J. Microbiol. Biotechnol.

(2012), 22(6), 736–741

http://dx.doi.org/10.4014/jmb.1112.12032
First published online March 19, 2012
pISSN 1017-7825 eISSN 1738-8872

Negative Role of wblA in Response to Oxidative Stress in Streptomyces

coelicolor
Kim, Jin-Su1, Han-Na Lee1, Pil Kim2, Heung-Shick Lee3, and Eung-Soo Kim1*
1
Department of Biological Engineering, Inha University, Inchon 402-751, Korea
2
Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi 420-743, Korea
3
Department of Biotechnology and Bioinformatics, Korea University, Jochiwon, Chungnam 339-700, Korea
Received: December 16, 2011 / Revised: January 23, 2012 / Accepted: January 25, 2012

In this study, we analyzed the oxidative stress response of
wblA (whiB-like gene A, SCO3579), which was previously
shown to be a global antibiotic down-regulator in Streptomyces
coelicolor. Ever since a WblA ortholog named WhcA in
Corynebacterium glutamicum was found to play a negative
role in the oxidative stress response, S. coelicolor wblA has
been proposed to have a similar effect. A wblA-deletion
mutant exhibited a less sensitive response to oxidative
stress induced by diamide present in solid plate culture.
Using real-time RT-PCR analysis, we also compared the
transcription levels of oxidative stress-related genes,
including sodF, sodF2, sodN, trxB, and trxB2, between S.
coelicolor wild type and a wblA-deletion mutant in the
presence or absence of oxidative stress. Target genes were
expressed higher in the wblA-deletion mutant compared
with wild type, both in the absence and presence of oxidative
stress. Moreover, expression of these target genes in S.
coelicolor wild type was stimulated only in the presence of
oxidative stress, suggesting that WblA plays a negative
role in the oxidative stress response of S. coelicolor, similar
to that of C. glutamicum WhcA, through the transcriptional
regulation of oxidative stress-related genes.
Keywords: wblA, Streptomyces coelicolor, oxidative stress
Streptomyces coelicolor is a high G+C Gram-positive
filamentous soil bacterium belonging to the order of
Actinomycetales. Previously, we identified and characterized
a novel global antibiotic down-regulator named whiB-like
gene A (wblA, SCO3579) in S. coelicolor as well as wblA
orthologs in several other Streptomyces species [12]. The
whiB was reported to be a developmental regulatory gene
essential for sporulation of aerial hyphae [7], and 11 whiB-
like genes have been identified in the chromosome of S.
*Corresponding author
Phone: +82-32-860-8318; Fax: +82-32-872-4046;
E-mail: eungsoo@inha.ac.kr
coelicolor [9]. Furthermore, whiB-like genes, which are
found only in the order Actinomycetales, have been identified
in other species; seven and four whiB-like genes in
Mycobacterium tuberculosis and Corynebacterium glutamicum,
respectively [15, 20]. The WhiB-like proteins are believed
to be putative transcription factors involved in the regulation
of diverse cellular processes, such as cell division,
differentiation, pathogenesis, starvation survival, and the
stress response. All WhiB-like proteins are known to
contain four conserved cysteine residues that are often
coordinated with redox-sensitive Fe-S clusters [5, 6, 11, 19],
implying that WhiB-like proteins could be reviewed in the
general context of redox regulation in actinobacteria [8].
It is well known that all aerobic bacteria inevitably
experience oxidative stress caused by reactive oxygen
species (ROS), which are generated as by-products of
aerobic respiration. To protect cells against ROS such as
superoxide anion or hydrogen peroxide, bacteria have
evolved complex oxidative defense and repair systems [8].
