Hydrometallurgy 127–128 (2012) 162–167

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Comparative genomics of the oxidative stress response inbioleaching microorganisms☆

Juan Pablo Cárdenas a,b,c, Francisco Moya b,d, Paulo Covarrubias b,c, Amir Shmaryahu b, Gloria Levicán d,David S. Holmes a,b,c, Raquel Quatrini a,b,c,⁎a Center for Bioinformatics and Genome Biology, Santiago, Chileb Laboratory of Microbial Ecophysiology, Fundación Ciencia & Vida, Santiago, Chilec Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chiled Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile

☆ This paper was originally presented at the Internatiposium (IBS), Changsha, China, 18-22 September 2011.⁎ Corresponding author at: Laboratory of Microb

Ciencia & Vida, Santiago, Chile. Tel.: +56 2 3672044; faE-mail addresses: juan.pablo.cardenas@gmail.com (J

moya.francisco@gmail.com (F. Moya), paulo.covarrubiasamir.shmaryahu@gmail.com (A. Shmaryahu), gloria.levidsholmes2000@yahoo.com (D.S. Holmes), rquatrini@ya

0304-386X/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.hydromet.2012.07.014

a b s t r a c t

a r t i c l e i n f o

Available online 3 August 2012

Keywords:BioleachingMicrobial consortiumOxidative stressComparative genomics

Bioleaching acidophiles inhabit environments with unusually high concentrations of iron that can potentiallycause oxidative stress via the Fenton reaction in which dangerous reactive oxygen species (ROS) are generated.ROS can cause damage to proteins, nucleic acids, lipids and other macromolecules and thus have deleteriouseffects on cell growth and survival. Many of these microorganisms are chemolithotrophs with unusually highoxygen consumption rates that may exacerbate the problem of oxidative stress. Although some knowledgehas been gained in recent years regarding the oxidative stress response in a few acidophiles, the general strate-gies used by them to face ROS challenges are still inadequately understood.Comparative genomics and multiple bioinformatic tools were used to explore 44 sequenced genomes ofacidophilic bacteria and archaea in order to reconstruct their individual oxidative stress responses and to identifyconserved strategies. The analyses revealed that acidophiles lack genes encoding typical oxidative stressresponse regulators and have an underrepresentation of classical ROS consumption enzymes (e.g. catalases)although they have a complete repertoire of repair systems for macromolecules (DNA, proteins and lipids).This suggests that stress mitigation is an active strategy in acidophiles confronting unavoidable ROS formationin their environment. Insights into the oxidative stress response in bioleaching acidophiles may contribute to abetter understanding of the aspects that influence fitness of the microbial consortium driving bioleaching.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Bioleaching acidophiles inhabit low pH environments with unusu-ally high concentrations of soluble metals, many of which areredox-active (e.g. iron and copper) (Demergasso et al., 2005). Ironand copper are critical cofactors for anumber ofmetalloenzymes involvedin relevantmetabolic functions, but only trace levels are required intracel-lularly. In excess, bothmetals are extremely toxic (Nies, 1999). The unify-ing factor in determining toxicity for these redox-active metals is thegeneration of reactive oxygen species (ROS) by a mechanism involvingFenton and Haber-Weiss reactions. By switching oxidation states metalions further activate species like hydrogen peroxide (H2O2) and superox-ide (O2•–) to the highly reactive hydroxyl radical (•OH) (Imlay, 2003). All

onal Biohydrometallurgy Sym-

ial Ecophysiology, Fundaciónx: +56 2 2372259..P. Cárdenas),@gmail.com (P. Covarrubias),can@usach.cl (G. Levicán),hoo.com.ar (R. Quatrini).

l rights reserved.

these ROS can cause damage to proteins, nucleic acids, lipids and other bi-ological macromolecules and thus have very deleterious effects on cellgrowth and survival (Fridovich, 1978). Recently, evaluations in tank re-actors have shown the negative influence of mineral-mediated ROSgeneration on bioleaching (Jones et al., 2011), suggesting the direct ef-fect of external ROS generation on bioleaching organisms. In addition tothe high metal concentrations present in their environment, unusuallyhigh oxygen consumption rates are associated with the aerobicmetabolism of several of these acidophilic chemolithotrophs further ex-acerbating the production of ROS. Both factors make this group of acid-ophiles highly susceptible to oxidative stress.

