Báo cáo khoa học: Identification and characterization of 1-Cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress pdf

Furthermore, we report the cloning, the expression and the characterization of the recombinant protein rBcp2 in order to shed light on its role in the detoxifi-cation process and on its c

from Sulfolobus solfataricus and its involvement in the response to oxidative stress

Danila Limauro1, Emilia Pedone2, Luciano Pirone2and Simonetta Bartolucci1

1 Dipartimento Biologia Strutturale e Funzionale, University of Naples ‘Federico II’, Complesso Universitario Monte S Angelo, Naples, Italy

2 Istituto di Biostrutture e Bioimmagini, C.N.R., Naples, Italy

Reactive oxygen species (ROS) are either generated

by incomplete oxygen reduction during respiration, or

by exposure to environmental factors such as light,

radiation or increased oxygen pressure ROS, notably,

superoxide (O2• )) and hydroxyl radical (OH•

), hydro-gen peroxide (H2O2) and singlet oxygen (1O2) cause

damage to all major classes of biological

macromole-cules, leading to protein oxidation, lipid peroxidation,

DNA base modifications and strand breaks [1] In

order to protect against toxic ROS, aerobic organisms

are equipped with a full array of defence mechanisms,

among which are antioxidative enzymes and

antioxid-ant molecules (e.g superoxide dismutases, catalases,

peroxidases, thioredoxins, and glutathione) which are developed by most cells [2–4] Most aerobes have multiple enzymes with overlapping ROS detoxification pathways but different expression and regulation times For example, many bacteria induce catalase or other protective enzyme expressions during the trans-ition from the exponential to the stationary growth phase, presumably as an adaptation to protect the genome and other cellular components against oxida-tion during a prolonged nongrowth phase In recent years, much attention has been given to peroxiredox-ins (Prxs) [5–7], a new family of thiol-specific anti-oxidant proteins These include alkyl hydroperoxide

Keywords

Archaea; Sulfolobus solfataricus; oxidative

stress; ROS; peroxiredoxin

Correspondence

S Bartolucci, Dipartimento di Biologia

Strutturale e Funzionale, Complesso

Universitario di Monte S Angelo, Universita`

di Napoli ‘Federico II’, Via Cinthia,

80126 Naples, Italy

Fax: +39 081679053

Tel: +39 081679052

E-mail: bartoluc@unina.it

(Received 9 November 2005, revised

14 December 2005, accepted 16 December

2005)

doi:10.1111/j.1742-4658.2006.05104.x

Bcp2 was identified as a putative peroxiredoxin (Prx) in the genome data-base of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus Its role in oxidative stress was investigated by transcriptional analysis of RNA isolated from cultures that had been stressed with various oxidant agents Its specific involvement was confirmed by a considerable increase in the bcp2transcript following induction with H2O2 The 5¢ end of the transcript was mapped by primer extension analysis and the promoter region was characterized bcp2 was cloned and expressed in Escherichia coli, the recombinant enzyme was purified and the predicted molecular mass was confirmed Using dithiothreitol as an electron donor, this enzyme acts as a catalyst in H2O2 reduction and protects plasmid DNA from nicking by the metal-catalysed oxidation system Western blot analysis revealed that the Bpc2 expression was induced as a cellular adaptation in response to the addition of exogenous stressors The results obtained indicate that Bcp2 plays an important role in the peroxide-scavaging system in S solfataricus Mutagenesis studies have shown that the only cysteine, Cys49, present in the Bcp2 sequence, is involved in the catalysis Lastly, the presence of this Cys in the sequence confirms that Bcp2 is the first archaeal 1-Cysteine per-oxiredoxin (1-Cys Prx) so far identified

Abbreviations

Bcp, Bacterioferritin comigratory protein; LB, Luria–Bertani; MCO, metal catalysed oxidation; Prx, peroxiredoxin; ROS, reactive oxygen species.

reductase, thiol-specific peroxidase, and

bacterioferr-itin comigratory protein (Bcp), which perform their

protective role in cells through antioxidant activity

(ROOH +2e–fiROH + H2O), i.e by reducing and

detoxifying H2O2, peroxynitrite and numerous organic

hydroperoxides They are ubiquitous enzymes found

in every domain of life such as eukarya, bacteria and

archaea Prxs use a redox-active cysteine to reduce

peroxides Based on the number of cysteinyl residues

involved in catalysis they can be divided into two

groups, 1-Cys and 2-Cys Prxs [8] Structural and

mechanistic data actually support the division of

2-Cys Prxs into two subclasses, ‘typical’ and ‘atypical’

