Microbial Copper Resistance: Importance in Biohydrometallurgy

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Microbial copper resistance: importance in

biohydrometallurgy
bal Martınez-Bussenius, Claudio A. Navarro
Cristo
microorganisms dissolve the mineral matrix occluding the
and Carlos A. Jerez*
metal to be extracted as it occurs in the case of gold (Kak-
Laboratory of Molecular Microbiology and Biotechnology,
sonen et al., 2014; Urbieta et al., 2015; Harrison, 2016).
Department of Biology, Faculty of Sciences, University of
These mineral–microbe interactions to recover metals from
Chile, Santiago, Chile.
ores have been widely used in small and large scales (Bri-
erley and Brierley, 2013; Norris et al., 2013; Harrison,
2016). An acidophilic microbial consortium is in charge of
Summary
metals mobilization. In practice, commercial biomining pro-
Industrial biomining has been extensively used cedures are mainly used for copper bioleaching and the
for many years to recover valuable metals such biooxidation of refractory gold ores (Brierley and Brierley,
as copper, gold, uranium and others. Further- 2013; Kaksonen et al., 2014; Urbieta et al., 2015). During
more, microorganisms involved in these pro- minerals bioleaching acid mine drainage is generated
cesses can also be used to bioremediate places which can cause serious environmental problems. How-
contaminated with acid and metals. These uses ever, bioremediation or removal of the toxic metals from
are possible due to the great metal resistance contaminated soils can be achieved by using abiotic meth-
that these extreme acidophilic microorganisms ods or the specific properties of the acidophilic microorgan-
possess. In this review, the most recent findings isms interacting with these elements (Johnson and
related to copper resistance mechanisms of bac- Hallberg, 2005).
teria and archaea related to biohydrometallurgy Industrial biomining operations are of several kinds
are described. The recent search for novel metal depending on the ore type and its geographical location, the
resistance determinants is not only of scientific metal content and the specific minerals present (metal oxi-
interest but also of industrial importance, as des and mostly secondary metal sulfides such as covellite,
reflected by the genomic sequencing of microor- chalcocite and others). Metal recovery processes can be
ganisms present in mining operations and the carried out by using tank, heap, dump and in situ leaching
search of those bacteria with extreme metal systems (reviewed in detail by Harrison, 2016). One of the
resistance to improve the extraction processes most used setups for the recovery of copper is the irrigation
used by the biomining companies. type of processes. These involve the percolation of leaching
solutions through the crushed ore that can be contained in a
column, heap or dump (Watling et al., 2010; Jerez, 2011;
Introduction Brierley and Brierley, 2013). The pregnant solution contain-
ing copper sulfate generated by the microbial solubilization
Biomining is used to enhance the recovery of metals from of the insoluble copper sulfides present in the ore, is sub-
ores such as sulfide minerals. Bioleaching solubilizes the jected to solvent extraction to obtain a highly concentrated
metal of interest by biological catalysis as in the case of copper sulfate solution from which the metal is recovered in
copper. On the other hand, during biooxidation an electrowinning plant to generate electrolytic copper of
high purity (Watling et al., 2010; Harrison, 2016).
Received 5 August, 2016; revised 30 September, 2016; accepted 3
On the other hand, as reactors are expensive to build,
October, 2016. *For correspondence. E-mail cjerez@uchile.cl; Tel. they are used with high-grade ores or with mineral concen-
562 22 9787376; Fax 22712983. trates. The advantages of tank reactors over heaps and
Microbial Biotechnology (2016) 0(0), 000–000.
doi:10.1111/1751-7915.12450
dumps, which are ‘open bioreactors’ is that in the tanks
Funding Information conditions can be controlled. The use of hyperthermophilic
The authors thank projects FONDECYT 1150791 and 1110214 for archaea for mineral sulfide processing has been studied
their research funding.
mainly because they appear to have greater tolerances to
ª 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
2 C. Martınez-Bussenius, C. A. Navarro and C. A. Jerez
high copper concentrations and solid particle concentra- A. thiooxidans are acidophilic mesophiles and together
tions that take place in bioreactors compared with other with the moderate thermophile, A. caldus, are some of
thermoacidophiles (Brierley and Brierley, 2013; Norris the most studied members of the biomining consortium.
et al., 2013; Harrison, 2016). Therefore, the search for key The role in biomining of many other acidophilic microor-
efficient mineral-oxidizing strains with very high metal resis- ganisms present in natural and man-made acidic envi-
tance may be of great importance to develop improved ronments has recently been reviewed (Hedrich and
industrial bioreactors for refractory minerals (Norris et al., Schippers, 2016). Although A. ferrooxidans has been
2013; Wheaton et al., 2015; Urbieta et al., 2015). described to oxidize metal sulfides even at 0°C (Lang-
Thermophilic archaea are able to oxidize mineral sul- dahl and Ingvorsen, 1997), no psychrophilic acidophiles
fides at temperatures higher than 60°C. Among these, (optimal growth temperature lower than 15°C) have been
Acidianus brierleyi, Sulfolobus metallicus and Metal- reported. Some psychrotolerant microorganisms such as
losphaera have been the most studied (Brierley and Acidithiobacillus ferrivorans have been also isolated that
Murr, 1973; Lindstro €m and Gunneriusson, 1990; Jones can catalyse metal dissolution from sulfide minerals at
et al., 2011; Norris et al., 2013; Wheaton et al., 2015). 5°C. These bacteria are of importance in biomining oper-
When a microbial community is used to start a series of ations as several of them are present at high altitudes
tanks for biomining, metal concentrations in the leachate and in Polar regions and therefore can be used for
increase and those species resistant to extremely high biotechnological operations in cold environments (Dop-
metal concentrations (as well as other compounds such son, 2016).
as organic carbon) are selected in the final tank (Okibe Chalcopyrite is the most abundant copper-containing
et al., 2003; Dopson and Holmes, 2014). This suggests mineral (around 70% of the world’s copper reserves).
that those microbial species with higher metal resis- However, it is the most difficult mineral to solubilize by
tances may be advantageous for the biomining process. mesophilic microorganisms, taking much longer time to
In this regard, a spontaneous mutant strain of Metal- recover copper. Therefore, there is actually great interest
losphaera sedula (CuR1) with supranormal metal resis- in developing processes by using thermophilic biomining
tance has been reported to leach copper from microorganisms for these recalcitrant minerals.
chalcopyrite (CuFeS2) at an accelerated rate (Maezato
et al., 2012) (see the archaeal section below).
Genetic manipulation and recent advances using
Several recent reviews related to biomining methods
OMICS to analyse biomining microorganisms
and the microorganisms involved are available (Brierley
and Brierley, 2013; Norris et al., 2013; Vera et al., 2013; It has been postulated that it is important to know the
Kaksonen et al., 2014; Wheaton et al., 2015; Zhuang copper resistance mechanisms of bioleaching microor-
et al., 2015; Ca rdenas et al., 2016; Harrison, 2016; ganisms in order to construct strains with high copper
Hedrich and Schippers, 2016). They cover bacteria and tolerance by using genetic modification (Wen et al.,
archaea involved in biohydrometallurgy, and describe 2014). Transformation of some biomining microorgan-
the new findings obtained by using Omics techniques, isms has been possible. Gene knockout systems were
such as genomic DNA sequencing of single species or previously reported in the case of A. ferrooxidans and
metagenomic sequencing of the microbial communities A. caldus (Liu et al., 2000; Van Zyl et al., 2008). These
involved, proteomics and metaproteomics and metabolo- transformations were performed by using a method
mics (Martınez et al., 2015). The recently published book based on marker exchange mutagenesis. Later on, an
‘Acidophiles: Life in Extremely Acidic Environments’ improved method of markerless gene replacement was
(Quatrini and Johnson, 2016) is also a reference of great established for A. ferrooxidans (Wang et al., 2012).
interest as most of its chapters are related to aspects However, the reported procedures are difficult to repro-
important for bioleaching. duce, very laborious and have very low efficiencies.