Oxidative stress results from an imbalance between the generation ofreactive oxygen species and their suppression by antioxidant defensemechanisms. The latter include (a) enzymatic prevention strategies pre-cluding the accumulation of ROS by transformation, quenching and/orconsumption of radicals, (b) systems for repair of damaged macromole-cules and (c) regulatory loops controlling the level of expression of thecomponents involved in different stages of the response. Althoughsome knowledge has been gained in recent years regarding the mecha-nisms and strategies that control metal homeostasis (Dopson et al.,2003; Orell et al., 2010; Osorio et al., 2008; Quatrini et al., 2007) and

163J.P. Cárdenas et al. / Hydrometallurgy 127–128 (2012) 162–167

oxidative stress in a few acidophiles (Cortés et al., 2011; Maaty et al.,2009), the general strategies used by these microbes to face ROS chal-lenge are still inadequately understood. Even less is known about the im-pact of this response on other aspects of the ecophysiology of bioleachingacidophiles (Cortés et al., 2011;Maaty et al., 2009). As in other well stud-ied microorganisms, the oxidative stress response of acidophiles isexpected to depend onmolecular mechanisms that mitigate the produc-tion of reactive oxygen species via redox cycling as a first line of defenseand secondly on enzymes to repair damaged chemical groups in proteins,DNA and lipids. In addition, oxidative stress master regulators are likelyto be involved in transcriptional control of the responses. The goals ofthis study were to characterize the potential molecular mechanismsused by this group of acidophiles to face ROS challenge using compara-tive genomic strategies and to gain insight into the particular adapta-tions these microorganisms may have developed to deal with theunique conditions of their acid and metal-rich environment.

2. Materials and methods

Genome sequences for 44 bacterial and archaeal acidophiles wererecovered from the NCBI, JGI and FCV databases. They included publicgenomic and metagenomic sequences and private environmental iso-lates. They were annotated using a bioinformatics pipeline consistingof combined in-house bioinformatics programs as AlterOrf (for the pre-diction of open reading frames, http://www.alterorf.cl) and externalprograms and databases. All genomes are listed in Table 1.

Predicted genes were catalogued using the COG database (http://www.ncbi.nlm.nih.gov/COG/), Pfam (http://pfam.sanger.ac.uk/) andSwissprot (http://ca.expasy.org/sprot/). Potential transmembrane re-gions and putative sub-cellular localization were predicted usingPSORTB (http://www.psort.org/psortb/) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). Categorizations were made usingPerl scripts and manual revision. Metabolic pathways were obtainedfrom KEGG (http://genome.ad.jp/kegg/kegg4.html), RAST (http://rast.nmpdr.org/) and Microbesonline (http://www.microbesonline.org/). Enzyme information was retrieved from BRENDA (http://www.brenda-enzymes.org/) and ENZYME (http://enzyme.expasy.org/). Extensive manual curation of this dataset was performed aposteriori, identifying candidates for different gene families andresponse stages of well characterized oxidative stress responses.

3. Results and discussion

3.1. ROS-specific enzymes in bioleaching genomes

Almost all genomes analyzed (Table 1) contained at least one copy(ortholog) of a superoxide dismutase-encoding gene (Fe or Mn-SOD,Cu/Zn-SOD or Ni-SOD). In contrast, none contained a neelaredoxin/desulfoferrodoxin gene, a protein with superoxide reductase activityfound in strict anaerobes (Abreu et al., 2001). This suggests that mostof the acidophiles are capable of transforming superoxide (O2•–) intohydrogen peroxide (H2O2) as a detoxification strategy using standardstrategies. Unexpectedly, only few microorganisms were predicted toencode a catalase (KatG, KatE, or Mn-dependent catalase) suggestingthat conversion of H2O2 to H2O occurs by an alternative pathway.Leptospirillum species did not contain any gene predicted to encodefor either of these enzymes. This raises the question as to how theseorganisms reduce their intracellular ROS concentrations in the absenceof an enzyme that prevents the formation of hydroxyl radicals (•OH)and/or the consumption of O2•–. Previouswork in Sulfolobus solfataricushas shown that the protein rubrerythrin functions as a H2O2 scavengingsystem, probably as an H2O2 reductase (Maaty et al., 2009). A bioinfor-matic search for orthologs of this protein in the bioleaching genomesrevealed that rubrerythrin is not only present in Sulfolobales, but alsoin A. thiooxidans, A. caldus, Acidithiomicrobium spp., Sulfobacillus spp.,Leptospirillum group II species and the alphabet–plasma A, E, I and G

