The alr2505 (osiS) gene from Anabaena sp. strain PCC7120
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

(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)bytwo hybrid (MI:0018)
Abbreviations
PLP, pyridoxal-5¢-phosphate.
FEBS Journal 277 (2010) 3715–3725 Journal compilation ª 2010 FEBS. No claim to original French government works 3715
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

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

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
OsiS: oxidative stress-induced cysteine desulfurase M. Ruiz et al.
3716 FEBS Journal 277 (2010) 3715–3725 Journal compilation ª 2010 FEBS. No claim to original French government works
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 A
390
(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

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
UCSF
CHIMERA software (http://www.cgl.ucsf.edu/chimera/download.
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
CLUSTAL W. Alr2505, Anabaena OsiS; Slr0387, Synechocystis PCC6803
(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

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 K
m
value was estimated to be approximately
0.057 ± 3.54 mm, and V
max
was 190 ± 5.6 nmol ala-
nineÆmin

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).
C
y
steine (
µ
M)
Velocity (nmole alanine·min
–1
·mg
–1
OsiS)
160
140
120

)1
)
)Apoferredoxin +Apoferredoxin
a
OsiS 123 ± 04 308 ± 06
OsiS C329S 0 Not determined
a
Additional details are provided in the text.
OsiS: oxidative stress-induced cysteine desulfurase M. Ruiz et al.
3718 FEBS Journal 277 (2010) 3715–3725 Journal compilation ª 2010 FEBS. No claim to original French government works
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

alr2494
sufD
alr2493
sufC
alr2492
sufB
all1457
SufE-likeSufA-likeNifU-like
isiA
rnpB
all1431
alr2385
alr3513
asr1309
alr0692
all4341
030 60
pkn22
prxQ-A
AB
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

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
suf operon [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

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.
OsiS: oxidative stress-induced cysteine desulfurase M. Ruiz et al.
3720 FEBS Journal 277 (2010) 3715–3725 Journal compilation ª 2010 FEBS. No claim to original French government works
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

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 path-
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

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
primer 5¢-
GTCGACTACCAAAGTTGCTTGTT-3¢ (SalI
site underlined). The PCR product was cloned into the
pBAD24 plasmid [47]. After DNA sequencing analysis,
recombinant plasmids were introduced in E. coli strains.
osiS expression was induced using arabinose.
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¢
M. Ruiz et al. OsiS: oxidative stress-induced cysteine desulfurase
FEBS Journal 277 (2010) 3715–3725 Journal compilation ª 2010 FEBS. No claim to original French government works 3721
(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
grown in ampicillin-supplemented medium until A
600
of
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.

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
30 °C in LB medium supplemented with 1 mm isopropyl
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)
and allowed to continue for 3 h at 37 °C. Reactions were
stopped by heating the mixtures at 99 °C for 10 min. 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
A
340
for NADH (e

4
)
2
,
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¢-to3¢)
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
OsiS: oxidative stress-induced cysteine desulfurase M. Ruiz et al.
3722 FEBS Journal 277 (2010) 3715–3725 Journal compilation ª 2010 FEBS. No claim to original French government works
PCR. All RT-PCR experiments were repeated three times,
and similar results were obtained consistently. The

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