A mini-review related to several metals resistance in Recently, a versatile and efficient markerless gene
acidophilic microorganisms has been published before knockout system for A. thiooxidans was reported and
(Dopson and Holmes, 2014). In the present review, we used to characterize a copper tolerance related multicop-
concentrate in critically analysing the most recent find- per oxidase (MCO) gene in this microorganism (Wen
ings related to copper resistance and its importance in et al., 2014).
copper sulfides bioleaching. Instead, some authors consider that the engineering
constraints of all forms of biomining operations (including
stirred tanks) means that it is not possible to prevent the
Microorganisms and bioleaching communities
release of microorganisms used in these processes to
The most studied leaching bacteria are from the the environment (Johnson, 2014). An alternative to this
Acidithiobacillia class. Acidithiobacillus ferrooxidans and situation is the selection of microorganisms from
Copper resistance in biohydrometallurgy 3
biomining areas that possess a better natural copper bioremediation in industrial processes (Jerez, 2013;
resistance together with a higher capacity to bioleach Martınez et al., 2015).
mineral sulfides. A. ferrooxidans strain ATCC 53993
might be an example of this kind of bacteria, as it is able
Copper resistance mechanisms
to resist high copper concentrations (10–12 g L 1 range)
(Orellana and Jerez, 2011) and capable of oxidizing both Acid-leaching solutions are characterized by concentra-
reduced inorganic sulfur compounds (RISCs) and ferrous tions of base and transition metals that are toxic to most
iron. In the case of A. thiooxidans and A. caldus, as they living organisms, and as might be expected, microorgan-
oxidize only RISCs, an additional strain such as Lep- isms that grow in mineral-rich environments are, in most
tospirillum ferrooxidans or a similar ferrous iron oxidizer cases, remarkably tolerant to a wide range of metal ions
would be required for suitable bioleaching of metal sul- and should possess robust metal resistance mecha-
fides. Some biomining companies such as BioSigma, nisms (Orell et al., 2012; Dopson and Holmes, 2014).
S.A. are currently using this approach and have isolated Biomining bacteria and archaea resist high levels of
native species from copper mines such as A. ferrooxi- copper (100–300 mM range) by using a few ‘canonical
dans strain Wenelen (DSM 16786) and others with systems’ such as active efflux or trapping of the metal
increased activity for copper leaching from mixed sulfide ions by metal chaperones. Nonetheless, gene duplica-
ores and high copper resistance (higher than 10 g L 1 tions, the presence of genomic islands (GI), the exis-
compared with 5 g L 1 of the type A. ferrooxidans ATCC tence of additional strategies such as passive
23270 strain) for their use in industrial processes (Sugio mechanisms for pH and cations homeostasis in aci-
et al., 2009; Latorre et al., 2016). dophiles, and an inorganic polyphosphate (polyP)-driven
Proteomics, metaproteomics, genomics and metage- metal resistance mechanism have also been proposed
nomics, together with metabolomics, have been widely (Chi et al., 2007; Orell et al., 2010, 2012; Orellana and
used in recent years to study the global regulatory Jerez, 2011; Navarro et al., 2013; Dopson and Holmes,
responses including metal resistance systems of all 2014; Wen et al., 2014; Wheaton et al., 2015). Next a
kinds of cells individually or in complex microbial com- selection of the most studied biomining microorganisms
munities (Gillan, 2016). By this approach it may be pos- is analysed regarding their copper resistance capacity.
sible to explore the new properties of biomining
microorganisms that arise from the interplay of genes, Copper resistance of biomining bacteria
proteins, other macromolecules, small molecules and the
Acidithiobacillus ferrooxidans
environment. These procedures enable to build meta-
bolic models, predict microbial interactions, defining Acidithiobacillus ferrooxidans ATCC 23270 can survive
genetic diversity and study microbial evolution. Some of under high copper concentrations. It does so by carrying
the findings obtained by using these molecular proce- in its genome more than 10 genes related to copper
dures for the study of biomining microorganisms and homeostasis in other bacteria (Rensing and Grass,
their metal resistance have been recently reviewed 2003; Nikaido, 2011). Three of these genes code for
(Martınez et al., 2015; Cardenas et al., 2016). Genomics putative ATPases related to the transport of copper
has been of great importance to define the biodiversity (copA1Af, copA2Af and copBAf), three other genes are
of biomining processes and has allowed developing both related to a system of the RND family, involved in the
conceptual and metabolic models for some of the aci- extraction of copper from the cell by using the proton
dophilic microorganisms involved. By using metabolo- motive force (PMF; cusAAf, cusBAf, cusCAf) and two
mics, some specific metabolites may be used as additional genes code for periplasmic chaperones for
markers to follow the evolution in time of industrial this metal (cusFAf and copCAf) (Navarro et al., 2009).
bioleaching operations (Martınez et al., 2013). On the These A. ferrooxidans copper resistance determinants
other hand, proteomics has been useful to study were found to be upregulated when this bacterium was
microorganisms–mineral interactions and biofilm forma- exposed to CuSO4 in the range of 5–25 mM and con-
tion (Martınez et al., 2015), including copper resistance ferred a greater resistance to copper when expressed in
as described in the next section. Escherichia coli compared with wild-type cells, support-
Global changes in gene expression and metabolite ing their functionality (Navarro et al., 2009).
levels allow exploring both cannonical and new possible Acidithiobacillus ferrooxidans and other acidophiles
mechanisms of metal resistance and also likely unknown live at an acid external pH (1–3) and their cytoplasmic
passive or active metal tolerance systems. It is expected pH is up to 5 units higher than the external pH. This
that a detailed knowledge of the responses that these generates an elevated pH gradient across the cytoplas-
environmental microorganisms use to adapt to their mic membrane that contributes to the PMF comprising
harsh niche will help to improve biomining and metal the membrane potential (DΨ) and the transmembrane
pH difference (DpH) (Baker-Austin and Dopson, 2007). Other of the ORFs found in the GI of A. ferrooxidans
The RND-type transporters are antiporters taking advan- ATCC 53993 corresponded to members of a putative
tage of the protons gradient for the efflux of copper with Cus system (cusA3Af, cusB3Af and cusC3Af). Two addi-
protons entrance to the cytoplasm. Due to its economy tional possible CusF-like chaperones previously
from the energetic point of view, the acidophilic microor- described in A. ferrooxidans ATCC 23270 (Navarro
ganisms would preferentially use these systems to et al., 2009) were found in this GI (Orellana and Jerez,
remove intracellular copper. A possible cytoplasmic acid- 2011). A comparison of the possible copper resistance
ification would be expected to take place if the microor- genes between these two A. ferrooxidans strains and
ganisms would excessively use these efflux pumps in several other selected biomining microorganisms is
the presence of high metal concentration. However, this shown in Figure 1.