(Table 1), indicating that this alternative pathway for H2O2 removal isindeedpresent in several of these acidophiles. Another protein, often in-volved in oxidative stress response is an “oxygen-detoxifying” NADHoxidase. It removes oxygen transforming it to water, using NAD(P)Has electron source (Kawasaki et al., 2004). Putative orthologs of thisprotein were found in the Acidithiobacilli (Table 1).

3.2. DNA repair systems

DNAprotection and repair is a very important aspect of the oxidativestress response. The hydroxyl radical (•OH) produces a complex patternof DNA modifications (Dizdaroglu, 1991) with both mutagenic andlethal effects. Three different DNA repair pathways are involved in theremoval of the oxidized bases in DNA and the mismatches generatedaccordingly (Czeczot et al., 1991; Lin and Sancar, 1989). These are baseexcision repair (BER), nucleotide excision repair (NER) and mismatchrepair (MMR).

All bacterial genomes analyzed possessed the mutL-mutS gene pair,encoding the minimal essential complex for mismatched bases repair(MisMatchRepair pathway). In addition, almost all these acidophilic bac-teria contained genes for the detection and removal of modified purineand pyrimidine bases (Base Excision Repair pathway), includingorthologs of the formamidopyrimidine-DNA glycosylase gene mutM,the A/G-specific adenine glycosylase mutY and the endonuclease IIIencoding gene nth. The endonuclease III gene was also present in someacidophilic archaea. The Sulfolobales and some Thermoplasmatales alsoencode for a deoxyinosine 3′ endonuclease (nfi), better known as endo-nuclease V. Genes for Nucleotide Excision Repair, a pathway for the re-moval of bulky lesions from the DNA duplex, were present in nearly allacidophilic bacteria. These included the UvrABC repair system and thetranscription-repair-coupling factor (Mfd protein) involved in blockingtranscription of damaged genes. In addition, the signature genes of theSOS response to DNA damage (Kelley, 2006), recA/radA was found inall organisms and lexA was found in all bacteria. In contrast, orthologsof the gene encoding the Dps protein, a ferritin-like protein involved inDNA protection (Ramsay et al., 2006), were predicted in the Sulfolobales,in agreement with Maaty and co-workers (Maaty et al., 2009), and alsoin members of Acidithiomicrobium, Alicyclobacillus and Sulfobacillus.

3.3. Lipid-specific repair systems

Organic groups such as alcohols, when attacked by ROS, produce or-ganic peroxides that in turn damage proteins and membranes. Thereare several systems, based in different redox transactions, involved inorganic peroxide removal, mainly peroxiredoxins of the Bcp and AhpCfamilies and peroxidases of the Dyp family. Genes potentially encodingperoxiredoxins are well represented in the acidophilic microorganismsanalyzed, often present inmultiple copies per genomes (Table 1). In thecase of the Ahp complex, the peroxiredoxin subunit (AhpC) waswidelyconserved, but the NADPH-transferring subunit (AhpF) was found onlyin Alicyclobacillus spp. AhpF contains an internal redox-active disulfidebridge, that is reduced by electron transfer from NADH via an internalFAD cofactor. This reduction triggers a cascade of disulfide-exchange re-actions (first intra-molecularly, then inter-molecularly) to the disulfidecysteine residues of the AhpC component, mediating the transforma-tion of an organic peroxide to its corresponding alcohol (Bieger andEssen, 2000). In contrast, redox-cycling of the Bcp protein is dependenton the thioredoxin system. Other proteins involved in organic peroxidescavenging, such as the haem-dependent peroxidase Dyp, which has awide range of specificities (Zubieta et al., 2007), has no recognizableortholog in any acidophile with the exception of the Leptospirilli. Also,a lipoyl-dependent organic hydroperoxide reductase, involved in theoxidative stress response of Xanthomonas campestris (Mongkolsuk etal., 1998), was found in A. cryptum and many Leptospirilli.

