal half rich in cysteine residues [133]; these cysteines form a copper-thiolate complex, similar to that described for yeast Cu-MT, which stabilizes ACEI for DNA binding [133]. A metal-activated transcription factor showing partial homology to the ACEI protein was reported in the yeast Candida glabrata [134]; this transcription factor showed ACEI properties when tested in S. ceret'isiae.

The CUP1 locus contains two open reading frames (ORFs), one of which codes for a cysteine-rieh protein analogous to the we[I-char- acterized mammalian MTs; the second O R F en- codes a protein with no function ascribed as yet [121,128].

Hetcrologous MT genes were shown to be fully functiona[ in yeast [135,136]. For example, c I )NA of the monkey cadmium MT gene fused to promoter sequences of the yeast CUP1 gcnc on a multicopy plasmid conferred copper resistance in a metal-sensitive S. ceret'isiae strain with a dele- tion in the CUPl gene [135]. Similar results were obtained by Eckcr et al. [136] with a multicopy chimeric plasmid consisting of the yeast MT promoter and a fusion of the yeast and monkey MT structural genes. It has also been observed that high levels ot" expression of yeast CUP1 gene lead to cadmium resistance [136,137] although a lower ( 'UP1 level was required to confer copper resistance [137]. The potential of yeasts containing the amplified CUI'I gene to be used for detoxifiea- lion of metal-polluted discharges has been recog- nized [138].

The copper-inducible MT of S. cerecisiae has been purified [139,140], its low molecular-mass and high content of cysteine, qua[flied it to be designated as copperthionein [140], copper chelatin [139], yeast MT, or simply Cu-MT [32].

Other fungi in which copper-inducible cysteine-rich proteins have been described include N. crassa [ 141 ] and Dactylium dendroides [ 142]. A pathogenic strain of Candida albicans was found to contain DNA sequences hybridizing with S. ceret'isiae CUP DNA and to synthesize copper-in-

ducible proteins similar to the S. ceret'isiae MT [128]. The pathogenic yeast ('. glahrata synthesizes a small protein in response to copper but no DNA sequence homology with the S. ceretisiae CUP gene was found [128]. Other workers were unable to detect hybridization of CUPI DNA probes with C. albicans DNA, although the presence of low-molecular mass copper-binding proteins was demonstrated in this yeast [33].

Cano-Cancho[a et al. [143] have shown that the dimorphic Zygomycetes Mucor racemosus and M. rmL~ii both contain DNA sequences which hybridize with CUP DNA from S. ceret'isme: they showed that in M. twuxii there exists a CUI' multigene family. It was also found that DNA methylation controls CUP expression during development of Mucor cells [143].

By using a histochemical procedure, Morselt et a[. [144] 15ave reported that tolerance to heavy metals in ectomycorrhizal fungi is based on the presence of MT-like proteins which probably pro- tect the host plant in metal-polluted areas. Cop- per-tolerant strains of Trichoderma ciride, isolated from a copper mine, contain a 25-kDa protein which is undetcctablc in Cu-sensitive strains, but no DNA homology was found with the ( 'UPI gene of S. cerecisiae (Cortt~s-Penagos, unpublished results).

Copper-inducible proteins, either containing or lacking cysteine, are produced in the fungus D. dendroides [142]. It is not clear whether they are similar to phytochelatins, metal-induced peptides of the general structure (y-Glu-Cys),,-Gly (n = 2- 11) that have been tkmnd in algae and higher phmts [145,146] and some fungi [33,147-149]. In the yeast C. glabrata, mctallothioneins and phytochelatins are produced in response to copper and cadmium, respectively [149]. Kneer ct al. [15(I] recently reported that the unre[ated fungi S. ceret'isiae and N. crassa, already known to synthesize MTs [32,33] also produce phy- toehclatins when exposed to cadmium; interestingly, the synthesis of phytochelatins in S. cere- t'isiae is induced by copper or zinc [15[)].

l:~tracellular chelation o f copper

It has been described that organic and i n o f ganic extrace[lular funga[ metabolites may detox-

ify copper and othcr metals by complexation or prccipitation [31]. Copper-tolerant fungi produce oxalate which precipitates the metal in the form of Cu-oxalate crystals [151]. The formation of lq~S in yeasts can also result in the precipitation of copper, which is then deposited in and around the cell wall [152]. However, the findings that H2S production by cells of a copper-resistant strain of S. cererisiae did not differ from that of a sensitive strain, and that copper tolerance can be developed by yeast strains lacking H2S-producing ability [152] cast doubts about the significance of H2S production as a mechanism of copper resistancc.