acidification could be diminished by the energetic meta- Acidithiobacillus ferrooxidans ATCC 53993 has a
bolism of the bacterium, as the oxidation of Fe(II) or much higher resistance to CuSO4 (>100 mM) than that
reduced sulfur compounds by molecular oxygen as the of strain ATCC 23270 (<40 mM) (Orellana and Jerez,
final electron acceptor consumes protons. As the RND 2011). As it occurs in Cupriavidus metallidurans (Van
systems are introducing protons from the culture medium Houdt et al., 2009), it was expected that the additional
to the cell during copper detoxification, an increase in multiplicity of possible copper resistance determinants in
the extracellular pH of the growth medium would be the GI of A. ferrooxidans ATCC 53993 would confer it a
expected in the presence of this metal. higher tolerance to the metal compared with strain ATCC
The genomic sequence of A. ferrooxidans ATCC 23270. Although both strains have a similar growth in
53993 contains all the copper resistance genes present the absence of copper, cell numbers of ATCC 23270
in A. ferrooxidans ATCC 23270 that have been experi- were reduced by sevenfold when grown in 100 mM
mentally confirmed as being expressed in the presence CuSO4, whereas those of ATCC 53993 were diminished
of copper, except that strain ATCC 23270 contains a by only twofold (Orellana and Jerez, 2011; Jerez, 2013).
second Cus-like operon interrupted by a transposase This additional capacity of strain ATCC 53993 to tolerate
gene which is absent in the genome of strain ATCC copper should confer it an adaptational advantage when
53993 (Luo et al., 2008; Navarro et al., 2009; Orell growing in a microbial consortium such as the one usu-
et al., 2010; Orellana and Jerez, 2011). These ORFs ally found in their habitat. If these two A. ferrooxidans
have 100% identity between their corresponding DNA strains were present in a bioleaching operation such as
sequences. However, A. ferrooxidans ATCC 53993 con- a heap or more likely in an industrial biooxidation reac-
tains several additional putative metal resistance ORFs tor, as copper concentration increases with the dissolu-
that confer it a higher metal resistance (Orellana and tion of the mineral, at the end of the operation strain
Jerez, 2011). These putative genes are clustered in a ATCC 53993 should predominate over ATCC 23270. A
160 kb GI that is absent in the genome of A. ferrooxi- preliminary test for this idea was carried out by growing
dans strain ATCC 23270 (Ca rdenas et al., 2010; Orell a mixture of approximately equivalent numbers of each
et al., 2010). The GI in A. ferrooxidans ATCC 53993 strain in the presence of copper. The relative proportions
contains genes encoding heavy metal resistance deter- of each kind of bacterium were estimated by means of
minants for copper, mercury detoxification and volatiliza- qPCR by using strain-specific primers. The results
tion, and the transcriptional regulator (merR), as well as obtained clearly indicated that A. ferrooxidans ATCC
a copper translocating P-type ATPase. On the other 53993 was able to outgrow strain ATCC 23270 in the
hand, strain ATCC 23270 has a 300 kb region that does presence of 50 mM CuSO4 (Orellana and Jerez, 2011;
not contain known metal resistance-related genes and is Jerez, 2013).
absent in the genome of strain ATCC 53993 (Ca rdenas Moreover, an upregulation of the transcriptional
et al., 2010; Bustamante et al., 2012). Obviously, the expression of most of the additional Cu resistance genes
presence of unknown metal resistance genes in this present in the GI of A. ferrooxidans ATCC 53993 was
300 kb GI cannot be excluded at present. observed when cells were grown in the presence of
Six ORFs possibly related to Cu resistance are pre- increasing CuSO4 concentrations. In addition, these
sent in the GI of strain ATCC 53993, including a P-type genes were functional when expressed in E. coli,
ATPase annotated as a possible Cu resistance determi- strongly supporting the functionality of the copper resis-
nant or copA3Af (Orellana and Jerez, 2011). All Cop-like tance determinants present in the GI of A. ferrooxidans
proteins found so far in A. ferrooxidans, including ATCC 53993 (Orellana and Jerez, 2011). These findings
CopA3Af contain the metal-binding motifs that are highly constituted the first experimental evidence for high cop-
conserved in Cop proteins from other microorganisms per resistance due to the expression of genes present in
(Orell et al., 2010). a GI of an acidophilic chemolithoautotrophic bacterium.
Fig. 1. Comparison of the main copper resistance determinants present in a selection of acidophilic bacteria and archaea usually found in bio-
mining environments. All the microorganisms chosen have their genomes sequenced and are publically available. The proteins selected for
comparison are those for which at least some experimental evidence as copper resistance determinants exists. Symbols: ( ) Gram negative;
(+) Gram positive; (C) Crenarchaeota; (E) Euryarchaeota. The Blastp program was used to align protein sequences by means of BLOSUM62
substitution matrix. The colours scale indicate the percentage of the obtained scores related to the maximum score value obtained. The
entrance protein sequences are in each column of the table and when more than one protein with a significant score was present in the same
microorganism the highest score was taken in each case. Af, Acidithiobacillus ferrooxidans; E. hi, Enteroccoccus hirae.
Interestingly, when A. ferrooxidans strains ATCC proteins changed in the former strain vs 120 proteins in
53993 vs ATCC 23270 subjected to 40 mM copper were the latter one (Martınez-Bussenius et al., 2016). This
analysed by differential quantitative proteomics, only 49 greatly diminished response to the metal is in good
agreement with the much higher copper resistance of By using high-throughput quantitative ICPL pro-
A. ferrooxidans ATCC 53993 already described. These teomics, it was found that A. ferrooxidans ATCC 23270
inter-population interactions may not only lead to grown in ferrous iron or elemental sulfur and in the pres-
changes in the capacities of the bacteria to adapt to their ence of 40 mM of copper sulfate changes the expres-
environment but may also help to select the more suit- sion of several proteins related to copper response, such
able microorganisms to enhance industrial biomining as the upregulation of a Cus system (Alma rcegui et al.,
operations. 2014a,b), confirming the previously reported transcrip-
Genomic strain variations have also been found in the tional analysis (Navarro et al., 2009). Furthermore, the
acidophilic Leptospirillum group II by deep metagenomic upregulation (almost 15-fold) of a protein coded by
genome sequence analysis (Simmons et al., 2008). It is AFE_1862, annotated in the genome of A. ferrooxidans
possible that horizontal gene transfer between A. fer- ATCC 23270 as a putative heavy metal-binding protein
rooxidans strains and other microorganisms are key ele- were also detected by the proteomic approach (Alma rce-
ments to supplement metal resistance and possibly gui et al., 2014b). This protein contains a possible heavy
other properties, thus conferring adaptational advantages metal-binding domain (MXCXXC) similar to the one
to all these microorganisms. found in CopZ-like proteins.