2-Cys Prxs All three Prxs classes are found to have

in common an initial catalytic step, at which

peroxid-atic cysteine (Cys-SpH), generally near residue 50,

attacks the peroxide substrate and is oxidized to

cys-teine sulfenic acid (Cys-SOH) The second step of the

peroxidase reaction, the resolution of cysteine sulfenic

acid, varies according to the class considered The

Cys-SOH of 1-Cys Prx, is presumably reduced by a

thiol-containing electron donor, although the

physio-logical partners have not ever been identified [9] The

main characteristic of 2-Cys Prxs, the largest Prx

class, is their second redox-active cysteine, the

resol-ving cysteine (Cys-SR) In typical 2-Cys, Sp at the N

terminus and SR at the C terminus of the protein

belong to different subunits and condense to form an

intersubunit disulfide bond; in atypical 2-Cys Prxs, Sp

and SR belong to the same subunit and establish an

intrasubunit disulfide bond The successive reduction

of 2-Cys Prxs involves a flavoprotein disulfide

reduc-tase and at least one additional protein or domain

with a CXXC motif, which is oxidized from the

di-thiol to the disulfide state during Prx reduction (e.g

thioredoxin reductase and thioredoxin) (Fig 1)

Two archaeal Prxs from Aeropyrum pernix APE2278

[10] and Pyrococcus horikoshii PH1217 [11,12] have

recently been characterized and subsumed within the 2-Cys family APE2278 was found to have a hexadeca-meric structure and it showed the ability to reduce

H2O2 using the NADPH⁄ thioredoxin reductase ⁄ thio-redoxin system as electron donor partner [13] Also PH1217 has peroxidase activity but the electron donor partner might be different from that found in A pernix because in the genome data base of P horikoshii nei-ther a homologue of A pernix thioredoxin-like nor other types of thioredoxins were found

In this study we examined the involvement of the peroxiredoxin Bcp2 in oxidative stress in the hyper-thermophilic aerobic archaeon Sulfolobus solfataricus Furthermore, we report the cloning, the expression and the characterization of the recombinant protein rBcp2 in order to shed light on its role in the detoxifi-cation process and on its catalytic mechanism

Results Identification of the bcp2 gene encoding putative peroxiredoxin

The analysis of the complete sequenced genome of

S solfataricus P2 [14] (http://www-archbac.u-psud.fr/ projects/sulfolobus/) revealed four ORFs homologues

of Prxs and annotated as Bcp1 (SSO2071), Bcp2 (SSO2121), Bcp3 (SSO225) and Bcp4 (SSO2613) The

S solfataricus Bcp which bears the greatest similarity

to other Prxs in the GenBank Database is Bcp2, which encodes a putative protein of 215 amino acids with a predicted molecular mass of 24744.79 Da and a theor-etical pI of 6.85 The deduced amino acid sequence shows 61% identity with the archaeal Prx (APE2278) from the aerobic hyperthermophilic archaeon A pernix [10], 61% with the putative bacterial Prx (Q9WZR4) derived from the hyperthermophilic bacterium Thermo-toga maritima, 57% with the Prx (PH1217) from the

XH 2

X + H 2 O

1-Cys Prx

S p OH

1-Cys Prx

S p H

H 2 O

H 2 O 2

RSH

Flavoprotein disulfide reductase

RSSR

2-Cys Prx

S p

2-Cys Prx S p H

H 2 O

H 2 O 2

S R

H 2 O 2-Cys Prx

S p OH

S R H B

S R H

A

Fig 1 Peroxiredoxin mechanisms (A) 1-Cys Prx (B) 2-Cys Prx S p peroxidatic cysteine;

X unidentified electron donor; SRresolving cysteine; RSH protein or domain with CXXC motif (e.g thioredoxin) In typical 2-Cys, S p

at the N terminus and SRat the C terminus belong to different subunits and condense

to form an intersubunit disulfide bond; in atypical 2-Cys, S p at the N terminus and S R

at C terminus, originate from the same subunit.