Table 1Oxidative stress-related genes in sequenced bioleaching acidophiles.

Bacteria

Acidithiobacillusspp.

Leptospirillumspp.

Acidimicrobiumspp.

Acidithiomicrobiumspp.

Acidiphiliumcryptum

Sulfobacillusspp.

Alicyclobacillussp.

# genomes 7 6 1 2 1 4 2

ROS scavenging Fe/MnSOD + − − − + + +Cu/Zn•SOD − − − − − − −Ni•SOD − − + + − − −Dfx/Nlr − − − − − − −KatG − (*) − + − + − −KatE − (*) − (*) − − + − +KatN − − − − − − +Rbr +(§) +(§) − + − + −

Protein repair TrxA/TrxB + + + + + + +Tpx − (*) − − + − +(§) −MsrA + + − − + + +MsrB + +(§) − − − + +Hsp33 + − − − + + +Dsb complex + + − − + − −

Lipid repair Bcp + + + + + + +AhpC + + + + + + +AhpF − − − − − − +Ohr − + − − + − −Dyp − + − − − − −

DNA repair UvrABC complex + + + + + + +MutL/ MutS + + + + + + +DNA glycosilases + + + + + + +AP endonucleases + + + + + + +Photolyases − (*) + − + + + +RecA/RadA + + + + + + +LexA + + + + + + +Dps − − + − − +(§) +

Regulators Fur family + + + + + + +PerR + + − − − + +Irr + − − − − − −OxyR − − − − + − −SoxRS − − − − − − −OhrR − − − − + − +Spx − − − − − − −Sta1 − − − − − − −

Abbreviations. SOD: superoxide dismutase; Dfx/Nlr: superoxide reductase, neelaredoxin-type; KatG, KatE, KatN: catalases; Rbr: rubrerytrhin; TrxA/TrxB: thioredoxin system; Tpx:thiol peroxidase; MsrA/B: methionine sulfoxide reductases; Bcp: peroxiredoxin; AhpC, AhpF: alkyl hidroperoxide reductase subunits; Ohr: organic hydroperoxide reductase; Dyp:dyp peroxidise; MutL/S: components of mismatch repair system; RecA/RadA: repair protein; LexA: SOS response regulator; Dps: DNA binding protein involved in protection; Furfamily: fur family protein; PerR, Irr, OxyR, SoxRS, OhrR, Spx: oxidative stress responsive proteins.*Absent in nearly all the microorganisms represented (see text).§Present in some of the microorganisms represented (see text).

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3.4. Protein-specific repair systems

Protein repair systems typically use redox reactions to restore theoriginal status of sulfur groups in cysteines or methionines in order toreestablish protein structure and function. The classical enzymaticmachineries involved in protein repair are: (i) the thioredoxin/thioredoxin reductase system, (ii) Tpx and (iv) the MsrA and MsrBmethionine sulfoxide reductases. Practically all the microorganismsexamined contained genes encoding components of thioredoxin sys-tem (Table 1), including one or more small thioredoxins that reactwith cysteine residues in oxidized proteins and the thioredoxin re-ductase which restores the redox status of the thioredoxins utilizingNAD(P)H (Farr and Kogoma, 1991). A thiol peroxidase (Tpx) original-ly described in E. coli, was also found in At. caldus, Acidithiomicrobiumsp. strains and Sulfobacillus acidophilus. Tpx is a periplasmic hydrogenperoxide scavenger, functioning as a thioredoxin system-dependentprotein antioxidant (Cha et al., 1995). To cope with oxidative damageof peptidyl and free methionine, MsrA and MsrB catalyze the revers-ible (thioredoxin-dependent) oxidation-reduction of methioninesulfoxides, the former being specific for the S- and the latter for theR-epimer (Ezraty et al., 2005). The msrA gene was well conserved inall the acidophilic bacterial species in the list, while themsrB orthologwas missing in the group II Leptospirilli and A. cryptum genomes.