Superoxide dismutase (SOD) is known to be involved
in the first stage of antioxidant defense via eliminating
superoxide anion. Specifically, it catalyzes the disproportionation
of superoxide anion to hydrogen peroxide and molecular
oxygen. S. coelicolor is reported to contain two types of
superoxide dismutase; Ni-containing SodN and Fe-containing
SodF [4]. Thioredoxins are also critical in the redox
regulation of protein function and signaling via thiol redox
control. The oxidized form of thioredoxin is reduced by
the trx gene product thioredoxin reductase, using NADPH
as a cofactor [1]. Subsequently, reduced thioredoxin provides
reducing equivalents to thioredoxin peroxidase, which breaks
down hydrogen peroxide to water, and ribonucleotide
reductase for DNA synthesis [16].
Recently, a WblA ortholog named WhcA in C. glutamicum
was found to play a negative role in the oxidative stress
response. In that study, cells overexpressing WhcA protein
not only showed retarded growth as compared with wild-
737 Kim et al.
type or whcA-deleted mutant cells but also increased
sensitivity to a variety of oxidants such as diamide, menadione,
and hydrogen peroxide [3]. Thioredoxin reductase activity
was also repressed in whcA-overexpressing cells, whereas
its activity in the whcA-deleted mutant strain was derepressed
regardless of oxidative stress [3]. Here, we show that S.
coelicolor wblA has a similar negative effect on the
expression of genes involved in the oxidative stress
response via transcriptional regulation of oxidative stress-
related genes, including SCO0999 (sodF2, superoxide
dismutase), SCO2663 (sodF, superoxide dismutase [Fe-
Zn]), SCO2554 (sodN, superoxide dismutase), SCO3890
(trxB, thioredoxin reductase NADPH), and SCO6834
(trxB2, thioredoxin reductase).
MATERIALS AND METHODS
Strains, Media, and Growth Conditions
All experiments were performed using S. coelicolor A3(2) strains
M145 and wblA mutant. The S. coelicolor ∆wblA mutant and the
wblA-overexpression strains have been described elsewhere [12]. The
wblA-overexpression mutant strain was constructed via amplification
of the wblA gene using the primer pair 5'-GGATCCTGTTGCGCT
GTGCCCA-3' and 5'-TCTAGAACCCGAGCTGCCCGAG-3'. E.
coli DH5α cells and the Streptomyces integrative expression vector
ermE*pSET152 were used for cloning. The E. coli ET12567/
pUZ8002 was used as the transient host for E. coli-Streptomyces
conjugation [2]. E. coli and Streptomyces strains were cultured at
30oC and 37oC, respectively. TSB (Tryptic Soy Broth) and modified
R5 liquid media were used as seed culture and main culture media,
respectively. Agar medium for Streptomyces strains was R2YE, and
the S. coelicolor wblA-overexpression mutant strain was cultured
with 50 µg/ml of apramycin when needed.
RNA Analysis by Real-Time RT-PCR
RNA was prepared using an RNeasy Mini Kit (Qiagen). The cDNA
conversion was carried out with a PrimeScript 1st strand cDNA
Synthesis Kit (TaKaRa) by following the manufacturer’s instructions.
Real-time RT-PCR was performed using TaKaRa SYBR Premix Ex
Taq (Perfect Real Time) with a Thermal Cycler Dice Real Time
System Single (code TP850) (TaKaRa). The following primers were
used for real-time RT-PCR: SCO0999_F, 5'-GCACATTCTGCA
CTCGATCT-3'; SCO0999_R, 5'-GTCGAAGACGAGGATCGG-3';
SCO2633_F, 5'-ACTCGATCTACTGGCACAACA-3'; SCO2633_R,
5'-GTCGAAGACGAGGATCGG-3'; SCO5254_F, 5'-CCGTCCAGG
AGAAGATGG-3'; SCO5254_R, 5'-GTCGATCTGGGCGATGTAG-
3'; SCO3890_F, 5'-ACCGAGGTGGAGAACTTCC-3'; SCO3890_R,
5'-AAGAAGAAGCCGTCACAGGT-3'; SCO6834_F, 5'-CCGAGC
TCATGGAGAACAT-3'; and SCO6834_R, 5'-CCTCCTCCATGG
CAGTGT-3'. All reactions were performed in duplicate. The PCR
conditions included activation for 10 min at 95oC, followed by 35
cycles of 30 s at 95oC, 30 s at 58oC, and 30 s at 72oC. Data were
collected during each 72oC step, and melting curve analysis was
performed at default settings from 60 to 95oC. The relative level of
amplified mRNA was normalized to the mRNA expression level of
the housekeeping gene S. coelicolor hrdB, which was amplified as
an internal control using the primer pair hrdB_F (5'-GCGGTGGAG
AAGTTCGACTA-3') and hrdB_R (5'-TTGATGACCTCGACCATG
TG-3').
