Tài liệu Báo cáo khoa học: The alr2505 (osiS) gene from Anabaena sp. strain PCC7120 encodes a cysteine desulfurase induced by oxidative stress docx

encodes a cysteine desulfurase induced by oxidative stress Marion Ruiz, Azzeddine Bettache, Annick Janicki, Daniel Vinella, Cheng-Cai Zhang and Amel Latifi Aix-Marseille Universite´ and

encodes a cysteine desulfurase induced by oxidative stress Marion Ruiz, Azzeddine Bettache, Annick Janicki, Daniel Vinella, Cheng-Cai Zhang and Amel Latifi Aix-Marseille Universite´ and Laboratoire de Chimie Bacte´rienne, IBSM-CNRS, Marseille, France

Introduction

Sulfur is present in a wide range of biomolecules with

various chemical features, including enzymes catalyzing

many important chemical reactions During the last

decade, considerable progress has been made towards

understanding sulfur-trafficking processes in various

organisms, with particular attention being paid to the

enzymes that catalyze the reactions involved Pyridoxal

5¢-phosphate (PLP)-dependent cysteine desulfurases

have been found to provide the sulfur required for a

wide range of cellular processes, such as the synthesis

of molybdopterin [1–3], thiamin [4,5], the

thionucleo-tides in tRNA [6–9] and the assembly of Fe-S clusters

[10–15] The first cysteine desulfurase to be discovered

was the NifS protein, which is involved in the

forma-tion of the nitrogenase Fe-S cluster in

Azotobac-ter vinnelandii [15] Subsequently, cysteine desulfurases

have been identified in many organisms and classified

on the basis of sequence similarities into two groups:

I and II [16] Group I contains the NifS proteins

themselves and other subsets of cysteine desulfurases that are not restricted to diaztrophic organisms, namely the ISC and NFS proteins All the members of this group have the consensus sequence SSSGSAC(T⁄ S)S in com-mon The members of group II, which includes the enzymes SufS, CsdA and CpNifS, have the consensus sequence -RXGHHCA- [16] The cysteine desulfurases

in both groups are homodimers that use PLP to cata-lyze the elimination of sulfur from l-cysteine, yielding alanine and either sulfane (S) or sulfide (S=), in the presence of a reducing agent This reaction involves the formation of a persulfide intermediate (R-S-SH) that is bound to an essential cysteine residue close to the C-terminus of the enzyme The existence of this intermediate was first established in NifS from A vinn-elandii and, subsequently, based on structural studies,

in SufS from Escherichia coli [17,18] The persulfide cleavage is the rate-limiting step in the processes of catalysis A new catalytic cycle can only occur once

Keywords

Anabaena; cysteine desulfurase; Fe-S

clusters; oxidative stress; sulfur transfer

Correspondence

A Latifi Laboratoire de Chimie Bacte´rienne,

IBSM-CNRS, 31 Chemin Joseph Aiguier,

13402 Marseille, Cedex 20, France

Fax: +33 4 91 71 89 14

Tel: +33 4 91 16 41 88

E-mail: latifi@ifr88.cnrs-mrs.fr

(Received 22 May 2010, revised 27 June

2010, accepted 12 July 2010)

doi:10.1111/j.1742-4658.2010.07772.x

NifS-like cysteine desulfurases are widespread enzymes involved in the mobi-lization of sulfur from cysteine The genome of the filamentous diazotrophic cyanobacterium Anabaena PCC 7120 contains four open reading frames potentially encoding NifS-like proteins One of them, alr2505, belongs to the pkn22 operon, which enables Anabaena to cope with oxidative stress The Alr2505 protein was purified and found to share all the features characteris-tic of cysteine desufurases This is the first NifS-like enzyme to be function-ally characterized in this bacterium On the basis of the transcriptional profiling of all nifS-like genes in Anabaena, it is concluded that alr2505 is the only cysteine desulfurase-encoding gene induced by oxidative stress The function of Alr2505, which was termed OsiS, is discussed

Structured digital abstract

l MINT-7966515 : osis (uniprotkb: Q8YU51 ) and osiS (uniprotkb: Q8YU51 ) physically interact ( MI:0915 ) by two hybrid ( MI:0018 )

Abbreviations

PLP, pyridoxal-5¢-phosphate.

