Báo cáo khoa học: Functional analysis of two divalent metal-dependent regulatory genes dmdR1 and dmdR2 in Streptomyces coelicolor and proteome changes in deletion mutants ppt

Functional analysis of two divalent metal-dependent
regulatory genes dmdR1 and dmdR2 in Streptomyces
coelicolor and proteome changes in deletion mutants
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´
Francisco J. Flores1, Carlos Barreiro2, Juan Jose R. Coque1,2 and Juan F. Martın1,2
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1 Area de Microbiologıa, Facultad de Ciencias Biologicas y Ambientales, Universidad de Leon, Spain
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2 Institute of Biotechnology of Leon, INBIOTEC, Parque Cientıfico de Leon, Spain

Keywords
iron metabolism; proteome changes;
regulatory proteins; Streptomyces
Correspondence
´ ´
´
J. F. Martın, Area de Microbiologıa, Facultad
´
de Ciencias Biologicas y Ambientales,
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´
Universidad de Leon, 24071 Leon, Spain
Fax: +34 987 291506
Tel: +34 987 291505
E-mail:

(Received 13 September 2004, revised 11
November 2004, accepted 29 November
2004)
doi:10.1111/j.1742-4658.2004.04509.x

In Gram-positive bacteria, the expression of iron-regulated genes is mediated by a class of divalent metal-dependent regulatory (DmdR) proteins.
We cloned and characterized two dmdR genes of Streptomyces coelicolor
that were located in two different nonoverlapping cosmids. Functional analysis of dmdR1 and dmdR2 was performed by deletion of each copy. Deletion of dmdR1 resulted in the derepression of at least eight proteins and in
the repression of three others, as shown by 2D proteome analysis. These 11
proteins were characterized by MALDI-TOF peptide mass fingerprinting.
The proteins that show an increased level in the mutant correspond to a
DNA-binding hemoprotein, iron-metabolism proteins and several divalent
metal-regulated enzymes. The levels of two other proteins – a superoxide
dismutase and a specific glutamatic dehydrogenase – were found to
decrease in this mutant. Complementation of the dmdR1-deletion mutant
with the wild-type dmdR1 allele restored the normal proteome profile. By
contrast, deletion of dmdR2 did not affect significantly the protein profile
of S. coelicolor. One of the proteins (P1, a phosphatidylethanolamine-binding protein), overexpressed in the dmdR1-deleted mutant, is encoded by
ORF3 located immediately upstream of dmdR2; expression of both ORF3
and dmdR2 is negatively controlled by DmdR1. Western blot analysis confirmed that dmdR2 is only expressed when dmdR1 is disrupted. Species of
Streptomyces have evolved an elaborated regulatory mechanism mediated
by the DmdR proteins to control the expression of divalent metal-regulated
genes.

Iron is an essential element for the growth of all living
organisms, but high intracellular concentrations of iron
are toxic for many cellular reactions, in part owing
to the formation (under aerobic conditions) of highly
reactive iron forms that may damage DNA and other
macromolecules. Therefore, the uptake of iron and the

biosynthesis of iron-metabolizing enzymes are strictly
controlled [1]. In Gram-negative bacteria the mechanism of control is mediated by the global regulatory
protein Fur [2,3], whereas in Gram-positive bacteria

the expression of iron-regulated genes is mediated
mainly by the DmdR (divalent metal-dependent)
family of regulatory proteins [4–6] including the
Corynebacterium diphtheriae DtxR (diphtheriae toxin
repressor), the DmdR of Corynebacterium (previously
Brevibacterium) lactofermentum [7,8] and Rhodococcus fascians [9], and the IdeR protein of Mycobacterium smegmatis and Mycobacterium tuberculosis [10].
Taking into account the industrial interest in several
Streptomyces strains for the production of secondary

Abbreviations
DmdR, divalent metal-dependent regulatory; DtxR, diphtheriae toxin repressor; MEY, maltose-yeast extract; YEME, yeast extract, malt extract.