anaerobic hyperthermophilic archaeon P horikoshii

[11,12], all of which belong to the 2-Cys Prx family; in

addition, Bcp2 reveals 40% of identity with 1-Cys

Human PRDX6 (Fig 2)

Following primary structure analysis, Bcp2 was

clas-sified as a 1-Cys Prx with only one conserved cysteine

residue (Cys49) in a consensus surrounding sequence

DFTPVCTTE which is also found both in prokaryotic

and eukaryotic Prxs It is the first of all Prxs analysed

so far in archaea that has only one cysteine residue in

the sequence

Transcriptional analysis of bcp2 under oxidative

stress and characterization of mRNA 5¢ end

In order to understand the involvement of bcp2 in

oxi-dative stress, the levels of bcp2 mRNA were assessed

after treatment of S solfataricus cells with paraquat,

which was used to generate O2• ), with H2O2 and

tert-butyl hydroperoxide as direct oxidants [15] To

estab-lish the concentrations of agents whose effect can be

to slow down or otherwise affect growth, in the

expo-nential phase the cells were treated with varying

amounts of stressors (data not shown) Therefore, the

S solfataricus P2 strain was grown until the early

exponential phase (0.3 OD600 nm) and then induced

with 0.05 mm H2O2, 0.1 mm paraquat or 0.05 mm tert-butyl hydroperoxide for different periods of time (Fig 3) As shown, the addition of stressors to the cul-tures inhibits growth without killing the cells

The hybridizing band in the northern analysis showed the expected size of about 680 bp indicating that the gene is transcribed as a monocistronic mRNA When S solfataricus cells were incubated with H2O2, paraquat and tert-butyl hydroperoxide, the bcp2

Fig 2 Multiple sequence alignment ( CLUSTAL

W 1.82) of Bcp2 from S solfataricus and

Prxs from A pernix (APE2278), T maritima

(Q9WZR4), P horikoshii (PH1217), and

human (PRDX6).

0 0,1 0,2 0,3 0,4 0,5 0,6

Time (h)

Fig 3 S solfataricus P2 cultures treated with different oxidative stress agents Sulfolobus solfataricus P2 cultures were grown until 0.3 OD600 nmthen the cultures were treated with 0.1 m M paraquat (n), 0.05 m M H2O2(m), 0.05 m M tert-butyl hydroperoxide(*), or con-trol (r) The arrow indicates the OD 600 nm value at which the anti-oxidant agents were added.

mRNA levels increased considerably (Fig 4A, B and C),

i.e a 10-fold increase in transcriptional levels was

observed 15 min after the addition of H2O2, and a

fourfold increase within 30 min after paraquat

treat-ment; when the tert-butyl hydroperoxide was used, the

induction observed was less marked To evaluate bcp2

expression in response to growth phases, the RNA

obtained from cultures harvested at 0.3, 0.6 and

1.0 OD600 nm corresponding to early, mid and

station-ary growth phases was analysed The data obtained

suggest that bcp2 transcriptional levels were

independ-ent of the growth phase (Fig 5)

Primer extension analysis was performed in order to

characterize the promoter region (Fig 6) Figure 6B

shows the nucleotide sequence 5¢ of the upstream

regu-latory region of the bcp2 gene A consensus sequence,

GGUG, with Shine–Dalgarno motifs of the Sulfolobus

species was observed upstream of the ATG start codon

[16] The 5¢ end of the bcp2 transcript begins with

an adenine and maps 10 nucleotides upstream of the

ATG translation start codon The presence of cis-act-ing regulatory sequences typical of archaeal promoters had been observed These sequences are part of the basal transcriptional apparatus, such as the TATA box, centered at)27 from the transcriptional start site, and the BRE motif, targets for the general transcrip-tion factors TBP and TFB, respectively [17]

Purification and characterization of recombinant Bcp2 (rBcp2)

In order to overproduce rBcp2, the gene was amplified

by PCR from S solfataricus genomic DNA, as des-cribed in Experimental procedures, and cloned into pET-30c(+), rBcp2 was highly overexpressed in Escherichia coli in soluble form, as a fusion with a C-terminal eight-residue histidine tag (LEHHHHHH) with a yield of 12.8% of homogeneous protein