One of the most abundant proteins in ferrous iron- The possible role of Rus and AcoP periplasmic cupre-
grown A. ferrooxidans is the cupredoxin rusticyanin doxins and of the putative cytoplasmic CopZ in copper
(Rus) as it occupies 21% of the total volume in the resistance in A. ferrooxidans was recently reported
periplasmic space (Li et al., 2015). This periplasmic (Navarro et al., 2016). Although CopZ is a known cyto-
component forms part of an iron-oxidizing/oxygen-redu- plasmic copper chaperone that binds and delivers cop-
cing supercomplex spanning both inner and outer mem- per to CopA in B. subtilis (Radford et al., 2003), the
branes in A. ferrooxidans (Yarza val et al., 2004; Castelle CopZ-like protein of A. ferrooxidans ATCC 23270 is the
et al., 2008). Rus and other components of the rus first cytoplasmic copper chaperone described in a bio-
operon have also been found expressed at lower levels mining bacterium with a role in copper resistance. Like-
in sulfur-grown cells (Ramırez et al., 2004; Yarza val wise, Rus and AcoP cupredoxins apparently function not
et al., 2004). However, the role for Rus in sulfur-grown only as components of the ferrous iron oxidation path-
cells is still not clearly defined (Yarza val et al., 2004). way in this acidophilic microorganism but they may also
Thus, the overexpression of Rus in sulfur-grown cells bind excess copper in the periplasm, most likely playing
exposed to copper supports the idea that this copper- a role in the considerably high copper resistance of
binding protein and possibly other components of the rus A. ferrooxidans (Navarro et al., 2016).
operon may have an additional role in A. ferrooxidans An increased time of growth has been observed for
copper resistance, as previously suggested (Felıcio ferrous iron-grown A. ferrooxidans cells in the presence
et al., 2003), and recently supported by Alma rcegui et al. of copper, suggesting that respiration is affected and the
(2014a). In this regard, the analysis of the A. ferrooxi- oxidation of ferrous iron occurs more slowly in the pres-
dans periplasm by high-throughput proteomics including ence of the metal (Alma rcegui et al., 2014b). It is well
high-resolution linear ion trap-FT MS allowed the identifi- documented in the literature that ferrous iron oxidation
cation of 131 periplasmic proteins (Chi et al., 2007). decreases in several A. ferrooxidans strains when cop-
Among these proteins, several were uncharacterized, per concentration increases (Leduc et al., 1997). The
including AFE_3151. This protein was later proposed to results recently reported by Navarro et al., 2016 showed
be part of the iron-oxidizing/O2-reducing system in A. fer- that the two proteins AcoP and Rus are upregulated in
rooxidans although its role has not been defined so far A. ferrooxidans cells in the presence of copper, suggest-
(Castelle et al., 2008, 2010). This periplasmic protein, ing they might have an additional role in copper toler-
now named AcoP, has recently been characterized and ance. Rus could be eventually saturated with the bound
it also contains a cupredoxin type copper-binding site copper. However, the extremely high concentration of
that binds one Cu atom per molecule and likewise forms Rus is in the range of copper sulfate that could be pre-
part of the rus operon and iron respiratory system sent under the usual growth condition for this acidophile.
(Roger et al., 2014). Furthermore, the levels of AcoP Rus copper-trapping would be only one of many other
also increased in sulfur-grown cells in the presence of components that respond to the presence of the toxic
copper (Alma rcegui et al., 2014a). Rus and AcoP have metal in the cells.
conserved copper-binding sites (HXCX2-4HX4M and It is known that in aerobic systems Cu(II) supplied is
HXCX4-8HX4M respectively) (Roger et al., 2014) such as reduced to Cu(I) upon contact with the respiratory chain
those described in CopC, a cupredoxin-like copper-bind- of bacteria (Thauer et al., 1977; Volentini et al., 2011).
ing protein from P. syringae involved in copper home- Almost certainly reduced quinones are the electron
ostasis (Arnesano et al., 2002). donors. As Cu(I) is much more toxic than Cu(II), it could
be exported from the periplasm to the outside by Cus- A working model including the possible role of Rus
like RND systems or oxidized back to Cu(II) by CueO/ and possibly AcoP in copper resistance is shown in
PcoA/CopA-like periplasmic copper-containing copper Fig. 2A. Obviously much more experimental work will be
oxidases. A. ferrooxidans has Cus-like RND systems required to support these proposals. Not all biomining
(Navarro et al., 2009) but it apparently lacks genes cod- microorganisms contain Rus (Fig. 1), but in those con-
ing for periplasmic copper-containing copper oxidases. A taining Rus-like proteins coding genes, such as A. fer-
new interesting speculative possibility for this acidophile rivorans, a similar role for this copper-binding protein
to prevent Cu(I) extreme toxicity would be to transfer an could be possible, especially considering that they pos-
electron from Cu(I) to the iron oxidation system (Navarro sess the same copper-binding site of type I found in
et al., 2016). For this it would be necessary that ApoRus A. ferrooxidans Rus. On the other hand, in sulfur-oxidiz-
and/or apoAcoP are able to bind Cu(I) at the periplasm. ing microorganisms such as A. thiooxidans and A. cal-
In this connection, it is known that folded aporusticyanin dus that lack rus genes (Fig. 1) and are less resistant to
rapidly binds Cu(I) with much higher affinity than Cu(II) copper compared with A. ferrooxidans, different compo-
(Alcaraz et al., 2007). As proposed by Li et al. (2015), nents for copper resistance may be expected.
the concentrated Rus in the periplasm would constitute a The copper-responsive protein complement of iron-
network where inter-protein electron transfer interactions grown A. ferrooxidans ATCC 23270 cells was recently
across the periplasmic space would effectively function evaluated by using ICPL proteomics (Alma rcegui et al.,
in connection with the respirasome. As previously sug- 2014b). In addition of the expected upregulation of RND-
gested, it is not unthinkable that this copper-binding pro- type Cus systems, some different RND-type efflux
tein may also constitute in part a barrier to trap excess pumps were overexpressed together with the upregula-
copper in a microorganism normally confronted with high tion of several genes involved in histidine and cysteine
concentrations of this metal (Navarro et al., 2016). synthesis. In response to copper, a downregulation of
Fig. 2. Summary working model of copper resistance determinants in biomining bacteria (A) and archaea (B). Dark grey, proteins that increase
their synthesis levels in cells subjected to copper; light grey those down expressed in the presence of copper.
the major outer membrane porin (Guiliani and Jerez, A. ferrivorans SS3 is the protein with the highest identity
2000) and some ionic transporters was observed in compared with Rus from A. ferroxidans and it forms part
A. ferrooxidans ATCC 23270 cells grown in ferrous iron of the rus operon from A. ferrivorans together with the
or sulfur (Almarcegui et al., 2014a,b). This result is an gene coding for the AcoP protein (Liljeqvist et al., 2013).
example of how a downregulation of outer membrane In the light of the recent report suggesting that Rus and
porins could cause a general decrease in the influx of AcoP are copper resistance determinants in A. ferrooxi-
the metal into the cell. dans ATCC 23270 (Navarro et al., 2016), it will be of
The native A. ferrooxidans strain Wenelen (DSM interest to study whether these proteins have a similar
16786) isolated from an industrial copper biomining oper- role in copper resistance in A. ferrivorans.
ation in Chile by Biosigma S.A. was recently reported to Gonza lez et al. (2014) reported the presence of a
have an extreme capacity to resist high concentration of ppk2 in a GI of A. ferrivorans SS3. The protein compar-
copper (over 10 g L 1) compared with the type strain isons shown in Fig. 1 support the presence of this PPK2
ATCC 23270 (5 g L 1). Furthermore, the global tran- (with 89% Blastp score) and also indicate that A. ferrivo-
scriptional response of this native strain showed the rans SS3 contains a PPX protein (85% Blastp score),
upregulation of several genes related with copper toler- suggesting that the polyP-dependent copper resistance
ance when the bacterium was grown in the presence of system may also be operative in this psychrotolerant
pyrite and CuFeS2 (Latorre et al., 2016). bacterium.