Orthologs of the hsp33 gene encoding a chaperone involved inprotein stabilization activated by a redox-dependent mechanism(Kumsta and Jakob, 2009) were found in members of bothgram-positive (Alicyclobacillus and Sulfobacillus) and gram-negativeacidophiles (Acidiphilium and Acidithiobacillus). Also, predictedorthologs of the periplasmic system for thiol:disulfide interchange(Dsb complex), were found to be present in the genomes of thegram-negative Acidithiobacillus spp., A. cryptum and Leptospirillum spp.

3.5. Low molecular weight thiols in bioleaching microorganisms

The use of low molecular weight (LMW) reactive thiol compounds isimportant for reestablishing the redox balance of proteins or lipids inmostmicroorganisms (Fahey, 2001). The best knownLMWthiol involvedin oxidative stress response is glutathione (GSH), a Gly-Cys-Glu tripeptide(Carmel-Harel and Storz, 2000). GSH can interact directly with proteinsreducing their disulfide bridges or indirectly through glutaredoxinswhich are thereafter reduced by GSH reductase using NAD(P)H. Pro-teins involved in the glutaredoxin system and glutathione reductasewere found only in the acidithiobacilli and A. cryptum. In addition, puta-tive orthologs encoding for the glutathione synthetase (gshB), thegamma-glutamyltranspeptidase (ggt) and the glutamate–cysteine li-gase (gshA) genes were also found exclusively in the acidithiobacilli

Archaea

Sulfolobusspp.

Metallosphaerasedula

Ferroplasmaspp.

A,E,G,I-plasma Picrophilustorridus

Thermoplasmaspp.

Microacrcheumspp.

Parvarcheumspp.

11 1 3 4 1 2 1 2

+ + + + + + + +− − − − − − − −− − − − − − − −− − − − − − − −− − − + − − − −− − − − − − − −− − − − − − − −+ + − + + − − −+ + + + + + + +− − − − − − − −− (*) + + + + − + +− − − − − − − −− − − − − − − −− − − − − − − −+ + + + + + + ++ + + + + + + +− − − − − − − −− − − − − − − −− − − − − − − −− − − − − − − −− − − − − − − −− − − − − − − −+ + + + + + + ++ + + +(§) + + − −+ + + + + + + +− − − − − − − −+ + − − − − − −+ + − + − + − −− − − − − − − −− − − − − − − −− − − − − − − −− − − − − − − −− − − − − − − −− − − − − − − −+ + − − − − − −

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and in A. cryptum, suggesting that the glutaredoxin system is onlypresent and functional in these microorganisms. However, someorthologs for glutaredoxin gene were found in the sulfobacilli andin Sulfolobales, yet these organisms do not encode for known genesinvolved in GSH biosynthesis, suggesting a potential use of externalGSH.

The limited presence of GSH biosynthesis and utilization genes raisedthe question whether the other organisms use different biothiolsfor redox homeostasis. The genes mshABCD, involved in mycothiolbiosynthesis, a LMW thiol typically present in Actinobacteria (Newton etal., 2008), were consistently predicted in the genomes of the Acidi(thio)microbium species. On the other hand, genes involved in the biosynthesisof bacillithiol (bshABC), a LMW thiol limited to the Bacilliales (Newton etal., 2009), were found exclusively in the Alicyclobacillus species.

Curiously, almost all acidophilic archaea analyzed and the bacteriaAcidi(thio)microbium spp. and Sulfobacillus acidophilus encode putativeorthologs of CDR (TIGR03385), the CoA Disulfide Reductase (Table 1).This enzyme catalyzes the reaction CoA\S\S\CoA+NAD(P)H+H+➔2CoA\SH+NAD(P)+, which is analogous to the glutathione/glutathione–disulfide redox reaction catalyzed by glutathionereductase. Co-A has been shown to play a role in thiol/disulfide redox bal-ance in some bacteria (delCardayre et al., 1998) and archaea (Harris et al.,2005). These findings suggest that CoA may play a role in thiol/disulfide

redox balance in acidophiles, in particular in those lacking the classicalglutathione based systems.