To purify the recombinant protein, the soluble frac-tion (140 mg) of the cell extract was heated at 80
C for 15 min; this heat treatment removed about 40% of

E coli proteins rBcp2 was purified to homogeneity in

a two-stage process using affinity chromatography on HisTrap HP and gel filtration on HiLoad Superdex 75 obtaining 30 mg and 18 mg, respectively The SDS⁄ PAGE of the final preparation revealed a single band with a molecular mass of 25 ± 1 kDa (Fig 7) The molecular mass of rBcp2, 25 678 Da, was deter-mined using mass spectrometric analysis as reported in Experimental procedures; the 131 shortfall compared

to the predicted 25 809 Da molecular mass suggested the removal of the N-terminal methionine

To assess the quaternary structure of the enzyme, analytical gel filtration on PC 75 and Biosep-SEC-4000

of purified rBcp2 were performed The protein was eluted at a volume accounting for a monomeric struc-ture, but it was observed that increasing protein

16S rRNA

bcp2 mRNA

0 15 30 45 60

C B

A

min

H2O2

0 15 30 45 60

paraquat

0 15 30 45 60

tert-butyl hydroperoxide

Fig 4 Northern hybridization analysis of S solfataricus P2 bcp2 transcripts: effect of oxidative stress agents Cultures of S solfataricus P2 were grown until the mid-exponential phase and treated with (A) 0.05 m M H2O2(B) 0.1 m M paraquat (C) 0.05 m M tert-butyl hydroperoxide RNAs were obtained from cultures harvested at time shown On the bottom 16S rRNA were reported as normalization.

A

B

16S rRNA

bcp2 mRNA

1 2 3

1 2 3

Fig 5 Northern hybridization analysis of S solfataricus P2 bcp2

transcripts at different growth phases (A) RNA was extracted from

cultures harvested at 0.3 OD 600 nm (1), at 0.6 OD 600 nm (2), at

1.0 OD600 nm(3) (B) 16S rRNA levels were used as a control.

concentration (0.4–2 lgÆlL)1) or prolonged storage

time (24 h at 4
C), produced changes in the

quater-nary structure with the appearance of the dimeric and

multimeric forms (data not shown)

Homogeneous rBcp2 was tested for its capacity to

serve as an antioxidant enzyme One of the most widely

used test for detecting Prx activity is the ability to

pro-tect plasmids against the metal catalysed oxidation

(MCO) system (DTT⁄ Fe3+⁄ O2); in the presence of an

electron donor, such as dithiothreitol (DTT), Fe3+

catalyses the reduction of O2to H2O2, which is further

converted to OH•

by the Fenton reaction [18] The MCO system causes damage to DNA by producing

OH•

, which in turn can nick the intact supercoiled

plas-mid DNA [19] as shown in Fig 8 (lane 4) The damage

was averted when rBcp2 was included in the reaction mixture, showing that the enzyme is an active Prx and can remove H2O2generated by the MCO system in vitro (Fig 8A) BSA was used as negative control

The antioxidant activity of rBcp2 was then tested for its ability to remove exogenously added H2O2 in a more quantitative in vitro spectrophotometric assay rBcp2 was capable of catalysing the removal of H2O2

in a concentration-dependent manner using DTT as the electron donor (Fig 8B)

In order to characterize the thermophilicity of rBcp2, peroxidase activity was investigated by measuring the

H2O2removal at increasing temperature rBcp2 showed maximum activity between 80 and 90
C, which is in the optimum temperature range for the growth of

A

B

Fig 6 (A) Primer extension analysis and sequence of the S solfataricus bcp2 gene Total RNA was isolated from a culture of S solfataricus P2 Primer extension was carried out as described in Experimental procedures, and the products were separated by electrophoresis under denaturing conditions alongside sequencing reactions with the same primer (B) Nucleotide sequence of bcp2 The transcriptional start point

is shown by the bent arrow above the underlined boldface A nucleotide The Shine–Dalgarno sequence is underlined by dotted line A puta-tive TATA box and BRE sequence are underlined The ATG start codon is in bold and the TAA stop codon is marked by an asterisk.