Current knowledge indicates that in addition to the key It is also important to consider that not only a higher
elements involved in A. ferrooxidans copper resistance presence of genes related to metal resistance is the only
already discussed, an abundant reserve of inorganic factor important for extreme metal tolerance but also
polyP used as a polyP-based copper resistance system metal speciation and pH are also important to consider
and a robust defensive response to oxidative stress are (Gonza lez et al., 2014). In addition, differences in cell
also important (Alvarez and Jerez, 2004; Remonsellez envelope components also may influence metal resis-
et al., 2006; Orell et al., 2010). Poly P is synthesized in tance in bacteria (Harrison et al., 2007; Alma rcegui
bacteria by a polyphosphate kinase (PPK) and degraded et al., 2014a).
by a polyphosphatase (PPX) (Kornberg et al., 1999).
The presence of copper in the cytoplasm would activate
Acidithiobacillus thiooxidans
PPX, which would degrade polyP to generate free phos-
phate (see Fig. 2). This phosphate can bind copper to Studies on copper tolerance mechanisms in A. thiooxi-
be eliminated to the periplasm or the extracellular med- dans have also been approached recently (Wen et al.,
ium by means of phosphate transporters such as PitA 2014). A putative MCO gene (cueO) was annotated in
as proposed before for E. coli (Keasling, 1997; Van Dien the genome of A. thiooxidans ATCC 19377 (Valdes
et al., 1997) and recently demonstrated for E. coli by et al., 2011). This gene is also present in Acidithiobacil-
Grillo-Puertas et al. (2014). Figure 2A summarizes a lus caldus ATCC 51756, Thiomonas arsenitoxydans and
general working model for copper resistance in less likely in Alicyclobacillus acidocaldarius LAA1 but is
A. ferrooxidans and other acidophilic bacteria. absent in A. ferrooxidans and all the other microorgan-
isms listed in Fig. 1. The MCO has been studied in detail
in E. coli (Grass and Rensing, 2001; Roberts et al.,
Acidithiobacillus ferrivorans
2002; Hoegger et al., 2006). The transcriptional level of
As previously described by Gonza lez et al. (2014), A. thiooxidans cueO was upregulated in response to
A. ferrivorans SS3 genome contains four possible cus 10 mM copper sulfate and a response to copper of a
clusters, three of them cusCBAF type and a fourth one putative cueO promoter was detected, suggesting it
of cusCBA type. All A. ferrivorans SS3 putative Cus sys- might be stimulated by a putative CueR protein (Wen
tems showed a high score of identity to the A. ferrooxi- et al., 2014). By using a modified markerless gene dis-
dans system (over 75% Blastp score). Furthermore, ruption system, these authors generated a cueO mutant
upstream of the genomic context of two of the Cus sys- of A. thiooxidans. This mutant was more sensitive to
tems of A. ferrivorans SS3, there is a putative copper external copper and this activity could be restored after
ATPase (Fig. 3) with 95% Blastp score with CopA from complementing the cueO gene, strongly suggesting the
A. ferrooxidans (Fig. 1). involvement of cueO in copper tolerance of this bac-
The A. ferrivorans SS3 genome contains two addi- terium (Wen et al., 2014).
tional copies of the rus gene (Liljeqvist et al., 2013) and Figure 1 shows that A. thiooxidans contains several
its three Rus proteins not only have a high Blastp score genes coding for proteins with a high identity Blastp
(>85%) to Rus from A. ferrooxidans but all of them also score (>70%) with those of the CusCBA systems from
conserve the same copper-binding site. RusA from A. ferrooxidans. Furthermore, A. thiooxidans may have
Fig. 3. Gene clusters of some copper resistance determinants as predicted by bioinformatics analysis of the indicated selected microorganisms
genome sequences. ct, cation transporter; hypr, hypothetical protein; hydC, cytochrome b561; pyk, pyruvate kinase C; merR, transcriptional
activators; pstS, phosphate ABC transporter substrate-binding protein; pstA, phosphate ABC transporter, permease protein; pstB, phosphate
ABC transporter ATP-binding protein; phoU, phosphate transport system regulatory; grx, glutaredoxins; mp, membrane protein; chlP, geranyl-
geranyl reductase; murl, glutamate racemase.
two Cus systems with similar genetic contexts and the metal-binding domains (HMA) and an ATPase E1-E2
characteristic domains of the previously characterized domain, which could be related with the incoming and
Cus systems (Fig. 3). exporting of copper.
Acidithiobacillus thiooxidans also showed a protein Moreover, Fig. 1 indicates that A. thiooxidans genome
with a high Blastp score (>65%) with CopB from A. fer- has a gene coding for a protein similar to the copper
rooxidans (Fig. 1). This putative copper ATPase shares chaperone CopZ from B. subtilis. The A. thiooxidans
several domains with CopBAf such as two heavy protein contains the same heavy metal-binding domain
present in CopZB. subtilis with conserved copper-binding (CusF), which has been described in E. coli (Loftin et al.,
amino acids (MXCXXC). In addition, the bioinformatics 2005) and in A. ferrooxidans ATCC strains 23270 and
analysis of its subcellular location predicted it to be a 53993 (Orell et al., 2010; Orellana and Jerez, 2011).
putative cytoplasmic copper chaperone, suggesting it The search in A. caldus of possible copper resistance
could be an A. thiooxidans CopZ. Upstream of the gene determinants similar to those of the CusCBAF system
coding for this protein, there is a putative heavy metals from A. ferrooxidans showed possible homologue pro-
transcriptional regulator that could regulate the expres- teins although in general they had 40–60% Blastp score
sion of this possible A. thiooxidans cytoplasmic metal (Fig. 1). These proteins possess the characteristic struc-
chaperone (Fig. 3). tural domains of these efflux systems and their corre-
As Fig. 1 shows, A. thiooxidans has a high Blastp sponding genes are contiguous in the genome (Fig. 3).
(>78%) with the genes coding for PPK and PPX from Most likely, they could be related to the transport of
A. ferrooxidans and they share basically the same struc- heavy metals such as copper, zinc or cadmium.
tural domains. Considering that A. thiooxidans accumu- In addition, A. caldus showed two ATPases with cop-
lates polyP granules as seen by TEM (Orell et al., per-binding sites (Interpro IPR006122) and with a high
2010), it is possible that a functional polyP-dependant Blastp score (>50%) when compared with CopB1 from
metal resistance system similar to that previously pro- A. ferrooxidans. Interestingly, a possible cytoplasmic
posed for A. ferrooxidans (Alvarez and Jerez, 2004) is copper chaperone with high similarity to CopZB. subtilis
also present in A. thiooxidans. was present in the same DNA region, close the two
The genome sequence of an A. thiooxidans Licanan- ATPases already mentioned (Fig. 3). Figure 3 also
tay strain isolated from a biomining operation suggested shows a glutaredoxin in the context of one of the
the presence of GIs (Travisany et al., 2014) as it has ATPases that could be related to maintain the redox
been previously reported for A. ferrooxidans ATCC state of the sulfhydryl groups present in cellular proteins
53993 strain (Orellana and Jerez, 2011) and for A. fer- damaged by the presence of copper (Alma rcegui et al.,
rivorans SS3 with a predicted GI in its genome 2014b).