3.6. Oxidative stress response transcriptional regulators

Specialized transcriptional regulators controlling gene expression inoxidative stress response in well characterized microbes include OxyR,SoxR, OhrR, Spx and PerR (Farr and Kogoma, 1991; Zuber, 2009). A bio-informatic search for candidate regulators in the acidophiles yieldedfew orthologs of these regulators. Virtual absence of orthologs for theresponse regulators against oxygen peroxide OxyR and superoxideSoxR (Farr and Kogoma, 1991), organic hydro-peroxide OhrR anddisulfide stress Spx (Zuber, 2009) suggests that other regulators haveassumed these responsibilities inmany acidophiles. Almost all microor-ganisms analyzed have at least one ortholog of the Fur family. Thisprotein family includes the well known peroxide response regulatorPerR (Zuber, 2009), the alphaproteobacterial regulator Irr (Yang et al.,2006), and other protein subfamilies displaying relevant roles in oxida-tive stress regulation in different model bacteria (e.g. Jittawuttipoka etal., 2010). A phylogenetic profile (data not shown) allow us to definethat PerR orthologs are present in many genera of acidophilic bacteria(Table 1) and, interestingly, Irr potential orthologs are found in the

Fig. 1. Overview of the oxidative stress response in acidophilic bioleaching microorganisms. Boxes represent the diverse functional modules involved in oxidative stress responses.Boxes represented with solid borders indicate predicted stress response proteins in acidophiles that are typically found in all/almost all the organisms; boxes with dotted bordersindicate predicted stress response proteins that are usually found in other organisms that are absent in acidophiles.

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Acidithiobacilli. We hypothesize that those orthologs may be candidateregulators for oxidative stress management in acidophiles.

In S. solfataricus Sta1 positively regulates a paralog of recA upon DNAdamage induction (Abella et al., 2007). A bidirectional BLAST-basedsearch uncovered candidate sta1 genes in all Sulfolobus species and inM. sedula (Table 1). No potential ortholog of sta1 was found in any ofthe acidophilic euryarchaea used in this work. The role of this regulatorin the overall oxidative stress response remains to be investigated.

4. Conclusions

Absence of catalase and the classical oxidative stress response regula-tors (OxyR, SoxR, OhrR) together with the sparseness of the glutathione/glutaredoxin systemcomponents are features that differentiate acidophil-ic micoorganisms from well characterized neutrophilic organisms. How-ever, alternative systems potentially undertaking most of these roles inacidophiles have been predicted and include the H2O2 scavengingrubrerythrin and the Fur family of regulators, respectively. In addition,the well-represented set of thioredoxins and peroxiredoxins suggeststhat acidophiles have built up a strong response to mitigate the effectsof ROS on organic molecules, augmenting the rather modest ROS direct

removal systems. DNA repair pathways involved in oxidative DNA dam-age mitigation are also conserved in this set of acidophiles. In turn, thelimited presence of GSH biosynthesis and utilization genes is compensat-ed through the use of alternative LMW thiols and/or the CoA/ CoA Disul-fide Reductase system. This study providesfirst insights into the oxidativestress response in several bioleaching acidophiles (Fig. 1) and contributesto a better understanding of the aspects that influence fitness of the mi-crobial consortium driving bioleaching.

Acknowledgments

Innova 08CM01-03, FONDECYT 1100887, FONDECYT 1090451,FONDECYT 1120746, FONDECYT 11085045, Conicyt Basal CCTE PFB16,CONICYT PhD Studies grant and UNAB DI-116-12/I.

References

Abella, M., Rodríguez, S., Paytubi, S., Campoy, S., White, M.F., Barbé, J., 2007. TheSulfolobus solfataricus radA paralogue sso0777 is DNA damage inducible and posi-tively regulated by the Sta1 protein. Nucleic Acids Res. 35 (20), 6788–6797.

167J.P. Cárdenas et al. / Hydrometallurgy 127–128 (2012) 162–167

Abreu, I.A., Saraiva, L.M., Soares, C.M., Teixeira, M., Cabelli, D.E., 2001. The mechanismof superoxide scavenging by Archaeoglobus fulgidus neelaredoxin. J. Biol. Chem.276 (42), 38995–39001.