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Two iron-dependent regulators in S. coelicolor

metabolites [11], and the genetic knowledge on Streptomyces coelicolor, including its full genome sequence
[12], it was of interest to study the possible existence,
in S. coelicolor, of a gene(s) encoding an iron-regulator
of the DmdR family. We report, in this article, the
presence of two different genes – dmdR1 and dmdR2 –
in the genome of S. coelicolor, both of which are functional as iron regulators.

Results
Two dmdR genes occur in S. coelicolor
As the genome sequence of S. coelicolor was not known
at the time that this work was started, a probe was
obtained by PCR using oligonucleotides FRBGL1 and
FRBGL2 or FRBGL1 and FRBGL3, based on the conserved sequences of dtxR homologous genes [1], and the
DNA of S. coelicolor as template. PCR products of
313 bp and 451 bp were obtained with each of the above
pair of primers. To confirm that the PCR products corresponded to the expected gene, they were cloned in
pBluescriptKS+ and sequenced. Both PCR products
showed high nucleotide sequence identity with a dtxRlike gene of S. lividans, named desR [13] and appear to
correspond to two different copies of the same gene.
Using, as probes, both the 451 bp PCR product and
the dtxR homologous gene of R. fascians, the John
Innes Research Center S. coelicolor cosmid library was
probed. Four cosmids (10A7, D10, D52 and 6F11)
were initially found to give a positive hybridization

F. J. Flores et al.

signal. After digestion of the cosmids with ApaI, KpnI
and PstI, an ApaI band of 4.0 kb from cosmid 10A7,
a 1.0 kb ApaI band from cosmid D10 and an 8.0 kb
PstI band from cosmid D52 gave a strong positive
hybridization. The three fragments were subcloned in
pBluescript KS(+); the resulting plasmids were named
pA7a, pD10a (Fig. 1) and pD52.
Initial insert DNA sequencing results indicated the
presence of two different dtxR-homologous genes,
because the insert cloned in pD10a was clearly different from that cloned in plasmid pA7a. Cosmids D10,

D52 and 6F11 are known to be overlapping (H. Kieser
and D. Hopwood, personal communication) [14],
whereas cosmid 10A7 (containing the dmdR2 gene
from which this gene was initially isolated) was different from the others and was later renamed 2 ⁄ 10A7
[12,14]. The two dtxR homologous genes that we isolated were named dmdR1 and dmdR2, respectively, as
they belong to the family of divalent metal-dependent
regulatory proteins (see below).
Both the dmdR1 and the dmdR2 genes were fully
sequenced. The dmdR1 gene encoded a protein of 230
amino acids with a deduced molecular mass of 25 192.
This sequence corresponds to the sco4394 ORF of the
S. coelicolor genome.
The dmdR2 gene encoded a protein of 238 amino
acids with a deduced relative molecular mass of 25 573,
starting at a GTG. This second dmdR gene corresponds
to ORF sco4017 in the S. coelicolor genome.
Comparative analysis by multiple alignment of both
DmdR proteins with proteins in the databases revealed

Fig. 1. Physical map of the Streptomyces coelicolor DNA regions in cosmids D10
(A) and 2 ⁄ 10A7 (B) containing the dmdR1
and dmdR2 genes. The arrows indicate the
location of the ORFs and the orientation in
each DNA fragment. The ApaI fragments of
cosmids D10 and 2 ⁄ 10A7, shown in the
figure, were subcloned in plasmids pD10a
and pA7a, respectively.

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Two iron-dependent regulators in S. coelicolor

Fig. 2. Comparative alignment of domains 1 (DNA–protein interaction), 2 (dimerization and metal binding) and 3 (containing a nonconserved
amino acid stretch), of the Streptomyces coelicolor DmdR1 and DmdR2 proteins, with other members of the DmdR (DtxR) family. (A) Note
the strong conservation (amino acids shown as white on black) of domains 1 and 2, and (B) the presence of an Ala- and Pro-rich segment
inserted in domain 3 of the S. coelicolor DmdR2 protein.