the sulfur atom is released from the persulfide and the

active site of the enzyme (i.e the cysteine residue)

becomes accessible [17] In vitro, this step can be

achieved by decomposing the persulfide into sulfide in

the presence of thiols, whereas, in vivo, it involves the

transfer of the persulfide-sulfur sulfane to a cysteine

residue of a sulfur acceptor protein In E coli, these

transpersulfuration reactions take the form of sulfur

being transferred from the cysteine desulfurases SufS

and CsdA to their sulfur acceptors, SufE and CsdE,

respectively [19,20]

In cyanobacteria, cysteine desulfurases have been

characterized in functional terms only in the case of

Synechocystis PCC 6803 This unicellular

nondiazo-trophic cyanobacterium possesses four ORFs, in which

the corresponding proteins show sequence similarities

with NifS (Slr0387, Sll0704, Slr5022 and Slr0077)

Slr0387, Slr5022 and Sll0704 belong to the group I

cys-teine desulfurases, whereas Slr0077 belongs to group

II The cysteine desulfurase activities of Slr0387 and

Sll0704 have been characterized in vitro [21–23] Both

proteins were found to be able to deliver sulfur to

apoferredoxin, and the finding that slr0387 and sll0704

mutants were obtained suggested that none of them

was essential for the viability of this cyanobacterium

[24] The cellular targets to which these proteins

trans-fer sulfur remain unknown

Slr0077 (SufS) appears to be the essential cysteine

desulfurase of Synechocystis PCC6803 because

attempts to obtain a fully-segregated mutant of this

gene have proved unsuccessful [24] Structural

charac-terization of this critical cysteine desulfurase suggested

that, similar to SufS and CsdA of E coli, it might

require an accessory sulfur acceptor protein [25] The

Slr0077 protein has been found to catalyse cysteine

desulfuration, as well as the conversion of cystine into

pyruvate, via a cystine lyase reaction that does not

require the conserved cysteine residue C372 [25,26]

The genome sequence of the filamentous

cyanobacte-rium Anabaena PCC7120 contains four ORFs, the

products of which (all1457, alr2495, alr3088 and

alr2505) show significant similarities to cysteine

desul-furases [27] The all1457 (nifS) gene has been reported

to be part of the nif operon, which is devoted to

nitro-gen fixation in this cyanobacterium [28], although, to

date, none of the cysteine desulfurase enzymes have

been characterized functionally In the present study,

which focused on the activity of Alr2505, it is

estab-lished that this enzyme effectively shows features

typi-cal of cysteine desulfurase The alr2505 gene is part of

the pkn22 operon, which also encodes the serine⁄

threo-nine kinase Pkn22 and the peroxiredoxine PrxQ-A In

a previous study, it was demonstrated that this operon

contributes importantly to the resistance of Anabaena

to oxidative stress [29,30] Because alr2505 is the only cysteine desulfurase-encoding gene induced in res-ponse to oxidative stress, we named the corresponding protein oxidative stress-induced cysteine desulfurase (OsiS)

Results

Alr2505 belongs to the NifS-like protein family

In a previous study, we began to investigate the contri-bution of the pkn22 operon to the response of

Anabae-na to oxidative stress The pkn transcriptional unit is composed of four ORFs: Alr2502, Alr2503, Asr2504 and Alr2505, the expression of which is specifically induced when Anabaena is exposed to oxidative condi-tions [30] We established that Alr2502 (Pkn22) is a serine⁄ -threonine kinase that regulates the CP43¢ (IsiA) protein [29], and that Alr2503 (PrxQ-A) is a proxire-doxin involved in defence against the oxidative stress

by reducing reactive oxygen species [30] The present study aimed to establish the function of the Alr2505 protein encoded by the last gene of this operon, which has been annotated as a putative aminotransferase in Cyanobase (http://www.kazusa.or.jp/cyano/Anabaena/ index.html) To further investigate this prediction, sequence alignment was performed on this protein, and the results obtained showed that Alr2505 demon-strates high levels of similarity with group I cysteine desulfurases, according to the classification of Mihara and Esaki [16] Not only the conserved amino acid ele-ments characteristic of this family of proteins (i.e the His-Lys motif required for PLP-cofactor binding and

an essential Cys residue at the active site) [15] are pres-ent in Alr2505, but also the consensus sequence around the active site (SSSGSACSS) is of the NifS-type (Fig 1B) The 3D structure of OsiS was predicted using the phyre server [31] The crystal structure of IscS [32] was consistently selected as a top candidate, with an e-value of 2.78· 10)43 The superposition of OsiS and IscS predicts an overall structure of OsiS monomer that is highly similar to that of IscS (Fig 1A), with a two-domain organization and the presence of both a-helices and b-strands On the basis

of these data and the fact that alr2505-expression is specifically induced under oxidative stress, we nemed this protein OsiS