Bieger, B., Essen, L.O., 2000. Crystallization and preliminary X-ray analysis of the cata-lytic core of the alkylhydroperoxide reductase component AhpF from Escherichiacoli. Acta Crystallogr. D: Biol. Crystallogr. 56 (Pt 1), 92–94.

Carmel-Harel, O., Storz, G., 2000. Roles of the glutathione- and thioredoxin-dependentreduction systems in the Escherichia coli and Saccharomyces cerevisiae responses tooxidative stress. Annu. Rev. Microbiol. 54, 439–461.

Cha, M.K., Kim, H.K., Kim, I.H., 1995. Thioredoxin-linked “thiol peroxidase” from peri-plasmic space of Escherichia coli. J. Biol. Chem. 270 (48), 28635–28641.

Cortés, A., Flores, R., Norambuena, J., Cárdenas, J.P., Quatrini, R., Chávez, R., Orellana, O.,Levicán, G., 2011. Comparative study of redox stress response in the acidophilicbacteria Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans. In: Qiu, G., et al.(Ed.), 19th International Biohydrometallurgy Symposium. Central South UniversityPress, Changsha, China, pp. 354–357.

Czeczot, H., Tudek, B., Lambert, B., Laval, J., Boiteux, S., 1991. Escherichia coli Fpg proteinand UvrABC endonuclease repair DNA damage induced by methylene blue plusvisible light in vivo and in vitro. J. Bacteriol. 173 (11), 3419–3424.

delCardayre, S.B., Stock, K.P., Newton,G.L., Fahey, R.C.,Davies, J.E., 1998. CoenzymeAdisulfide re-ductase, the primary lowmolecularweight disulfide reductase from Staphylococcus aureus.Purification and characterization of the native enzyme. J. Biol. Chem. 273 (10), 5744–5751.

Demergasso, C.S., Galleguillos P., P.A., Escudero G., L.V., Zepeda A., V.G., Castillo, D.,Casamayor, E.O., 2005. Molecular characterization of microbial populations in alow-grade copper ore bioleaching test heap. Hydrometallurgy 80 (4), 241–253.

Dizdaroglu, M., 1991. Chemical determination of free radical-induced damage to DNA.Free Radic. Biol. Med. 10 (3–4), 225–242.

Dopson, M., Baker-Austin, C., Koppineedi, P.R., Bond, P.L., 2003. Growth in sulfidic min-eral environments: metal resistance mechanisms in acidophilic micro-organisms.Microbiology 149 (8), 1959–1970.

Ezraty, B., Aussel, L., Barras, F., 2005. Methionine sulfoxide reductases in prokaryotes.Biochim. Biophys. Acta 1703 (2), 221–229.

Fahey, R.C., 2001. Novel thiols of prokaryotes. Annu. Rev. Microbiol. 55, 333–356.Farr, S.B., Kogoma, T., 1991. Oxidative stress responses in Escherichia coli and Salmonella

typhimurium. Microbiol. Rev. 55 (4), 561–585.Fridovich, I., 1978. The biology of oxygen radicals. Science 201 (4359), 875–880.Harris, D.R., Ward, D.E., Feasel, J.M., Lancaster, K.M., Murphy, R.D., Mallet, T.C., Crane III,

E.J., 2005. Discovery and characterization of a Coenzyme A disulfide reductase fromPyrococcus horikoshii. Implications for this disulfide metabolism of anaerobichyperthermophiles. FEBS J. 272 (5), 1189–1200.

Imlay, J.A., 2003. Pathways of oxidative damage. Annu. Rev. Microbiol. 57, 395–418.Jittawuttipoka, T., Sallabhan, R., Vattanaviboon, P., Fuangthong, M., Mongkolsuk, S.,

2010. Mutations of ferric uptake regulator (fur) impair iron homeostasis, growth,oxidative stress survival, and virulence of Xanthomonas campestris pv. campestris.Arch. Microbiol. 192 (5), 331–339.