extensive homology with the DtxR protein of C. diphtheriae and with the homologous proteins of C. lactofermentum, R. fascians, M. tuberculosis, M. leprae,
M. smegmatis, R. erythropolis, R. equi, S. pilosus and
S. lividans. The cloned dmdR1 gene showed 99% identity at the nucleotide level to the known S. lividans
desR gene, confirming that it corresponds to the
S. coelicolor homologous gene, whereas dmdR2 showed
77% identity with the S. lividans desR gene.
A characteristic common to both DmdR1 and
DmdR2 proteins is the high conservation of the N-terminal region, particularly domains 1 and domain 2,
when compared with other DtxR-like proteins
(Fig. 2A). The high conservation of these domains
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agrees with the important role of domain 1 on
DNprotein interaction and of domain 2 in the protein
dimerization and metal binding (see the Discussion).
There are important differences between DmdR1 and
DmdR2 proteins in a Pro- and Ala-rich eight amino
acid stretch that occurs in DmdR2 but is absent in

DmdR1 and in the rest of the proteins of this family
(domain 3, Fig. 2B).
Disruption of dmdR1 alters significantly
the protein profile in S. coelicolor
Disruption of the dmdR1 gene was achieved by using a
9.6 kb PstI fragment (cloned from cosmid D10)
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Two iron-dependent regulators in S. coelicolor

F. J. Flores et al.

containing dmdR1, as indicated in Fig. 3. In this construction, the dmdR1 gene was inactivated in vitro by
insertion of the apramycin-resistance gene [aac(3)IV]
prior to recombination. Eleven transformants were
isolated that were resistant to apramycin and sensitive
to thiostrepton.
Hybridization results with probes containing the
dmdR1 gene (1 kb ApaI) or the apramycin-resistance
gene (aac) (1.5 kb PstI–EcoRI) showed a hybridization
pattern that was different from that of the host S. coelicolor (Fig. 3B, lane 6), indicating that the dmdR1 has
been partially deleted and replaced with the apramycin-resistance gene (Fig. 3). One of the disrupted transformants (all of which showed identical hybridization
patterns) was randomly selected and named S. coelicolor dmdR1::aac(3)IV. The disrupted transformants
showed a slow rate of spore formation, but otherwise
were similar to the parental strain.
Proteome of the wild type and of the dmdR1
strain: proteins regulated by DmdR1
As the DmdR1 protein is a transcriptional regulator
[1], it was of interest to characterize the S. coelicolor

proteins that show an increased or decreased level in
response to dmdR1 gene disruption. As shown in
Fig. 4B, the concentration of eight proteins (P1 to P8)
clearly increased in the dmdR1 mutant when compared
with the parental wild-type strain (Fig. 4A), whereas
the concentration of three other proteins (P9 to P11)
decreased in this mutant.

These 11 proteins were characterized by MALDITOF peptide mass fingerprinting and identified with
full confidence (Table 1 and 2). Several of these proteins correspond to Fe2+- or Zn2+-dependent metalloenzymes, indicating that the formation of these
enzymes is under control of the divalent metal regulator, DmdR1. One interesting example is the Zn2+dependent fructose 1,6-biphosphate aldolase (proteins
P6 and P10 in Fig. 4). The P10 protein is modified and
changes its isoelectric point in the dmdR1 mutant,
switching from the P10-form to the P6-form.
Protein P2 (putative DpsA), which shows an
increased level in the dmdR1 mutant, is a DNA-binding
protein with domains typical of the ferritin superfamily.
This protein might be involved in a cascade of iron regulation in response to DmdR1 (see below). In other
micro-organisms this DNA-binding haemoprotein confers resistance to peroxide damage during periods of oxidative stress and long-term nutrient limitation [15,16].
One of the more interesting dmdR1-regulated proteins is a hypothetical phosphatidylethanolamine-binding protein (P1), which is encoded by a gene (ORF3
in Fig. 1B; located upstream of the dmdR2 gene) that
encodes the second iron regulator. Both P1 and
DmdR2 appear to be formed from a bicistronic transcript, as both ORFs are nearly overlapping. This
result suggests that expression of the dmdR2 gene is
negatively regulated by DmdR1, and its expression is
enhanced in response to dmdR1 inactivation, probably
as a backup system, to ensure the supply of a DmdR
regulator.