(Gonza lez et al., 2014). Most likely, horizontal gene Figure 1 shows the possible presence of a MCO in
transfer in mining environments probably generates A. caldus. This putative protein contains the same
these genomic elements. domains described for E. coli CueO as noted before
(Navarro et al., 2013).
Although a small score (5–10%) indicated the possible
Acidithiobacillus caldus
presence of Rus and AcoP in A. caldus (Fig. 1), a
Regarding copper tolerance of A. caldus, a copper MIC detailed analysis of the amino acid sequences of these
of 24 mM has been reported for strain DSM8584 (Watkin two putative periplasmic proteins indicated that none of
et al., 2008). The response to copper stress of an A. cal- them have the domains and copper-binding sites charac-
dus strain isolated in China has been analysed (Xia teristic of the respirasome-related proteins from A. fer-
et al., 2010). The authors found a negative effect on the rooxidans.
activity of some of the enzymes involved in sulfur meta- As seen in Fig. 1, A. caldus shows the likely presence
bolism, such as sulfite oxidase and APS reductase. Nev- of PPX and PPK similar to those from A. ferrooxidans
ertheless, no direct evidence was reported to support (Blastp score >70%), both involved in polyP metabolism
the idea of overexpression of a Cu-specific inducible and the polyP-related metal resistance system reported
ATPase pump; the authors speculated that would be before for A. ferrooxidans (Alvarez and Jerez, 2004;
present under their conditions of study. Orell et al., 2010). It has been reported that A. caldus
Acidithiobacillus caldus ATCC 51756 (Mangold et al., accumulates polyP granules as many other acidophilic
2013) (see Fig. 1) and strain SM-1 (You et al., 2011) microorganisms with high metal resistance (Orell et al.,
contain a variety of genes coding for putative copper 2012). Furthermore, both A. ferrooxidans and A. caldus
resistance-related proteins as they showed a great have a putative Pho regulon, which includes ppx in its
degree of identity (between 50% and 90%) with those genomic context (Vera et al., 2003; Mangold et al.,
known for A. ferrooxidans (Navarro et al., 2009; Orellana 2013; Navarro et al., 2013). It is possible then that both
and Jerez, 2011). These two strains of A. caldus share acidophilic bacteria would respond in a similar way to
several of these genes in their chromosomal DNA, such the presence of copper (Navarro et al., 2013).
as the components of an RND-type efflux pump
(CusABC), two P-type ATPases (CopB), two cytoplasmic
Leptospirillum ferriphilum
metalochaperones (CopZ) and a copper oxidase (CueO)
(see Fig. 1). Interestingly, strain SM-1 contains a gene Leptospirillum ferriphilum ML-04 has some identity (20
coding for a putative periplasmic copper chaperone to 50% depending on the Cus protein) with a
CusCBAF system from A. ferrooxidans (Fig. 1) and

Acidimicrobium ferrooxidans
two copies of this system are present in its genome.
As seen in Fig. 3, both Cus systems have a genetic The bioinformatics analysis in Fig. 1 indicates that A. fer-
context similar to that described for E. coli, except that rooxidans DSM 10331 has a possible copper ATPase
CusF is neighbour of CusA. Nevertheless, all of the with a BlastP score higher than 20% when compared
components of these putative Cus systems contain the with CopA1 from A. ferrooxidans. Both proteins share
characteristic domains of these metal efflux pumps. On several important domains, such as the metal-binding
the other hand, a copper ATPase with some identity domain. Interestingly, next to the gene for this putative
to CopAAf and a gene coding for a protein with 30% ATPase the gene coding for a putative small cytoplasmic
identity to CopZB. subtilis were also found. However, this chaperone similar to CopZ with a copper-binding site
last protein would rather correspond to a mercury (MXCXXC) characteristic of this protein was found by
reductase and it contains a metal-binding domain simi- using the CELLO v.2.5 program (Fig. 3). Being a Gram
lar to that present in CopZ. Finally a putative PPX positive, this bacterium does not contain genes with
coding gene was found in L. ferriphilum but in a differ- identity to the Cus system of A. ferrooxidans in Fig. 1.
ent genomic context. In addition, a putative PPK2 was Acidimicrobium ferrooxidans also has genes coding for
also found in this bacterium but with a low score when putative PPX and PPK both possessing the characteris-
compared with the equivalent enzyme from A. ferrooxi- tic domains of these enzymes. Although the presence of
dans. Without current information of the existence of inorganic polyP has not been reported in this bacterium,
polyP in L. ferriphilum and the low identity score found most likely it exists as it occurs in other biomining bacte-
for both possible enzymes involved in polyP metabo- ria to play an important role for oxidative stress response
lism, at present it is not possible to speculate on their and metal resistance (Orell et al., 2010; Navarro et al.,
putative role in metal resistance in this bacterium. 2013).
Leptospirillum ferrooxidans Sulfobacillus thermosulfidooxidans
The iron oxidizer L. ferrooxidans shows several putative This Gram (+) bacterium is not expected to have a com-
copper resistance determinants similar to those in A. fer- plete Cus system. Figure 1 shows the gene for one pro-
rooxidans (Fig. 1). However, its iron-oxidizing system tein with a low identity to CusA1 from A. ferrooxidans
lacks rusticyanin (Blake et al., 1993). As seen in Fig. 1, and no other possible components of a Cus system.
the iron oxidizer L. ferrooxidans contains a possible However, this possible protein does not contain any of
CusCBAF in which its CusA has a Blastp score over the domains present in CusA1. In contrast, S. thermosul-
50% when compared with CusA1 from A. ferrooxidans. fidooxidans showed four possible ATPases with similar-
In addition, the genetic context of the L. ferrooxidans is ity to CopA1 and CopB1 proteins from A. ferrooxidans.
in general similar to those described for these Cus sys- All these putative copper ATPases possess metal bind-
tems in other bacteria (Fig. 3). Compared with L. fer- ing (HMA), E1-E2_ATPase and ZntA domains, all of
riphilum, L. ferrooxidans has only one Cus system. In them important for the function of the ATPases and
addition, L. ferrooxidans genome contains a possible which also form part of CopAAf and CopBAf. As seen in
metal ATPase with a Blastp score higher than 20% other bacteria (Radford et al., 2003; Navarro et al.,
when compared with A. ferrooxidans CopA. Although 2016), one of these ATPAses is found contiguous to a
this is not a very high score value, this protein conserves putative CopZ having an MXCXXC copper-binding site
the metal binding (HMA), E1-E2_ATPase and ZntA characteristic of these cytoplasmic chaperones (Fig. 3).
domains typical of CopA. As seen in Fig. 1, S. thermosulfidooxidans has a gene
Regarding the possible existence of a polyP-depen- coding for a protein with 20–30% Blastp score compared
dent metal resistance system in L. ferrooxidans, the bac- with AcoP from A. ferrooxidans. There are three copies
terium showed two proteins with a low Blastp score of this gene coding for a protein that contains a cupre-
(over 10%) compared with the A. ferrooxidans PPX. doxin domain to bind copper. However, although these
These two possible PPX enzymes have the Ppx/GppA cupredoxin protein genes are neighbours of cyto-
(IPR003695) domain characteristic of these proteins. On chromes, no rus gene is present in this Gram (+) iron
the contrary, no PPK similar proteins were found. This oxidizer (Blake et al., 1993).
evidence and the lack of information for the presence of Finally, as seen in Fig. 1, clearly Gram (+) bacteria do
polyP do not allow us to suggest the existence of a not contain the proteins related to the Cus systems due
metal resistance system dependent on polyP in this to their different envelope structure compared with the
microorganism. Gram ( ) bacteria. On the other hand, most Gram (+)
bacteria, as expected do not show the presence of The genome of Ferroplasma acidarmanus contains a
periplasmic Rus. cop operon (copYZB) with similar structure and response
to copper as for copTMA (Baker-Austin et al., 2005). All
the putative ATPases identified in the genomes of F. aci-
Copper tolerance of biomining Archaea
darmanus, T. acidophilum, T. volcanium, P. torridus and
Concerning archaeal copper resistance mechanisms, a A. cupricumulans in Fig. 1 contain TRASH domains
few metal efflux pumps have been identified from (data not shown). On the other hand, all the possible
sequenced genomes of some members of this domain CopA found in archaeal genomes in Fig. 1 contained
(Pedone et al., 2004; Wheaton et al., 2015; see Fig. 1). YHS or HMA metal-binding domains (data not shown).