S solfataricus We also analysed the thermoresistance

of the enzyme by incubating rBcp2 for varying periods

of time at 80, 90, and 95
C and then assaying the

resid-ual peroxidase activity Following incubation for 6 h at

80
C, the activity retained was 63%; the enzyme

dis-played a half-life of 3 h at 90
C, while after 30 min at

95
C the activity measured was 30% (Fig 9)

Bcp2 expression in S solfataricus

We investigated how the expression of Bcp2 was

induced by H2O2, paraquat and tert-butyl

hydroper-oxide A polyclonal rBcp2-specific rabbit antiserum was used to conduct a quantitative analysis of the Bcp2 expression in S solfataricus The Bcp2 anti-serum was used in a western blot analysis on cytoplas-mic extracts from cells harvested in the exponential growth phase before and after addition of the stressor agents at the identical concentrations utilized in the northern analysis (Fig 10) Twenty-five kDa signals corresponding to Bcp2 were detected in noninduced and induced cells The increased amount of Bcp2 in the cytoplasmic fraction of S solfataricus correlated with increased bcp2 mRNA level after treatment with the stressors In particular, the maximum Bcp2 expres-sion) a sevenfold increase compared to that monit-ored for controls ) was observed after H2O2 addition

as in the northern analysis

Fig 7 SDS ⁄ PAGE of different steps in the purification of rBcp2.

Lane 1, E coli BL21-CodonPlus (DE3)-RIL ⁄ pETBcp2 cellular extract

not induced by IPTG; lane 2, E coli BL21-CodonPlus

(DE3)-RIL ⁄ pETBcp2 induced by 1 m M

isopropyl-thio-b-d-galactopyrano-side; lane 3, heat-treated sample; lane 4, molecular weight

markers; lane 5, sample after affinity chromatography; lane 6,

sam-ple after size-exclusion chromatography.

Fig 8 (A) rBcp2 assayed as antioxidant enzyme: DNA cleavage protection assay performed by rBcp2 Supercoiled pUC19 plasmid was exposed to the MCO system (DTT ⁄ Fe 3+ ⁄ O 2 ) alone and with different rBcp2 concentrations Nicked form (NF) and supercoiled form (SF) of pUC19 are indicated on the left by arrows (B) rBcp2 was assayed for its ability to remove H2O2in an in vitro assay system in the presence (r) and absence (d) of DTT Peroxidase activity was measured at 80
C using the ferrithiocyanate complex as described in experimental procedures The nonenzymatic removal of H 2 O 2 by heat was performed in parallel.

Fig 9 Thermoresistance was measured as residual peroxidase activity with 50 lgÆmL)1 of rBcp2 after incubation for different times at 80
C (n), 90
C (r), 95
C (m).

Role of the conserved cysteine residue in rBcp2

To investigate the catalytic role of the Cys residue we

constructed a mutant enzyme in which the cysteine at

position 49 was replaced by serine (C49S) The mutant

Bcp2 protein was expressed in E coli BL21-CodonPlus

(DE3)-RIL cells and purified from the soluble fraction

of bacterial cells as described in Experimental

proce-dures The yield of C49S was the same of that

obtained for the wild-type protein

The activity of C49S was tested by peroxidase

(Fig 11A) and DNA cleavage protection (Fig 11B)

assays and compared to that of the wild-type protein

In both cases the mutant showed no peroxidase or

plasmid DNA protection activity These findings

indi-cate that Cys49 is required for the proceeding of the

enzymatic reaction and that Cys49-SOH can be

conver-ted back to Cys-SH using DTT, and that Cys49-SH is

responsible for scavenging the OH•

induced by the MCO system

Discussion The natural environment in which S solfataricus lives is strongly oxidative, in addition ROS can be generated by naturally occurring phenomena such as ultraviolet irradiation of water, autoxidations and aeration turbulence Consequently, to survive in this harsh habitat S solfataricus should have developed antioxidant enzymes and molecules that protect it from ROS At the present time the investigation of response to oxidative stress in S solfataricus is at an initial stage and has been focused mainly on the superoxide dismutase (Fe-SOD) (EC 1.15.1.1) [20,21] This enzyme represents the primary defense against

O2• ) as suggested by its ubiquitous location in the membrane and in the cytoplasm [22], by its constitu-tive level [21] and by the long half-life (2 h) of the mRNA [23] The dismutation of O2• ) by SOD deve-lops H2O2 that can go freely through the membrane

C B

A

Fig 10 Bcp2 expression in S solfataricus cells in response to H2O2(A), paraquat (B) and tert-butyl hydroperoxide (C) Twenty micrograms

of cytoplasmic proteins extracted from nonexposed and exposed culture for 30, 60 and 120 min were analysed by western blot with anti-rBcp2 IgG.