Spectrophotometric properties of OsiS

To confirm the activity of OsiS predicted from the sequence-alignment data presented above, the osiS

gene was overexpressed in the heterologous host

E coli Purification of the OsiS protein was difficult

because it tended to aggregate into inclusion bodies

Because the histidine-tag can influence the solubility

and the folding of the protein in some cases, we also

purified the OsiS protein without any tag, using a

mono-Q purification column This method of

purifica-tion did not improve the solubility of the protein or

affect its activity; therefore, we continued our

investi-gation using the His-tagged protein Among the

vari-ous experimental approaches tested, solubilization by

urea was found to be the most efficient method of

purification, and this method was used to obtain the

enzyme followed by removal of urea and refolding

Pure OsiS showed the characteristic yellow colour

observed in the case of other PLP-containing enzymes,

including all the NifS-like proteins studied to date UV visible spectra showed an optimum for A390 (Fig 2A), which is consistent with the presence of PLP associated with the protein moiety The addition of 0.1 mm cyste-ine to the protein sample induced a shift in the major peak in the range 390–420 nm, which indicates that some interaction occurred with the cysteine substrate (Fig 2A) These spectral changes were not observed when 0.1 mm l-cystine was added instead of l-cyste-ine Therefore, tt is unlikely that l-cystine serves as a substrate of OsiS (data not shown)

OsiS–OsiS interactions Because all NifS-like proteins are homodimers, we aimed to assess whether OsiS also shows this feature The interactions between OsiS monomers were ana-lyzed using a bacterial two-hybrid system originally described by Karimova et al [33] This system exploits the fact that the catalytic domain of adenylate cyclase

C-term

N-term

A

B

Alr2505 Ana VSSGSACSSTKTAPSHVLTA 342

Slr0387 Syn LSSGSACSSYRTEASHVLYA 339

IscS A.Vin VSSGSACTSASLEPSYVLRA 341

IscS E.coli VSSGSACTSASLEPSYVLRA 341

IscS V.fis VSSGSACTSASLEPSYVLRA 356

AtNfsl Ara VSSGSACTSASLEPSYVLRA 262

Nifs Ana ASSGSACTSGSLEPSHVLRA 337

Fig 1 Sequence analysis (A) Prediction of the tertiary structure of

OsiS was performed using the PHYRE server (http://www.sbg.bio.ic.

ac.uk/phyre/) and the results were analyzed and visualized using

html) [53] The tertiary structure of IscS monomer was predicted

using the PHYRE server The two structures were then superposed.

The IscS monomer is shown in red and the OsiS monomer in yellow.

(B) Proteins similar to Anabaena OsiS (alr2505) were aligned using

(accession number NC_000911.1); IscS, Azotobacter vinelandii

(accession number AAC24472) IscS, E coli str K-12 (accession

number AAT48142); IscS Vibrio fischeri (accession number

YP_002155374.1) AtNFSI, Arabidopsis thaliana (accession number

NP_001078802); NifS Anabaena (accession number AAA22006).

Only the region surrounding the catalytic cysteine is shown.

0.21 0.22

0.16 0.17 0.18 0.19 0.2

0.12 0.13 0.14 0.15

Wavelength per nm

180

80 100 120 140 160

0 20 40 60

A

B

Fig 2 Physical characteristics of OsiS (A) Absorption spectra of OsiS were recorded before (bold line) and after (discontinued line) adding 0.1 m M of L -cysteine (B) Protein–protein interactions between OsiS monomers OsiS–OsiS interactions were detected using an E coli two-hybrid approach OsiS was fused to the T18 and T25 fragments The T18 ⁄ T25 combination was used as the negative control b-Galactosidase activities are expressed in Miller units (MU) Values shown are the means of three independent experiments.