A Cu resistance (cop) loci has been described in In silico studies have further identified a CPx-ATPase,
Archaea, which includes genes encoding for a new type which most likely mediates the efflux of heavy metal
of archaeal transcriptional regulator (CopT), a putative cations in the biomining archaeon M. sedula (Auernik
metal-binding chaperone (CopM) and a putative et al., 2008). This putative protein has significant identity
Cu-transporting P-type ATPase (CopA) (Ettema et al., to a P-type ATPase from S. solfataricus (CopA) (Ettema
2006) (see Fig. 2B). et al., 2006). Moreover, M. sedula contains ORFs with
Single unit, membrane class 1B heavy metal translo- significant similarity to both CopM (Msed0491) and CopT
cating P-type ATPases are involved in homeostasis and (Msed0492) from S. solfataricus (Auernik et al., 2008).
resistance to copper as well as to other metals (Palm- Maezato et al. (2012) reported a genetic approach to
gren and Nissen, 2011). Although Sulfolobus solfataricus investigate the specific relationship between metal resis-
is not a biomining microorganism, it has been consid- tance and lithoautotrophy during biotransformation of
ered a model for archaeal studies. The copTMA operon CuFeS2 by M. sedula. The functional role of its copTMA
from S. solfataricus P2 (i.e., copRTA in S. solfataricus operon was demonstrated by cross-species complemen-
98/2 as reported by Villafane et al., 2009) contains a tation of a Cu-sensitive S. solfataricus copR mutant
copper-responsive regulator (CopT), a copper-binding (Maezato et al., 2012).
protein (CopM) that includes the metal coordinating The previously published genetic context of several
ligands within the so-called TRASH domain (‘Trafficking, archaeal putative CopMA and CopT showed that all of
Resistance and Sensing of Heavy metals’) (Ettema them have the same organization (Ettema et al., 2006;
et al., 2003). Maezato et al., 2012; Orell et al., 2013). Some of the
CopT usually has an HTH (‘helix–turn–helix’) DNA archaea analysed in Fig. 1 also possess the same
binding domain in its N- terminal end and a TRASH genetic organization, indicating that this copper resis-
metal-binding domains in their C-terminal ends. On the tance system is highly conserved in most archaea and it
other hand, both CopA and CopM may have metal-bind- could be one of the most relevant ones.
ing domains such as TRASH, YHS (tyrosine, histidine, Regarding the polyP-based copper resistance mecha-
serine) or HMA (heavy metal-associated) (Gitschier nism, S. metallicus is known to tolerate very high con-
et al., 1998). centrations of copper and accumulates high amounts of
CopA and CopT copper resistance systems are pre- polyP granules. Furthermore, the levels of intracellular
sent in most of the selected archaea as shown in Fig. 1. polyP are greatly decreased when this archaeon is either
In several cases, there was an overlap between CopA grown in 200 mM Cu or shifted to 100 mM Cu and an
and CopM at the genomic level and therefore they are increase in exopolyphosphatase (PPX) activity and Pi
not annotated in the protein databases. For this reason, efflux due to the presence of Cu suggests a metal toler-
only CopA from S. solfataricus was included for compar- ance mechanism mediated through polyP in this
ative purposes in Fig. 1. archaeon (Remonsellez et al., 2006; Orell et al., 2010,
CopA is known to impart copper resistance to S. solfa- 2012).
taricus (Ettema et al., 2006), being an effective copper Acidithiobacillus ferrooxidans (Alvarez and Jerez,
pump at low copper concentrations. Other archaeal aci- 2004) and S. metallicus (Remonsellez et al., 2006)
dophiles also contain versions of the cop operon (Fig. 1). apparently do not contain PitA in their genomes but they
The search for possible CopT transcriptional regulators have Pho84-like phosphate transporters similar to Pho-
in the genomes of the archaea included in Fig. 1 was 84 from Saccharomyces cerevisiae which transports
done by comparison with CopT from S. solfataricus. All metal-phosphate complexes at acid pH (Fristedt et al.,
archaea where CopT was identified showed high amino 1999) (see Fig. 1).
acid sequence identity compared with the S. solfataricus Figure 1 clearly shows that all Chrenarchaoeta anal-
regulator and all of these putative transcriptional regula- ysed had a high Blastp score for their putative PPX
tors contained the TRASH and HTH domains character- when compared with the PPX from S. solfataricus. All of
istic of CopTS. solfataricus. these putative PPX enzymes contain the characteristic
domains of this PPX (data not shown). Interestingly, all had a BlastP score of around 20% when compared with
Euryarchaeota in Fig. 1 do not show a protein with iden- the blue-copper protein Rus from A. ferrooxidans. Other
tity to PPX from S. solfataricus. This is due to the fact microorganisms such as S. metallicus showed a lower
that Euryarchaeota apparently do not have PPX genes identity (10%). All these archaeal proteins have been
identified in their genomes and Crenarchaeota have annotated as possible rusticyanin, having a type I copper-
PPX genes but no genes coding for PPK. As they do binding site and a characteristic cupredoxin domain. In
synthesize polyP (Orell et al. (2012), a different unknown M. sedula, one of these proteins has been suggested to
PPK yet to be identified should be present in these be involved in iron oxidation (Auernik et al., 2008). The
archaeons. proteins from M. sedula, S. acidocaldarius, S. tokodai and
It was recently reported that the wild-type M. sedula S. solfataricus having identity with A. ferrooxidans Rus are
(DSM 5348T) is a pitA mutant, as M. sedula strain sulfocyanins which also have a copper-binding site. The
CuR1, a spontaneous mutant, had a supranormal metal genetic context for the genes coding these proteins is not
resistance and was able to leach copper from CuFeS2 at identical to that of rus gene from A. ferrooxidans but are
an accelerated rate (McCarthy et al., 2014). This mutant close to cytochromes and iron-sulfur protein genes. As
contained a gene orthologue of the bacterial pitA. mentioned before, some experimental evidence supports
M. sedula PitA was a low affinity, high velocity sec- the role of Rus as a possible copper chaperone related
ondary transporter implicated in copper resistance and with copper resistance in A. ferrooxidans (Navarro et al.,
arsenate sensitivity. This archaeal PitA protein would 2016). However, whether some of the putative Rus pro-
then be a key element for the increased metal resistance teins from Fig. 1 other than Rus from A. ferrooxidans are
of CuR1 and its increased capacity to leach copper implicated in copper resistance in other bacteria and
(McCarthy et al., 2014). This archaeal Pit transport sys- archaea clearly remains to be demonstrated.