Fig 11 Effect on peroxidase activity of replacement of Cys 49 of rBcp2 with serine (A) At different enzyme concentrations the peroxidase activity was assayed as previously reported H 2 O 2 removal by rBcp2 (r) and C49S (m) was measured over a range of concentrations (0–

100 lgÆmL)1) (B) Effect on protection against DNA cleavage of the replacement of Cys49 of rBcp2 with serine Lane 1, pUC19; lane 2, pUC19 and 10 m M DTT ; lane 3, pUC19 and 3 l M FeCl3; lane 4, pUC19, 10 m M DTT, and 3 l M FeCl3; lane 5, pUC19, 10 m M DTT, 3 l M FeCl3, and 50 lgÆmL)1rBcp2; lane 6, pUC19, 10 m M DTT, 3 l M FeCl 3 , and 50 lgÆmL)1C49S.

in the cell, where it must be scavenged to prevent

damage of biological molecules

In S solfataricus the peroxide detoxification system

has not yet been studied Genome analysis has shown

the absence of putative catalases and the presence of

four putative Bcps proteins: Bcp1, Bcp2, Bcp3, Bcp4

whose roles should be clarified in detail and could play

a key role in the detoxification processes

In this study we examined the role of Bcp2 in order

to increase the knowledge of the enzymatic activity

involved in the oxidative stress in S solfataricus

To detect differences in the response to various

agents we induced oxidative stress with paraquat,

an O2• ) generating compound, H2O2 and tert-butyl

hydroperoxide, an alkyl hydroperoxide Our results

show that compounds acting both indirectly and

directly as oxidants can induce transcription of bcp2

Data reveal that transcription of bcp2 in S solfataricus

is upregulated by the various stressors, and the

differ-ent kinetics in response to these agdiffer-ents imply that

sev-eral regulatory mechanisms or at least variations on

the same mechanism could be involved in controlling

the expression of bcp2 Western analysis performed

after treatment with H2O2 showed a slower and more

slight increase in protein level than mRNA level This

could imply that post-transcriptional processes, such as

lower rate of translational or protein instability caused

by oxidative stress conditions, are important in

deter-mining the level of Bcp2 protein Similar results are

observed for genes and related proteins involved in

oxidative stress in other microrganisms [24] Moreover,

the basal level of bcp2 transcript in the early and

mid-exponential, and the stationary phase of growth

suggests that bcp2 is not involved in the control of

endogenous peroxides that are produced during

aero-bic respiration

In contrast with Prxs discovered in the aerobic

hyperthermophilic archaeon A pernix and the

anaer-obic hyperthermothilic archaeon P horikoshii, the

ana-lysis of the primary structure of Bcp2 shows only one

cysteine (Cys49) This residue is positioned inside the

DFTPVCTTE sequence in the N-terminal region of

the protein that is conserved both in 1-Cys and 2-Cys

classes of Prxs Site-directed mutagenesis showed that

Cys49 is required for peroxidase activity Both

func-tional data and analysis of homologous sequences

sup-ported the classification of Bcp2 in the family of

peroxiredoxin in the 1-Cys Prx class and Bcp2 could

be considered the first ancient Prx developed in the

early stages of evolution

The results indicate that Bcp2 displays peroxidase

activity with a temperature optimum between 80 and

90
C which is the temperature range for growth of

S solfataricus The enzyme appears to be less thermo-stable (60% of activity after 15 min at 90
C) than the Prx of P horikoshii that retains full activity on heating

at 90
C for 20 min; this difference reflects the differ-ence in the optimum growth temperature between the two organisms The enzyme can function at 37
C as verified by the protection of DNA in MCO system and

by removal of H2O2 (data not shown) but it has the maximum activity in the range 80–90
C The peroxi-dase activity of Bcp2 is DTT dependent, suggesting a mechanism in which Cys49residue is firstly oxidized by