Biochemical Analysis
The sensitivity of the S. coelicolor wild-type and wblA mutant strains
to diamide, which is a thiol-specific oxidant, was assessed on plate
culture. All strains were spread on R2YE plates. A total of 3.44 mg
(20 µl of 1 M) of diamide (N,N,N',N'-tetramethylazodicarboxamide)
was then applied to paper disks, which were positioned on the
center of the plate. The plate was then incubated at 30oC for 72 h
until the formation of a clear zone had completely occurred.
RESULTS
Analysis of wblA Gene and Its Effect on Cell Growth
In the genome of S. coelicolor M145, there are 11 wbl
(whiB-like) genes, and five (whiB, whiD, wblC, wblA, and
wblE) of them are well conserved in other actinobacteria
[9]. Whereas wblA has been determined to be a novel
antibiotic down-regulator, the biological functions of most
wbl genes remain to be determined [7, 12]. In the genome
of C. glutamicum ATCC13032, four ORFs (NCgl0275,
NCgl0574, NCgl0711, and NCgl0734) encoding homologs
of S. coelicolor WhiB protein have been identified.
Among them, whcA (NCgl0275) has been proposed to
play a negative role in the oxidative stress response of C.
glutamicum [6, 15]. WblA exhibits relatively high identity
scores of 64% with WhcA, due to its conserved redox-
sensitive domains, including Cys-X21-Cys-X2-Cys-X5-
Cys and Helix-Loop-Helix (HLH) motifs (Fig. 1) [8, 10,
20]. Based on these similar properties, we hypothesized
Streptomyces gene wblA to be a homolog of corynebacterial
gene whcA.
To determine the physiological significance of wblA, we
monitored the growth patterns of S. coelicolor M145 wild-
type, ∆wblA mutant, and wblA-overexpressing strains (Fig. 2).
Each strain was inoculated into modified R5 medium after
2-day seed culture, followed by dry-cell weight measurement
for 5 days. These experiments were performed independently
in triplicate. As shown in Fig. 2, all three strains grew
comparably and showed no significant differences throughout
the 5-day culture period, suggesting that deletion or
overexpression of wblA did not affect normal cell growth
under the culture conditions in this study. Previously, C.
glutamicum wild-type and ∆whcA mutant strains were
shown to exhibit almost identical growth rates, whereas
the whcA-overexpressing strain exhibits a retarded growth
rate [3].
Response of Mutant Cells to Oxidative Stress and wblA
Expression
Assuming that wblA plays a role in the stress response, we
challenged the wild-type, ∆wblA mutant, and wblA-
 WBLA RESPONSE TO OXIDATIVE STRESS IN STREPTOMYCES COELICOLOR 738

Fig. 1. (A) Phylogeny of whiB-like proteins in S. coelicolor (SCO3579, SCO3034, SCO6715, SCO6922, SCO5240, SCO7106,
SCO7306, SCO6965a, SCO4767, SCO5046, SCO5190) and C. glutamicum (NCgl0275, NCgl0711, NCgl0734, NCgl0574) and (B)
alignment of Wbl proteins with S. coelicolor and C. glutamicum.
The CXCXCXC motif is shown and marked with a thick line. The helix-loop-helix DNA-binding motif is shown and marked with a broken line.

overexpressing cells with an extracellular stress inducer.