A

B

728

C

Fig. 3. Disruption of dmdR1. (A) Strategy for
disruption. Plasmid pHZD10HAM was constructed to inactivate the dmdR1 gene by
inserting the aac(3)IV (apramycin resistance)
gene in the opposite orientation into
dmdR1. (B) Hybridization of ApaI-digested
total DNA of different transformants with a
dmdR1 probe (1 kb ApaI fragment). Note
the size change of the hybridizing band with
respect to the control (lane 6). (C) Hybridization with an aac(3)IV probe (1.5 kb PstI–
EcoRI fragment). Lane 6, control Streptomyces coelicolor A3(2). Lanes 1–5, 7–11 and
12, S. coelicolor transformants. The dmdR1
probe cross-hybridized with dmdR2. The
opposite is not true because the dmdR2
probe contains a region that is missing in
the dmdR1 genes and does not give crosshybridization.

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F. J. Flores et al.

A

Two iron-dependent regulators in S. coelicolor

B

C

D

Fig. 4. Two-dimensional protein resolution of the wild-type (A), the dmdR1 mutant (B) and the complemented strain (dmdR1 mutant complemented with the wild-type allele) (C). The proteins that either increase or decrease in concentration in the dmdR1 mutant are encircled.
(D) Enlarged sections of (A) and (B) showing the changes in proteins P1 to P11 (arrowheads). Note that the levels of proteins P1 to P8
increase significantly in the mutant, whereas the levels of proteins P9 to P11 decrease in the mutant (see Tables 2 and 3 for identification
of the proteins).

In addition to P10 (putative Zn2+-dependent fructose 1,6-biphosphate aldolase) two other proteins (P9
and P11) show a decreased concentration in the dmdR1
mutant. P9 corresponds to the well-known Fe2+- or
Mn2+-dependent superoxide dismutase, whereas P11
appears to correspond to a divalent metal-dependent
glutamate dehydrogenase.
Disruption of dmdR2 does not significantly
affect the protein profile in S. coelicolor
The dmdR2 gene was disrupted in the S. coelicolor
genome by replacement with the kanamycin-resistance
gene (aphII) inserted in the XhoI site of dmdR2
(Fig. 5). A transformant was first obtained that was
resistant to both kanamycin and thiostrepton, indicating that a single recombination, resulting in chromosomal integration of the plasmid, had occurred. When
this transformant was allowed to sporulate, a clone
was selected that was resistant to kanamycin and sensitive to thiostrepton. In subsequent replicas, 100% of
the clones obtained from spores were kanamycin-resistant and thiostrepton-sensitive, confirming that a double recombination with deletion of the dmdR2 gene
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had occurred (Fig. 5). One of these recombinants was
selected and named S. coelicolor dmdR2::aphII.
SDS ⁄ PAGE gels and 2D-gel proteome analysis of
the dmdR2-deleted mutants showed no major protein
differences with the parental S. coelicolor strain (data
not shown), suggesting that this second copy of the
dmdR gene has probably very little effect on the
expression of iron-regulated proteins when the dmdR1
allele is intact.
Complementation of the S. coelicolor dmdR1
mutant restores the proteome to that of the
wild type
A 9233 bp BamHI–HindIII fragment, containing the
dmdR1 gene and adjacent regions, was cloned in the
pHZ1351 vector, which has an unstable replication origin [17], to obtain pHZBH9. This plasmid was used to
transform the S. coelicolor dmdR1 and one transformant was selected at random. Cultures of this transformant were grown in liquid yeast extract, malt extract
(YEME)-sucrose medium for 36 h in the absence of
antibiotics, and aliquots were plated in maltose-yeast
extract (MEY) medium with or without apramycin.
729


730
Fructose
1,6-biphosphate aldolase
(Zn2+-dependent)
Hydrolase activity of the
SGHN superfamily
Glu-tRNA Gln amidotransferase

(subunit B)
1175 nt
41 719 Da
1514 nt
54 485 Da

1031 nt
36 926 Da

773 nt
26 914 Da
989 nt
34 642 Da
1088 nt
37 949 Da

563 nt
20 052 Da

533 nt
17 926 Da

Size

Best matches in S. avermitilis and
M. tuberculosis to hydrolases
Homologous to subunit B of
Glu-tRNAGln amidotransferases