H2O2 and successively reduced by DTT that could be the electron donor partner in vitro The physiological partner has not yet been found Recently, a

thioredox-in reductase has been characterized thioredox-in S solfataricus; the presence in the genome of two thioredoxins and two other thioredoxin reductases suggest their involve-ment as physiological partners as electron donor These speculations require experimental evidence and studies are underway in our laboratory

Finally size-exclusion chromatography has shown that the protein can shift from a monomer to a multi-meric form depending on the protein concentration and the temperature On the basis of the data repor-ted in the literature on the structure of Prxs [25] ionic interactions play an important role in oligomerization; further analyses are in procress to define completely the quaternary structure of the enzyme

Experimental procedures Strains, media and growth conditions

Company, Franklin Lakes, NJ, USA), 0.1% tryptone (Oxoid, Basingstoke, Hampshire, UK) and 0.2% sucrose (TYS medium) in an orbital shaker Oxidative stresses

hydro-peroxide at a final concentration of 0.05 mm, 0.1 mm and 0.05 mm, respectively, to S solfataricus cultures in early

DNA manipulation, E coli XL1-Blue (Invitrogen SRL, Milan, Italy) was used to transform the mutagenesis

(Stratagene, La Jolla, CA, USA) was used for expression of the recombinant Bcp2 These strains were cultivated in

maintain plasmids

RNA extraction

hydroperoxide Aliquots were collected at different times by

was extracted by the guanidinium isothiocyanate method as

described in Sambrook et al [26] The integrity and

concen-tration of total RNA were verified by electrophoretic

analy-sis by separating total RNA on 1% agarose gel containing

formaldehyde

Northern hybridization

Northern blot analysis was used to quantify the amount of

the size of the specific transcript Genomic DNA from S

of bcp2 using HF Taq DNA polymerase (Roche Applied

Science, Monza, Italy) and the following primers: forward

primer (the inserted NdeI restriction site is underlined)

5¢-CTAGGTGAACATATGAGTGAGGAAAGAATTCC-3¢

and the reverse primer 5¢-GGAGCTGGATTAATGCTC

GAGTCTCCTATTAG-3¢ (the inserted XhoI restriction site

is underlined) The PCR products obtained were purified

from agarose gel, and the NdeI–XhoI fragment was labelled

kit (Roche)

Primer extension

Primer extension analysis was carried out with avian

myelo-blastosis virus reverse transcriptase (Roche) as described

in Limauro et al [27] using the synthetic oligonucleotide

to nucleotides 1801–1822 of DB source AE006819 The

sequencing reaction of the corresponding DNA fragment

cloned, which had been primed with the same synthetic

oligonucleotide, was used as a marker to locate the

Construction and expression of recombinant

protein

Genomic DNA of S solfataricus was prepared as described

by Arnold et al [28] bcp2 was amplified by PCR using

chromosomal DNA as template and the same two primers

as used to generate the northern blot probe Amplification

DNA polymerase (Roche) The PCR product was purified

with QIAquick PCR purification kit (Quiagen Spa, Milan,

Italy) and cloned in pGEMTeasy vector (Promega Italia

srl, Milan, Italy) The nucleotide sequence of the inserted

gene was determined to ensure that no mutations were pre-sent in the gene Then, the NdeI–XhoI fragment was cloned into pET-30c(+) (Novagen, Darmstadt, Germany) giving the recombinant plasmid pETBcp2 that was used to trans-form the E coli BL21-CodonPlus (DE3)-RIL

Purification of the recombinant Bcp2 protein (rBcp2)

Induc-tion was carried out by the addiInduc-tion of 1 mm isopropyl-thio-b-d-galactopyranoside to the medium and growth was continued for 12 h Cells were harvested by centrifugation,

ultrasonication (Sonicator Ultrasonic liquid processor; Heat System Ultrasonics Inc., Plainview, NY, USA) The suspen-sion was ultracentrifugated at 160 000 g for 30 min The

the denaturated proteins were removed by centrifugation (15 000 g for 30 min) The extract was concentrated (Am-icon, Millipore Corp., Bedford, MA) and loaded on a Hi-sTrap HP (Amersham Biosciences Europe GmbH, Milan,