of Bordetella pertussis consists of two complementary

fragments, T25 and T18 These fragments are not

active when physically separated However, if these

fragments are fused to interacting proteins, homo- or

heterodimerization of the resultant hybrid proteins

reconstitutes an active adenylate cyclase The cAMP–

catabolite activator protein complex can than activate

the transcription of target genes (e.g those of the lac

operon) Thereby, b-galactosidase activities obtained

during two hybrid reconstitution reflect the

dimeriza-tion of the proteins fused to the T25 and T18

frag-ments [33] Two-hybrid reconstitution systems have

been used to assess the dimerization of SufS and CsdA

cysteine desulfurases from E coli [20,34], as well as

NifS from Rhodobacter capsulatus [3] The fact that the

b-galactosidase activities, obtained with T15-OsiS and

T28-OsiS, were significantly higher than those of the

negative controls confirmed the specificity of the OsiS–

OsiS interactions (Fig 2B), strongly suggesting that

OsiS is able to form homodimers

Cysteine desulfurase activity of OsiS

To determine whether OsiS displays cysteine

desulfur-ase enzymatic activity, its ability to catalyze the

production of alanine from cysteine was tested

Apparent kinetic parameters were obtained, and the

enzyme showed Michaelis–Menten behaviour (Fig 3)

The Km value was estimated to be approximately

0.057 ± 3.54 mm, and Vmax was 190 ± 5.6 nmol

ala-nineÆmin)1Æmg protein)1, which is consistent with the

kinetic parameters of SufS-type cysteine desulfurases,

rather than those of IscS [25,35] When the cysteine

desulfurase activity of OsiS was monitored in the

pres-ence of apoferredoxin and iron, 2.5-fold more alanine

was produced (Table 1) The stimulation of OsiS

activ-ity in the presence of apoferredoxin suggests that OsiS

is able to transfer sulfur to apoferredoxin in vitro

The three cysteine desulfurases from E coli have

been found to catalyze abortive transamination

reac-tions, which convert the l-cysteine into pyruvate and

the PLP into pyridoxamine 5¢-phosphate, thus

inacti-vating the enzyme [35] The production of pyruvate

from cysteine by OsiS was measured and found to be

approximately 32 nmol pyruvateÆmin)1Æmg OsiS)1

Pyruvate can also be produced from cysteine by

cyste-ine lyase enzymes via a b-elimination reaction [26]

We therefore wanted to establish whether pyruvate

production by OsiS resulted from a lyase-type reaction

Accordingly, pyruvate formation catalyzed by OsiS

was measured in the presence of b-chloroalanine,

which is known to inhibit only the desulfurase

reac-tion Because no pyruvate was produced under these

conditions, it was concluded that OsiS is able to cata-lyze abortive transamination processes

Cys329 residue is likely the catalytic residue in OsiS

The sequence alignment data obtained indicate that the Cys329 residue is strictly conserved in all NifS-like proteins We therefore constructed a mutant of the OsiS protein (OsiS329S) in which the Cys329 residue was replaced by a serine residue This mutant protein was subsequently purified from E coli using the same procedure as that employed for OsiS OsiSC329S proved to be unable to sustain any cysteine desulfurase activity (Table 1), which strongly suggests that Cys329 would be the catalytic residue The replacement of this cysteinyl residue with a serine neither affected the spec-troscopic properties of OsiS, nor abolished its ability

to form dimers (data not shown)

Cysteine (µM)

–1 ·m

160 140 120 100 80 60 40 20 0

0 20 40 60 80 100 120 140 160 180

Fig 3 Cysteine desulfurase activities of OsiS Graph showing the rate-dependency of the reaction on substrate concentrations Assays were performed at 37 C in 25 m M Tris-HCl (pH 7.5),

50 m M NaCl, 10 m M dithiothreitol in the presence of 1 l M OsiS and L -cysteine as the substrate The production of alanine was determined as described in the Experimental procedures The line gives the best fits generated with ORIGIN 6.1 software (OriginLab Corporation Northhampton, MA, USA), based on the equation

V = Vmax[S] ⁄ (K m + [S]).

Table 1 Cysteine desulfurase activity of OsiS and OsiS C329S.

Enzyme

Cysteine desulfurase activity (nmol alanineÆmin)1Æmg protein)1) )Apoferredoxin +Apoferredoxina

a Additional details are provided in the text.