Other mechanisms of copper tolerance in fungi

Copper tolcrancc in yeasts correlates with large incrcascs in thc amount of supcroxidc dismutase [161], an enzymc which uses coppcr as an inorganic eofactor. This finding is consistent with thc observation that copper addition to Cu-depletcd yeast cells rcsults in an increased lcvcl of super- oxidc dismutase m R N A [162]. In P. ochro-chlo-

CW-

CM

Copper ions ' " ' " ' °

i~=! i!i!ii!!K ~ ~ .................................

] 00001000

I M e t a l l o - . ~ ~:~: Gamma- !thioneins peptides

Fig. 4. Schematic representation of the principal mechamsms of copper resistance in fungal cells. 1, binding of copper ions by cell wall (CW) components" 2, cell membrane (CM) copper uptake and efflux: 3, intraccllular copper chelation by mctal- Iothionein proteins and glutathionc-dcrivcd y-peptidcs; 4. extracellular sequestration of copper by secreted metabolites.

Modified from Sos~l-l.una. unpublished.

131

ron, high levcls of intracellular glyccrol, induced by high external copper, were shown to be in- volvcd in thc cxclusion of the metal [163]. Resis- tance to copper in Phycomyces blakesleeanus was accompanied by an altered rcspiration rate and by changcs in spore morphology and viability [153]; it was also shown that all thcsc phenotypic alterations were of cytoplasmic naturc, suggcsting a mitochondrial origin of the correspondent mutation [153]. This intcrprctation was supported by studics in N. crassa [154] and C. blakesleeana [104] showing mitochondrial damagc after culturing the cells in toxic conccntrations of copper.

A summary of the more clearly understood mechanisms used by fungal cell to cope with high lcvcls of copper is diagrammcd in Fig. 4.

Concluding remarks

Bacteria have evolved diffcrcnt mechanisms encoded by either chromosomal or plasmid genes to overcome copper toxicity. These include reduced copper transport, enhanced effiux of the metal, and copper complexing by bacterial components. More than one mechanism may function in bacterial cells to ensure adequate intraccllular copper concentrations. As is truc for other essential, but toxic mctal ions (i.e. zinc, nickcl, cobalt), bacterial control of cytoplasmic copper [cvcls is probably achieved by regulating the rate of membrane coppcr uptakc a n d / o r effiux. In E. coli, two pathways appear to control cytoplasmic copper levels: a chromosomal system functioning at low copper and a plasmid system enabling cells to tolerate toxic coppcr conccntrations. Thesc two systems probably intcract and perhaps also share control mechanisms.

Two distinct bacterial plasmid copper resistance determinants have been analyzed to date at the molccular level. Interestingly, although the predicted gcnc products of these systems show significant scqucncc homology, thcy sccm to in- volve rathcr different approachcs for avoiding copper toxicity, namely: (i) intracellular cfflux of cupric ions, and (ii) metal complcxing by pcriplas- mic and membranc proteins. Understanding of the functioning of the plasmid systems and their

132

relationship to chromosomal genes is still under way.

Similarly to bacteria, fungi have developed a variety of copper resistance mechanisms to sur- vive in the presence of toxic concentrations of cupric ions. These mechanisms include copper complexing by cell wall components, changes in membrane copper transport, synthesis of mtracel- lular copper-binding metallothioneins and phytochelatins, and production of extraccIlular copper-complexing or -precipitating metabolites. With the exception of some yeasts, the underlying genetic or molecular basis for copper resistance remains mostly unknown. Among these different copper-resistance mechanisms, only metallothionein production has been tested through the employment of molecular genetic approaches.

Acknowledgements

We thank S. Silver for helpful suggestions on the manuscript and for making available unpublished inh)rmation, and L. Sosa-Luna for helpful comments. Work in the authors' laboratories was supported by Coordinaci6n de lnvestigaci6n Cientffica (Universidad Michoacana), DIGICSA (SEP), and CONACYT (0944-N9111 ).

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Copper resistance mechanisms in bacteria and fungi · lar copper storage/carrier proteins (CutE and CutF), also described by Lee et al. [351, may be responsible for protecting the