High homology with several fructose

1,6-biphosphate aldolases

Contains domains typical of the ferritin-like
superfamily, such as: DNA-binding ferritinlike protein, ferritins and bacterioferritins
Putative dpsA gene
Best match in S. avermitilis and
M. tuberculosis with a gas vesicle protein
High homology to malate dehydrogenases
from different micro-organisms
Homology with several
Zn2+-dependent dehydrogenases

Phosphatidylethanolamine-binding
proteins in other bacteria

Homology

High score with GatB
Probable Glu-tRNA Gln amidotransferase

Enzymes of the SGHN hydrolase superfamily

This gene appears to form part of a large operon encoding
at least 10 proteins
Contains a malate dehydrogenase active site signature
(Prosite PS00068)
In addition, the protein has domains homologous
to methylases.
It is probably a Zn2+-dependent dehydrogenase with a
methyltransferase domain

Enzymes with zinc-binding ability

Gene located upstream of the iron regulator dmdR2
Both P1 and DmdR2-encoding genes appear
to form an operon
The Dps protein family includes DNA-binding hemoproteins
in several bacteria

Remarks

a
P6 is the same protein as P10 (Table 2) but with different isoelectric points. P6 increases in the mutant, whereas P10 is more abundant in the parental strain.
nt, nucleotide.

sco5501

sco0604

P7
Large increase
P8
Medium increase

Zn2+-dependent
dehydrogenases

sco0741

sco3649

Malate dehydrogenase

sco4827

P6a
Large increase

Gas-vesicle protein

sco6501

P3
Medium increase
P4
Small increase
P5
Medium increase

DNA-binding protein
of the ferritin family

sco0596

P2
Medium increase

Phosphatidylethanolaminebinding protein

sco4018

Name

P1
Large increase

Proteins

GenBank
accession
no.

Table 1. Protein changes in the proteome of the dmdR1 mutant as compared to the wild type: proteins that increase in level in the dmdR1 mutant. nt, Nucleotide.

Two iron-dependent regulators in S. coelicolor
F. J. Flores et al.

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Table 2. Protein changes in the proteome of the dmdR1 mutant as compared to the wild type: proteins that decrease in level in the dmdR1
mutant. nt, Nucleotide.

Proteins

GenBank
accession

no.
Name

P9
sco0999
Large decrease
(disappeared)
P10a
sco3649
Large decrease
(disappeared)
P11
sco4683
Medium decrease
(almost disappeared)

Size

Homology

Remarks
Fe2+ or Mn2+-dependent superoxide
dimutase Putative scdF2 gene

Superoxide dismutase
647 nt
High homology with other
(Fe2+ or Mn2+-dependent) 23 599 Da superoxide dismutases
Same as P6a

Specific glutamate
dehydrogenase

1385 nt
High homology with other
Contains a GLFV dehydrogenase
49 480 Da glutamate dehydrogenases. active site, similar to that of GdhA.
Putative gdhA gene
Probably requiring divalent metals

a

P10 is the same protein as P6 (Table 1) but with different isoelectric points. P6 increases in the mutant, whereas P10 is more abundant in
the parental strain.

One of the 1350 clones tested had a double recombination and was sensitive to both apramycin and thiostrepton. In this recombinant the Southern
hybridization pattern agreed with the substitution of
the mutant dmdR1 by the wild-type allele.
The complemented dmdR1 mutant showed the phenotype of the wild-type S. coelicolor strain. As shown
in Fig. 4C, the proteome of the complemented strain
did not differ from that of the parental wild-type
strain, and the protein changes observed in the dmdR1
mutant were reverted.
DmdR2 protein levels increase drastically
in response to dmdR1 disruption
The increase in the P1 protein (phosphatidylethanolamine-binding protein), encoded by ORF3 located

upstream of dmdR2, (Fig. 1) in the dmdR1-disrupted
mutant prompted us to study the levels of DmdR2
and DmdR1 by Western blot analysis. As shown in