Expression and genomic organization of cysteine

desulfurase-encoding genes

In addition to osiS, analysis of the genome of

Anabae-na PCC 7120 [27] reveals the presence of three other

putative nifS-like genes (alr3088, alr1457 and alr2495)

The transcript levels of these four genes in Anabaena

were investigated under either standard growth

condi-tions or oxidative stress The latter was obtained by

treating the cyanobacterial culture with methyl

violo-gen As a positive control, we also investigated the

level of expression of the isiA transcript, which was

previously found to be up-regulated under oxidative

stress conditions [36,37] The transcription of all these

genes was assessed using the semi-quantitative

RT-PCR approach, as described in the Experimental

procedures The increase observed in the level of isiA

transcript confirmed the establishment of oxidative stress conditions in the cells under the present experi-mental conditions (Fig 4A) In response to methyl viologen treatment, only alr2505 (osiS) expression was induced, whereas the weak expression of alr2495 was constitutive The all3088 gene encoding a putative group I cysteine desulfurase was not expressed under our experimental conditions Lastly, as expected, the all1457 (nifS) gene was not expressed under either of the two conditions tested (Fig 4A) The nifS gene has been reported to be part of the nifB operon, the expression of which occurs only after a DNA-arrange-ment event induced during heterocyst differentiation [28] It has been suggested that the nifB, nifS and nifU genes, in view of their similarity with the correspond-ing genes from other diazotrophs, may be involved in the maturation of nitrogenase [28]

Group 1

all1457

nifS

all1516

fdxN

all1517

nifB

all1456

nifU

alr2505 asr2504

alr2495 alr2505

Group 2

alr3088 alr3088

alr2495

sufS

alr2494

sufD

alr2493

sufC

alr2492

sufB

all1457

SufE-like SufA-like

NifU-like

isiA

rnpB

all1431 alr2385

alr3513 asr1309

alr0692

all4341

pkn22 prxQ-A

C

Cysteine desulfurase Ferredoxin NifB:FeMoco core assembly Scaffold Energy producing system

A-type carriers Sulfur acceptor protein

Fig 4 Expression and genomic organization of cysteine desulfurase-encoding genes (A) RT-PCR analysis of cysteine desulfurases and cyst(e)ine lyase genes RNA was collected from cells grown in BG11 medium (line 0) or in BG11 incubated for 30 min (line 30), or 1 h (line 60) with 50 l M of methyl viologen One microgram of RNA was used in each experiment Samples were collected at the exponential phase

of the PCR All RT-PCR experiments were repeated twice, and similar results were obtained consistently Expression of the rnpB gene was used as the control assay All the primers used in the experiment were initially checked in PCR reactions with Anabaena genomic DNA as the template, at the same annealing temperatures as those used in the RT-PCR experiments (B) Organization of cysteine desulfurase-encoding genes in Anabaena genome The classification of the corresponding enzymes is indicated The ORFs surrounding these genes are indicated (C) Anabaena genome search for ORFs relevant to the Fe-S cluster assembly.

In addition to the Nif system, which is specifically

devoted to nitrogenase maturation, two other Fe-S

assembly systems exist in bacteria The role of these

mechanisms has emerged mainly from studies on

E coli [38,39] The ISC system (Fe-S cluster

forma-tion) comprises the housekeeping Fe-S assembly

sys-tem, whereas the SUF (sulfur mobilization) system

appears to be required under oxidative stress or iron

starvation conditions The three systems (NIF, ISC

and SUF) have in common the involvement of a

cyste-ine desulfurase, a scaffold protein that serves as a

con-struction site for Fe-S clusters before their transfer to

the apoproteins, and an A-type scaffold protein that

serves as a Fe-S cluster carrier [40] In addition, the

ISC and SUF systems include ATP-hydrolyzing

pro-teins such as the SufBCD complex This ATPase

com-plex in the SUF system has been shown to have a

scaffold function in E coli [41]

Among the four cysteine desulfurase genes from

Anabaena, only two are co-localized with genes involved

in sulfur transfer processes: the nifS gene, as explained

above, and the alr2495 (sufS) gene (Fig 4B) In

addi-tion to the sufS gene, the former cluster includes ORFs

which are similar to the SufBCD proteins Furthermore,

our bioinformatic analysis on the Anabaena genome

demonstrated the presence of ORFs showing similarities

with the NifU scaffold, the A-type scaffold SufA and

the sulfur acceptor SufE (Fig 4C) The relevance of this

analysis in terms of OsiS function is discussed below

IscR maturation in an iscS sufS mutant of E coli

expressing osiS

To assess whether OsiS could be involved in the

bio-genesis of Fe-S clusters, we investigated whether it

could compensate for the lack of cysteine desulfurase

in E coli We used the DV1247 strain (iscS sufS)

mutant that also harbours an iscR::lacZ fusion [42]