tem would support increased phosphate efflux and metal Finally, proteomic studies of S. metallicus subjected to
symport through this Pit-like system (see Fig. 2B). Previ- copper, showed 23 downregulated and 30 upregulated
ous studies on metal resistance in S. metallicus pro- proteins, suggesting they are possibly involved in copper
posed that another phosphate transporter (Pho84 like) resistance (Orell et al., 2013). A similar behaviour was
would be present (Remonsellez et al., 2006). Like Pit, also observed in ‘Fp. acidarmanus’ challenged by either
Pho84 belongs to the family of phosphate-proton sym- arsenite or copper (Baker-Austin et al., 2005; Baker-Aus-
porters and the major facilitator superfamily (MFS). As tin and Dopson, 2007). Most of these proteins are
suggested by McCarthy et al. (2014) perhaps PitA and related to the production and conversion of energy,
Pho84 comprise dual phosphate transporters that are amino acids biosynthesis and stress responses. Within
inherent to these thermoacidophilic archaeons and are proteins upregulated in S. metallicus cells exposed to
critical for their extreme metal resistance. copper, a putative ATP synthase subunit B was detected
All archaeons analysed had proteins with around 20% (Orell et al., 2013). When cells are subjected to some
identity to Pho84. Most of these putative proteins are stressing conditions such as the presence of heavy met-
annotated as major facilitator superfamily (MFS) trans- als, a greater cellular demand for energy has been
porters as they contain an MFS domain. In addition, all reported to occur (Baker-Austin et al., 2005).
of them contain a domain annotated as ‘phosphate:H+ Several previous reports have suggested that oxidore-
symporter’. Considering that both domains are present in ductases contribute to an oxidative protection in both
Pho84 and that there is evidence for the presence of bacteria and archaea in response to heavy metals (e.g.
Pho84-like and PitA orthologues (Fig. 1) in some archae- Rodriguez-Montelongo et al., 2006; Williams et al.,
ons (Remonsellez et al., 2006; McCarthy et al., 2014), 2007). Some proteins involved in oxidative damage
the possible function of polyP in metal resistance in repair, such as NADH-dependent oxidases and thiore-
these analysed microorganisms cannot be discarded. doxin reductases, were expressed in cells of ‘Fp. acidar-
Actual evidence suggests that polyP may provide manus’ exposed to arsenite (Baker-Austin and Dopson,
mechanistic alternatives in tuning microbial fitness for 2007). The expression of this group of proteins has also
the adaptation under stressful environmental situations been observed when the same microorganism was
such as oxidative stress may be of crucial relevance exposed to copper (Baker-Austin et al., 2005).
among extremophiles. This is supported by the recent
report assigning the universally conserved biopolymer
Concluding remarks
general roles as a protein-protective chaperone and in
regulating general stress response pathways (Gray Current knowledge indicates that in addition to passive
et al., 2014; Gray and Jakob, 2015). adaptations such as a positive membrane potential and
Figure 1 shows that proteins from some archaeal a very high number of basic proteins in the periplasm of
microorganisms (M. sedula, T. acidophilum, T. volcanium) Gram ( ) bacteria that make difficult cation
displacements, key active elements involved in metal Acidithiobacillus ferrooxidans. Appl Environ Microbiol 70:
resistance in environmental acidophilic microorganisms 5177–5182.
appear to be a wide repertoire of known regulated cop- Arnesano, F., Banci, L., Bertini, I., and Thompsett, A.R.
(2002) Solution structure of CopC: a cupredoxin-like pro-
per resistance determinants similar to those present in
tein involved in copper homeostasis. Structure 10: 1337–
all microorganisms. The duplications of these copper 1347.
resistance genes also confer extra metal tolerance to the Auernik, K.S., Maezato, Y., Blum, P.H., and Kelly, R.M.
microorganisms containing them. Global OMICS proce- (2008) The genome sequence of the metal-mobilizing,
dures have allowed determining the presence of novel extremely thermoacidophilic archaeon Metallosphaera
copper-chaperones and other possible metal resistance sedula provides insights into bioleaching-associated meta-
determinants by their overexpression in the presence of bolism. Appl Environ Microbiol 74: 682–692.
Baker-Austin, C., and Dopson, M. (2007) Life in acid: pH
the tested metal. The study of novel metal resistance
homeostasis in acidophiles. Trends Microbiol 15: 165–
determinants in environmental microorganisms will also 171.
help with the functional annotation of these genes in the Baker-Austin, C., Dopson, M., Wexler, M., Sawers, R.G.,
increasingly available genomic sequences from environ- and Bond, P.L. (2005) Molecular insight into extreme cop-
mental extremophilic bacteria and archaea. per resistance in the extremophilic archaeon Ferroplasma
Horizontal gene transfer by means of mobile genetic acidarmanus Fer1. Microbiology 151: 2637–2646.
elements such as GIs plays an important role in increas- Blake, R.C. II, Shute, E.A., Greenwood, M.M., Spencer,
G.H., and Ingledew, W.J. (1993) Enzymes of aerobic res-
ing the adaptability and versatility of microorganisms liv-
piration on iron. FEMS Microbol Rev 11: 9–18.
ing in environments with high metal concentrations. Most Brierley, C.L., and Brierley, J.A. (2013) Progress in
importantly, is to consider that studies of metals resis- bioleaching: part B: applications of microbial processes by
tance in individual microorganisms do not necessarily the minerals industries. Appl Microbiol Biotechnol 97:
reflect some interspecific bacterial cooperation that takes 7543–7552.
place during metal resistance in complex communities. Brierley, C.L., and Murr, L.E. (1973) Leaching: use of a ther-
Therefore, future studies on metal resistance determi- mophilic and chemoautotrophic microbe. Science 179:
488–490.
nants in biomining microorganisms should also include
Bustamante, P., Covarrubias, P.C., Levica n, G., Katz, A.,
detailed analysis of the consortia actually living in indus- Tapia, P., Holmes, D., et al. (2012) ICEAfe1, an actively
trial bioleaching operations to thoroughly understand the excising genetic element from the biomining bacterium
mechanisms that acidophilic bioleaching microorganisms Acidithiobacillus ferrooxidans. J Mol Microbiol Biotechnol
use to adapt to their extreme environments. This knowl- 22: 399–407.
edge should eventually improve biomining or metal rdenas, J.P., Valde
Ca s, J., Quatrini, R., Duarte, F., and
bioremediation processes if the most fitted bacteria are Holmes, D.S. (2010) Lessons from genomes of extremely
acidophilic bacteria and archaea with special emphasis
involved in these industrial processes.
on bioleaching microorganisms. Appl Microbiol Biotechnol
88: 605–620.
Conflict of interest rdenas, J.P., Quatrini, R., and Holmes, D.S. (2016)
Ca
Genomic and metagenomic challenge and opportunities
None declared. for bioleaching: a mini-review. Res Microbiol 167: 529–
538.
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Microbial Copper Resistance: Importance in Biohydrometallurgy

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Microbial copper resistance: importance in

CusCBAF system from A. ferrooxidans (Fig. 1) and

Leptospirillum ferrooxidans Sulfobacillus thermosulﬁdooxidans

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