(buffer A) After the column was washed with buffer A containing 20 mm imidazole, proteins were eluted with the same buffer A supplemented with 250 mm imidazole The active fractions were pooled and dialyzed against 20 mm

Amersham) connected to an FPLC system (Amersham) and

Determination of quaternary structure

The molecular mass of the protein was determined by

cm, Phenomenex Inc., St Torrance, CA, USA) connected to AKTA system (Amersham) Protein was eluted with buffer

(48.9 kDa), chymotrypsinogen (22.8 kDa) and the

RNA-se A (15.6 kDa) were uRNA-sed as molecular weight standards

Analytical methods for protein characterization

Protein concentration was determined using BSA as the standard [29] Protein homogeneity was estimated by

SDS⁄ PAGE [30] using a 12.5% (w ⁄ v) acrylamide resolving

gel and a 5% acrylamide stacking gel Samples were heated

and run in comparison with molecular weight standards

Gels were stained with the Coomassie blue

The molecular mass of the protein was also estimated

using electrospray MS recorded on a Bio-Q triple quadrupole

instrument (Thermofinnigan, San Jose, CA, USA) Samples

using a Phoenix syringe pump Spectra were collected and

elaborated using MASSLYNX software provided by the

manufacturer Calibration of the mass spectrometer was

performed with horse heart myoglobin (16.9 kDa)

Assay of peroxidase activity rBcp2 was tested for its

concentration of 0.2 mm to the reaction mixture

different concentrations of Bcp2 in a final volume of

and stopped by the addition of 0.9 mL trichloroacetic

from the amount of peroxide remaining, which was

detec-ted by measurement of the purple-coloured

ferrithiocya-nate complex developed after the addition of 0.2 mL

com-plex was determined by absorbance at 490 nm The

obtained without Bcp2

Thermophilicity and thermoresistance

Thermophilicity was evaluated in the temperature range

ferrithio-cyanate method rBcp2 thermoresistance was estimated by

differ-ent times

DNA cleavage assay by the MCO system

The ability of Bcp2 to protect DNA from oxidative

was determined as described previously [19]

DTT for the thiol MCO system, 100 mm Hepes pH 7.0,

different concentrations of rBcp2 or BSA as a negative

control The reaction was initiated incubating the mixture

and developed for an additional 4 h at the same

Site-directed mutagenesis

The mutant rBcp2 C49S was obtained by following the pro-tocol outlined in the QuickChange II site directed mutagen-esis kit (Stratagene) using primers complementary to the coding and noncoding template sequence (pET30Bcp2) con-taining a double mismatch To generate the C49S mutant,

CTACGGAGTTCTAC-3¢ and a complementary reverse pri-mer were used (the underlined letter indicates the base pair mismatch) The reaction (50 lL) contained 50 ng template DNA (pET30Bcp2), 125 ng each primer, 200 lm dNTP and 2.5 U Pfu Ultra HF DNA polymerase Twelve cycles of

for 2 min To digest methylated template, each reaction

Muta-genesis products were transformed into XL-1 Blue cells

kanamicin, and isolated plasmid DNA was sequenced throughout the coding region at Primm (DNA sequencing service Naples, Italy) The plasmid pETBcp2Mut containing the mutation inserted was used to transform BL21-Codon-Plus (DE3)-RIL competent cells C49S was expressed and purified with the same procedure reported above for rBcp2

Western blot analysis

0.05 mm tert-butyl hydroperoxide for 30, 60, and 120 min Cells were harvested by centrifugation, suspended in 20 mm

electrophoretically transferred to polyvinylidene difluoride membranes The membranes were blocked for 1 h with 5%

pH 7.5, 0.9% NaCl) and then incubated with rBcp2-specific rabbit antibodies (Igtech, Paestum, Salerno, Italy) for 2 h, followed by peroxidase-conjugated secondary antibodies for 1 h Antibodies to rBcp2 were detected by enhanced chemiluminescence using ECL Plus western blotting Detec-tion system (Amersham Biosciences)

Acknowledgements This work was supported by grants from MIUR (PRIN 2002)

References

1 Halliwell B & Gutteridge JMC (1989) Free Radicals in Biology and Medicine Oxford: Clarendon Press