IscR is a Fe⁄ S protein encoded by the first gene of the

isc operon, and it is involved in the transcriptional

regulation of both the isc and the suf operons In its

holoform, IscR represses the isc operon transcription,

whereas, in its apoform, it acts as an activator of the

sufoperon [43,44] In the iscS mutant, the IscR

regula-tor is not maturated [43] and can hence activate the

suf operon transcription To avoid a possible effect of

SufS over-expression as a result of the iscS mutation,

we used the double mutant iscS sufS rather than a

simple iscS mutant The activity of the iscR::lacZ

fusion was assessed in the genetic backgrounds

reported in Fig 5 As expected, in the absence of the

IscS and SufS cysteine desulfurases, the activity of the

iscR::lacZ fusion was de-repressed compared to

the wild-type context (MG1655 strain) However, when the expression of the osiS gene was induced from the pBAD promoter, the fusion showed the same activity

as in the wild-type background This result suggests that, when osiS is over-expressed, the maturation of the IscR repressor occurred sufficiently to allow it to fulfil its function Furthermore, the observation that the effect of OsiS was strictly dependent upon the pres-ence of arabinose strongly confirms the above conclu-sion Because the maturation of IscR might need a supply of sulfur specifically from IscS (D Vinella, unpublished results), we conclude that, in the DV1247 strain, OsiS over-production compensates for the absence of IscS with respect to IscR maturation

Discussion

In the present study, the first functional characterization

of a cysteine desulfurase in Anabaena 7120 is presented OsiS is encoded by the last gene of the pkn22 operon, which contributes to the response to oxidative stress, as previously described [29,30] OsiS shows all the proper-ties and features typical of NifS-enzymes: (a) its amino acid sequence includes the consensus sequence of the group I cysteine desulfurases; (b) OsiS is a homodimer and binds to a PLP cofactor; and (c) it catalyzes the formation of alanine and sulfide, using l-cysteine as a substrate The results of site-directed mutagenesis

A

1400 1200 1000 800 600 400 200 0

Fig 5 b-galactosidase activities of the IscR::lacZ fusion The MG1655 (iscR::lacZ) or the DV1247 strain and its recombinant derivatives were grown overnight in LB medium as explained in the Experimental procedures Cultures were used to inoculate fresh LB medium supplemented (or not) with arabinose The b-galactosidase activities were measured as described in the main text The data are the means of values obtained from three independent clones The experiment was repeated twice A, MG1655 (iscR::lacZ);

B, DV1247 ⁄ pBAD24; C, DV1247 ⁄ pBAD24:osiS plus arabinose;

D, DV1247 ⁄ pBAD24:osiS minus arabinose.

showed that the cysteine residue C329 is the binding site

of the persulfide intermediate

The kinetic properties of OsiS indicate that it has a

weak cysteine desulfurase activity Similar activities

have been reported in the case of cysteine desulfurases

from other organisms In some cases, it was concluded

that this inefficiency reflects the involvement of an

accessory factor that accepts the sulfur from the

enzyme, and thus enhances its activity This was found

to be the case, for example, with SufS of E coli, which

is stimulated by the SufE protein [25,34,35,45]; sufS

and sufE genes belonging to the same operon The

pkn22 operon lacks a sufE-like gene (Fig 4B)

How-ever, the Anabaena genome contains a putative ORF

(Alr3513) encoding a protein showing 31% identity

with SufE (Fig 4C) Whether this SufE-like protein

may act as a sulfur acceptor for OsiS will be the

sub-ject of future studies The stimulation of the OsiS

activity by the presence of apoferredoxin is consistent

with the suggestion that sulfur transfer to an acceptor

protein may facilitate the turnover of OsiS and

enhance its activity (Table 1)