When oxidative stress was induced by treating cells with a
thiol-specific oxidant, diamide, on a paper disk, the growth
of ∆wblA mutant exhibited higher resistance with a smaller
clearing zone compared with that of wild type (Fig. 3).
Moreover, wblA-overexpressing cells showed a more sensitive
phenotype against diamide, implying that S. coelicolor
wblA indeed plays a negative role in the oxidative stress
response similar to C. glutamicum whcA.
Now knowing that the wblA gene is negatively involved
in the regulatory response to thiol-specific oxidative stress,
we investigated whether or not the expression of the wblA
gene is affected by oxidative stress. For this, S. coelicolor
wild-type cells were cultured for approximately 40 h to
early stationary phase and then left to grow for an
additional 8 h after addition of 1 mM diamide. The cells
challenged with or without diamide were harvested separately,
after which wblA transcription levels were determined by
real-time RT-PCR. Although both wblA transcripts were
extremely low, no significant stimulation or reduction in
wblA transcription was observed upon diamide treatment.
These results suggest that the expression of wblA gene was
not directly regulated by the oxidant (data not shown).

Effects of Oxidative Stress on Expression of sod and

Fig 2. Growth of S. coelicolor strains in modified R5 medium for
5 days; wild type ( ▼ ), ∆wblA ( ○ ), wblA-overexpression ( ● ).
All cultures were performed in triplicates.
trxB Genes
To investigate the effects on the oxidative stress response,
we compared the expression levels of target genes between
S. coelicolor wild type and the ∆wblA mutant strain via
739 Kim et al.
Fig 3. Sensitivity of the S. coelicolor strains to diamide.
A paper disk containing diamide was placed on each plate containing
lawns of S. coelicolor M145 wild type or wblA mutants, followed by
incubation at 30oC for 3 days.
real-time RT-PCR. Based on previous studies, the target
genes related to oxidative stress response were selected to
be three superoxide dismutase genes (sodF, sodF2, and
Fig 4. Real-time RT-PCR analysis of various target genes.
S. coelicolor M145 wild type or ∆wblA mutants were grown in modified R5 liquid medium to early stationary phase and exposed for 8 h to 1 mM diamide.
(A) Real-time analysis. The y-axis scale represents the expression value relative to that of hrdB, a housekeeping sigma factor, which was set to 1. (B)
Detection of mRNA by RT-PCR. The hrdB was used as a control.
sodN) and two thioredoxin reductase genes (trxB and
trxB2) [8]. As stated above, sodF (SCO2633) and sodF2
(SCO0999) are Fe-containing superoxide dismutases,
whereas sodN (SCO5254) is a Ni-containing superoxide
dismutase [14]. In addition, two of the genes encode
thioredoxin reductase (SCO3890, trxB), whereas another
encodes a putative thioredoxin reductase (SCO6824, trxB2).
Although not much information is available on the
mechanism of the oxidative stress response in S. coelicolor,
sigR, which encodes a RNA polymerase sigma factor, is
believed to be involved in the thioredoxin response to thiol-
specific oxidative stress [8, 17]. Therefore, the expression
levels of these five sod and trx genes were quantitatively
measured via real-time RT-PCR.
The mRNA levels of sodF and sodF2 in the ∆wblA
mutant strain were significantly higher than those in wild
type (Fig. 4). Transcription of trxB was also induced in the
∆wblA mutant strain, even though the induction level was
much lower. Meanwhile, no noticeable differences in sodN
and trxB2 expression were detected owing to their intrinsically
low levels of expression between the S. coelicolor wild-
type and ∆wblA mutant strains (Fig. 4). These results
suggest that wblA plays a negative role in the oxidative
stress response through transcriptional regulation of the
sodF, sodF2, and trxB genes. We also investigated whether
or not the expression of the sod and trx genes would be
affected by an oxidizing agent. Diamide challenge with the
S. coelicolor wild type significantly stimulated the expression
of the sodF, sodF2, and trxB genes, implying that addition
of an oxidizing agent to the S. coelicolor culture led to the
stimulation of wblA-dependent oxidative stress response
genes. Interestingly, however, the expression levels of the
 WBLA RESPONSE TO
sodF, sodF2, and trxB genes were reduced in the ∆wblA
mutant strain upon diamide challenge, suggesting that the
wblA-dependent oxidative stress response might be controlled
by some additional regulatory systems in S. coelicolor.