Jones, G.C., Corin, K.C., van Hille, R.P., Harrison, S.T.L., 2011. The generation of toxic re-active oxygen species (ROS) from mechanically activated sulphide concentratesand its effect on thermophilic bioleaching. Miner. Eng. 24 (11), 1198–1208.

Kawasaki, S., Ishikura, J., Chiba, D., Nishino, T., Niimura, Y., 2004. Purification and char-acterization of an H2O-forming NADH oxidase from Clostridium aminovalericum:existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria.Arch. Microbiol. 181 (4), 324–330.

Kelley, W.L., 2006. Lex marks the spot: the virulent side of SOS and a closer look at theLexA regulon. Mol. Microbiol. 62 (5), 1228–1238.

Kumsta, C., Jakob, U., 2009. Redox-regulated chaperones. Biochemistry 48 (22),4666–4676.

Lin, J.J., Sancar, A., 1989. A new mechanism for repairing oxidative damage to DNA:(A)BC excinuclease removes AP sites and thymine glycols from DNA. Biochemistry28 (20), 7979–7984.

Maaty, W.S., Wiedenheft, B., Tarlykov, P., Schaff, N., Heinemann, J., Robison-Cox, J.,Valenzuela, J., Dougherty, A., Blum, P., Lawrence, C.M., Douglas, T., Young, M.J.,Bothner, B., 2009. Something old, something new, something borrowed; how thethermoacidophilic archaeon Sulfolobus solfataricus responds to oxidative stress.PLoS One 4 (9), e6964.

Mongkolsuk, S., Praituan, W., Loprasert, S., Fuangthong, M., Chamnongpol, S., 1998.Identification and characterization of a new organic hydroperoxide resistance(ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonascampestris pv. phaseoli. J. Bacteriol. 180 (10), 2636–2643.

Newton, G.L., Buchmeier, N., Fahey, R.C., 2008. Biosynthesis and functions of mycothiol,the unique protective thiol of Actinobacteria. Microbiol. Mol. Biol. Rev. 72 (3),471–494.

Newton, G.L., Rawat, M., La Clair, J.J., Jothivasan, V.K., Budiarto, T., Hamilton, C.J.,Claiborne, A., Helmann, J.D., Fahey, R.C., 2009. Bacillithiol is an antioxidant thiolproduced in Bacilli. Nat. Chem. Biol. 5 (9), 625–627.

Nies, D.H., 1999. Microbial heavy-metal resistance. Appl. Microbiol. Biotechnol. 51 (6),730–750.

Orell, A., Navarro, C.A., Arancibia, R., Mobarec, J.C., Jerez, C.A., 2010. Life in blue: copperresistance mechanisms of bacteria and Archaea used in industrial biomining ofminerals. Biotechnol. Adv. 28 (6), 839–848.

Osorio, H., Martinez, V., Nieto, P.A., Holmes, D.S., Quatrini, R., 2008. Microbial iron man-agement mechanisms in extremely acidic environments: comparative genomicsevidence for diversity and versatility. BMC Microbiol. 8, 203.

Quatrini, R., Lefimil, C., Veloso, F.A., Pedroso, I., Holmes, D.S., Jedlicki, E., 2007. Bioinformaticprediction and experimental verification of Fur-regulated genes in the extremeacidophile Acidithiobacillus ferrooxidans. Nucleic Acids Res. 35 (7), 2153–2166.

Ramsay, B., Wiedenheft, B., Allen, M., Gauss, G.H., Lawrence, C.M., Young, M., Douglas,T., 2006. Dps-like protein from the hyperthermophilic archaeon Pyrococcusfuriosus. J. Inorg. Biochem. 100 (5–6), 1061–1068.

Yang, J., Panek, H.R., O'Brian, M.R., 2006. Oxidative stress promotes degradation of theIrr protein to regulate haem biosynthesis in Bradyrhizobium japonicum. Mol.Microbiol. 60 (1), 209–218.

Zuber, P., 2009. Management of oxidative stress in Bacillus. Annu. Rev. Microbiol. 63,575–597.

Zubieta, C., Joseph, R., Krishna, S.S., McMullan, D., Kapoor, M., Axelrod, H.L., et al., 2007.Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA. Proteins 69 (2), 234–243.