It is likely that the in vitro catalysis of

holoferredox-in reconstitution holoferredox-in the presence of iron is ability

com-mon to most cysteine desulfurases This property has

often been considered to reflect the involvement of

these enzymes in the biogenesis of Fe-S clusters

Indeed, such a conclusion can only be made with great

caution because this in vitro finding simply means that

these enzymes are able to deliver sulfur, and does not

necessarily reveal the nature of the targets to which

the sulfur is transferred The question remains

con-cerning the role of OsiS in vivo Because the level of

expression of the osiS gene is undetectable under

stan-dard growth conditions, it can be concluded that OsiS

is not required for housekeeping purposes The most

likely hypothesis appears to be that this protein

con-tributes to cell defence against oxidative stress It has

been established that some reactive oxygen species can

react with Fe-S clusters and thus induce their

oxida-tion [46] The repair and⁄ or synthesis of damaged Fe-S

clusters under oxidant conditions therefore represents

an important challenge It is tempting to speculate that

OsiS may contribute to these processes by providing

sulfur, when Anabaena is exposed to an oxidative

threat The fact that the over-expression of OsiS

com-plements the iscS mutation for the maturation of IscR

confirms this hypothesis However, OsiS could not

res-cue the auxotrophy of the iscS mutant for thiamine

(data not shown) Whether this result would mean that

OsiS could deliver sulfur only to Fe-S biogenesis

ways or also to other sulfur-using biosynthetic

path-ways in Anabaena remains unanswered

On the basis of the data obtained for the Anabaena genome presented in Fig 4, it is tempting to speculate that the SUF system [alr2492(SufB), alr2493(SufC), alr2494(SufD), alr2495(SufS)] might be the housekeep-ing Fe-S assembly system in this cyanobacterium Indeed, Anabaena lacks a counterpart of the ISC sys-tem, and a group 2 cysteine desulfurase was found to

be the essential cysteine desulfurase in Synechcoystis PCC6803 [25] Because the alr3088 gene is not expressed under normal growth conditions (Fig 4A), it

is likely that its product (a group 1 cysteine desulfur-ase) may be required under stress or starvation condi-tions that still remain to be identified The characterization of OsiS constitutes the starting point for the study of the basic mechanisms involving sulfur transfer in Anabaena, as well as other cyanobacteria in general because little is known so far about these mechanisms

Experimental procedures

Bacterial strains, plasmids and growth conditions

grown in the LB medium were used to express the cysteine desulfurase OsiS and its mutant derivative OsiS C329S The BHT101 E coli [33] strain was used for the two-hybrid

DsufS::kan zdj-925::Tn10 MVA+) [42] was grown in LB medium supplemented with mevalonate (1 mm),

required

The expression of the osiS gene in E coli strains was per-formed as following: a DNA fragment corresponding to the entire coding region of alr2505 was amplified by PCR using the Osi top primer 5¢-GAATTCATGTCTAATCGTCCTA-TATATC-3¢ (EcoRI site underlined) and the Osi bottom

site underlined) The PCR product was cloned into the pBAD24 plasmid [47] After DNA sequencing analysis, recombinant plasmids were introduced in E coli strains

Expression and purification of recombinant proteins

A DNA fragment corresponding to the entire coding region

of alr2505 was amplified by PCR using the Osi forward primer 5¢-GAATTCATGTCTAATCGTCCTATATATC-3¢

(EcoRI site underlined) and the Osi reverse primer 5¢-CTC

GAGTACCAAAGTTGCTTGTT-3¢ (XhoI site underlined)

The PCR product was cloned into the pET22 vector

(Novagen) A clone confirmed by DNA sequencing was

0.3–0.4 was reached, and protein expression was induced by

adding 1 mm isopropyl thio-b-d-galactoside for 4 h After

sonication, OsiS protein aggregated into inclusion bodies

Urea solubilization was performed using a final

urea-con-centration of 6 m The extracts were then dialyzed in a

three-step experiment (3, 1 and 0 m urea) to eliminate urea

The recombinant proteins were purified using Hitrap

columns in accordance with the manufacturer’s instructions

(Amersham Pharmacia, Piscataway, NJ, USA) Imidazol

was removed from purified proteins using PD10 columns

(Amersham Pharmacia) Proteins were concentrated on

Vivaspin columns (Sartorius, Gottingen, Germany) and

used for subsequent analyses Proteins were separated by

procedure (Euromedex, Mundolsheim, France)