DISCUSSION
In this study, we performed functional analysis of a
specific whiB-like gene, wblA of S. coelicolor. The WhiB-
like proteins carry a common CXXC motif that might be
involved in redox sensing [8, 10]. In addition, in the C-
terminus of the protein, a helix-loop-helix motif that plays
a putative role in DNA-binding is present. However, DNA-
binding of WhiB-like proteins to target DNA sequences as
transcription factors has never been demonstrated
experimentally. Despite the presence of common characteristics,
whiB-like genes have been shown to participate in diverse
cellular processes. Consistent with this trend, we have
found that the wblA gene played a distinctive negative role
in the regulation of the oxidative stress response network
of S. coelicolor.
Growth experiments indicate that the wblA gene is
dispensable under ordinary growth conditions. The finding
that genes encoding superoxide dismutase and thioredoxin
reductase are under the negative control of wblA indicates
that this gene may function in a stress-response network.
Similarly, thioredoxin reductase, NADH oxidase, alcohol
dehydrogenase, and quinone reductase have been found to
be under the negative control of whcA in C. glutamicum.
Previously, we reported that the wblA gene of S. coelicolor,
which is a homolog of whcA, functions as a down-regulator
of genes involved in the production of antibiotics and
morphological differentiation [12]. Overexpression of
wblA in S. coelicolor resulted in broad down-regulation of
activator genes and inhibited the production of antibiotics.
Although their target genes are different, C. glutamicum
whcA and S. coelicolor wblA genes may function similarly.
Although the detailed oxidative stress response mechanism
of wblA needs to be further elucidated, it may play a
similar negative role as that of whcA of C. glutamicum.
Recently, C. glutamicum WhcA was revealed to specifically
bind to SpiA (stress protein interacting with WhcA) and
participate in the oxidative stress mechanism [18].
Considering that S. coelicolor also has a spiA ortholog in
its choromosome, WblA might behave similarly to WhcA
in that it undergoes a specific protein-protein interaction
to cope with oxidative stresses in the cell.
Acknowledgment
This work was supported by a National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MEST 2011-0027683).
OXIDATIVE STRESS IN STREPTOMYCES COELICOLOR 740
REFERENCES
1. Arner, E. S. and A. Holmgren. 2000. Physiological functions of
thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267:
6102-6109.
2. Choi, S. U., C. K. Lee, Y. I. Hwang, H. Kinoshita, and T.
Nihira. 2004. Intergeneric conjugal transfer of plasmid DNA
from Escherichia coli to Kitasatospora setae, a bafilomycin B1
producer. Arch. Microbiol. 181: 294-298.
3. Choi, W. W., S. D. Park, S. M. Lee, H. B. Kim, Y. Kim, and H.
S. Lee. 2008. The whcA gene plays a negative role in oxidative
stress response of Corynebacterium glutamicum. FEMS
Microbiol. Lett. 290: 32-38.
4. Chung, H. J., E. J. Kim, B. Suh, J. H. Choi, and J. H. Roe.
1999. Duplicate genes for Fe-containing superoxide dismutase
in Streptomyces coelicolor A3(2). Gene 231: 87-93.
5. Crack, J. C., C. D. den Hengst, P. Jakimowicz, S. Subramanian,
M. K. Jhonson, M. J. Buttner, et al. 2009. Characterization of
[4Fe-4S]-containing and cluster-free forms of Streptomyces
WhiD. Biochemistry 48: 12252-12264.
6. Crack, J. C., L. J. Smith, M. R. Stapleton, J. Peck, N. J.
Watmough, M. J. Buttner, et al. 2011. Mechanistic insight into
the nitrosylation of the [4Fe-4S] cluster of WhiB-like protein. J.