Site-directed mutagenesis

The mutation of the cysteine residue at position 329 of OsiS

into a serine residue was performed by PCR using the

megaprimer strategy [48] The primers used were: forward

mut primer 5¢-GAATTCATGTCTAATCGTCCTATATA

TCTTGACT-3¢ (EcoRI site underlined), internal mut primer

5¢-TCCGCTTCTTCCTCCA-3¢ and reverse mut primer

underlined) The PCR product was cloned into the vector

pET22 Recombinant plasmids were confirmed by DNA

sequencing The mutant protein was expressed and purified

using the same procedure as for the wild-type protein

E coli two-hybrid assays

To fuse the C- and N-terminals of OsiS to adenylate

cyclase, the osiS gene was amplified by PCR using the

primers OsiS-TH forward 5¢-CTC GAG CTA TAC CAA

AGT TGC TT-3¢ (XhoI site underlined) and OsiS-TH

reverse 5¢-GAA TTC ATG GTT CAA TTT ATC CCA-3¢

(EcoRI site underlined) The PCR fragments were cloned

into the XhoI and EcoRI sites of vectors pT25-zip and

pT18-zip [33] BHT101 strain was co-transformed with the

pT18- and pT25-based plasmids and incubated overnight at

thio-b-d-galactoside b-galactosidase activity was assayed

and expressed in Miller units [49] Plasmids pT25 and pT18

were used as negative controls

Cysteine desulfurase assay

The cysteine desulfurase activity was quantified by

deter-mining the amount of alanine formed from l-cysteine

(Sigma, St Louis, MO, USA) The standard reaction mix-ture in a final volume of 100 lL was: 25 mm Tris, pH 7.5,

100 mm NaCl, 10 mm dithiothreitol and 100 lm PLP Final protein concentrations were 1 lm of OsiS or OsiSC329S Reactions were initiated by adding variable concentrations

of l-cysteine (final concentration in the range 0–250 lm)

Dena-tured proteins were removed by centrifugation, and the supernatant was analyzed to determine its alanine content

by performing an alanine dehydrogenase assay [50] The alanine content of assay mixtures was determined based on

desulfurase activity is expressed in units corresponding to

using 0.12 lmol NADH and 10 lg of lactate dehydroge-nase (Sigma) in a final volume of 0.5 mL Oxidation of

Apoferredoxin preparation and Fe-S cluster reconstitution

The Fe-S cluster was incorporated into apoferredoxin using apoferredoxin from Spinach (Sigma) as substrate Apoferre-doxin was obtained from holoferreApoferre-doxin as described previ-ously [51] The reconstitution experiment of the Fe-S cluster was carried out in 50 mm Tricine–NaOH (pH 7.5)

10 mm dithiothreitol, 0.1 mm PLP, 1 lm apoferredoxin and

1 lm of OsiS cysteine desulfurase The proteins were han-dled under anaerobic conditions Cysteine desulfurase activ-ity was measured as described above

Semi-quantitative RT-PCR experiments RNA was extracted as described previously [29] One microgram of RNA was used in each RT-PCR experiment Samples were collected at the exponential phase of the Table 2 Sequences of the primers used in the RT-PCR experiments Gene Primers (5¢- to 3¢)

rnpB Forward: AGG GAG AGA GTA GGC GTT GC

Reverse: GGT TTA CCG AGC CAG TAC CTC T isiA Forward: GCC CGC TTC GCC AAT CTC TC

Reverse: CCT GAG TTG TTG CGT CGT TA alr2495 Forward: AAA ACG GCT GCA GTT CTC A

Reverse: CCC AAT TGC AGG TGT ACC alr3088 Forward: GTT TTA GTT TCT GTT ATT TAC GGT CAA

Reverse: TTC TCT GTC GCC GGT GGG GAT alr1457 Forward: AAT ATC GCC GTT AAC TTC GC

Reverse: GCC TTG GTG ACA ATT ATG TA alr2505 Forward: GTT GCA ACA CAC CAA TTT CG

Reverse: CAA GCA CGG GAA ATT TTA GC

PCR All RT-PCR experiments were repeated three times,

sequences of all the primers used in this experiment are

Analytical methods

Protein was assayed as described by Bradford [52], using

BSA as the standard Absorption spectra were recorded in

a Varian Cary 50 Bio UV-visible spectrophotometer

(Var-ian Inc., Palo Alto, CA, USA) The reaction mixtures

were used to record the respective baselines

Acknowledgements

We are grateful to B Py for valuable discussions We

thank the members of the Gudicci–Orticoni

Labora-tory for their help with the anaerobic experiments We

thank Jessica Blanc for revising the English

manu-script This work was supported by an ‘Environnement

et sante´’ grant (AFSSE)

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