Am. Chem. Soc. 133: 1112-1121.
7. Davis, N. K. and K. F. Chater. 1992. The Streptomyces
coelicolor whiB gene encodes a small transcription factor-like
protein dispensable for growth but essential for sporulation.
Mol. Gen. Genet. 232: 351-358.
8. den Hengst, C. D. and M. J. Buttner. 2008. Redox control in
actinobacteria. Biochem. Biophys. Acta 1780: 1201-1216.
9. Goldsworthy, K. F., B. Gust, S. Mouz, G. Chandra, K. C.
Findlay, and K. F. Chater. 2011. The actinobacteria-specific
gene wblA controls major development transition in Streptomyces
coelicolor A3(2). Microbiology 157: 1312-1328.
10. Gladyshev, V. N. 2001. Thioredoxin and peptide methionine
sulfoxide reductase: Convergence of similar structure and
function in distinct structural folds. Protein 46: 149-152.
11. Jakimowicz, P., M. R. Cheesman, W. R. Bishai, K. F. Chater, A.
J. Thomson, and M. J. Buttner. 2005. Evidence that the
Streptomyces developmental protein WhiD, a member of the
WhiB family, binds a [4Fe-4S] cluster. J. Biol. Chem. 280:
8309-8315.
12. Kang, S. H., J. Huang, H. N. Lee, Y. A. Hur, S. N. Cohen, and
E. S. Kim. 2007. Interspecies DNA microarray analysis
identifies WblA as a pleiotropic down-regulator of antibiotic
biosynthesis in Streptomyces. J. Bacteriol. 189: 4315-4319.
13. Kim, E. J., H. J. Chung, B. Suh, Y. C. Hah, and J. H. Roe.
1998. Expression and regulation of the sodF gene encoding
iron- and zinc-containing superoxide dismutase in Streptomyces
coelicolor Miller. J. Bacteriol. 180: 2014-2020.
14. Kim, E. J., H. P. Kim, Y. C. Hah, and J. H. Roe. 1996.
Differential expression of superoxide dismutases containing Ni
and Fe/Zn in Streptomyces coelicolor. Eur. J. Biochem. 241:
178-185.
15. Kim, T. H., J. S. Park, H. J. Kim, Y. Kim, P. Kim, and H. S.
Lee. 2005. The whcE gene of Corynebacterium glutamicum is
important for survival following heat and oxidative stress.
Biochem. Biophys. Res. Commun. 337: 757-764.
741 Kim et al.
16. Mustacich, D. and G. Powis. 2000. Thioredoxin reductase.
Biochem. J. 346: 1-8.
17. Paget, M. S., J. G. Kang, J. H. Roe, and M. J. Buttner. 1998.
SigmaR, an RNA polymerase sigma factor that modulates
expression of the thioredoxin system in response to oxidative
stress in Streptomyces coelicolor A3(2). EMBO J. 19: 5776-
5782.
18. Park, J. S., S. Shin, E. S. Kim, P. Kim, Y. Kim, and H. S. Lee.
2011. Identification of SpiA that interacts with Corynebacterium
glutamicum WhcA using a two-hybrid system. FEMS Microbiol.
Lett. 322: 8-14.
19. Singh, A., L. Guidry, K. V. Narasimhulu, D. Mai, J. Trombley,
K. E. Redding, et al. 2007. Mycobacterium tuberculosis WhiB3
responds to O2 and nitric oxide via its [4Fe-4S] cluster and is
essential for nutrient starvation survival. Proc. Natl. Acad. Sci.
USA 104: 11562-11567.
20. Soliveri, J. A., J. Gomez, W. R. Bishai, and K. F. Chater. 2000.
Multiple paralogous genes related to the Streptomyces coelicolor
developmental regulatory gene whiB are present in Streptomyces
and other actinomycetes. Microbiology 146: 333-343.