Fig. 6, DmdR1 and DmdR2 cross-react with specific
antibodies raised against each of these proteins, but
they differ in their electrophoretic mobility, which was
slightly higher for DmdR2.
Results of the Western blot analysis indicated that
DmdR2 is not detected in the parental S. coelicolor
strain under standard growth conditions. In the
dmdR1-disrupted mutant, DmdR1 is absent, but there
are much higher levels of DmdR2, as detected with
either anti-DmdR2 (Fig. 6B, lane 4) or anti-DmdR1
(Fig. 6A, lane 4).
By contrast, the dmdR2-disrupted mutant did not
show any alteration of DmdR1 levels (Fig. 6A,B, lane
5). These results confirm that the synthesis of DmdR2

A

Fig. 5. Disruption of dmdR2. (A) Strategy for
disruption. Plasmid pHZA7AKM was constructed by inserting the kanamycin-resistance (aphII) gene in the 5¢ region of the
dmdR2 gene. Transformants were detected
as containing the aphII gene and having a
partially deleted dmdR2 gene. (B) Hybridization with a dmdR2 probe (XhoI-SacII fragment) and (C) hybridization of ApaI-digested
total DNA with an aphII (XbaI-HindIII
fragment) probe. Lane 1, Streptomyces coelicolor A3(2). Lanes 2, 3, 4 and 5, S. coelicolor
transformants. Note the endogenous dmdR2
band in S. coelicolor (arrow) and the change
of the hybridizing band in different disrupted
clones.

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B

C

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Two iron-dependent regulators in S. coelicolor

A

F. J. Flores et al.

B

Fig. 6. Western blot analysis of DmdR1 and DmdR2 levels in the parental Streptomyces coelicolor strain and in the dmdR1- or dmdR2-disrupted mutants. (A) Immunodetection with anti-DmdR1. (B) Immunodetection with anti-DmdR2. Lane 1, prestained molecular mass markers
(in kDa, between the two panels); lane 2, pure DmdR1 (100 ng); lane 3, S. coelicolor A3(2) extract (100 lg); lane 4, S. coelicolor dmdR1
mutant (100 lg); lane 5, S. coelicolor dmdR2 mutant (100 lg); lane 6, pure DmdR2 (200 ng). In (B) the lanes are as described for (A), except
that 200 ng of pure DmdR1 (lane 2) was used to permit better detection with anti-DmdR2.

is under the control of DmdR1, as occurs also with
the P1 protein, i.e. expression of the ORF3-dmdR2 is
controlled negatively by DmdR1.
A cascade mechanism of iron regulation
in S. coelicolor?
The S. coelicolor DmdR1 and DmdR2 regulators are
known to bind to iron boxes (see the Discussion).
Computer analysis of the nucleotide sequences
upstream of the genes encoding proteins P1 to P11

failed to detect consensus iron boxes. As iron boxes
have been identified in 10 genes of the S. coelicolor
genome [1], the available evidence indicates that proteins P1 to P11 are probably controlled by transcriptional regulators that respond to DmdR1, i.e. by a
cascade mechanism. In addition, protein P10 is
modified post-translationally in the dmdR1 mutant,
where it disappears and is converted into protein P6,
which accumulates.

Discussion
The finding of two dmdR genes similar to the dtxR
gene of C. diphtheriae [18,19], the dmdR genes of
C. lactofermentum [7,8] and R. fascians [9], and the
ideR gene of Mycobacterium spp. [10], indicates that
the dmdR family of iron (or other divalent metals) regulatory proteins is common in Gram-positive bacteria
[13]. A related protein family, SirR, occurs in Staphylococcus epidermidis [20].
A detailed analysis of the amino acid sequences of
the DmdR1 and DmdR2 proteins in comparison with
those of other actinomycetes revealed a strong conservation of motifs in domains 1 and 2 ( 70% identical
residues), particularly in the DNA-binding region
732

(domain 1) which contains an HTH motif [21] and the
metal-binding and dimerization domains (domain 2)
[22,23].
Despite their similarities, the DmdR2 protein shows
important differences from DmdR1 and the known
members of this group; namely DmdR2 contains a
Pro- and Ala-rich stretch of eight amino acid residues
at the beginning of domain 3, which is absent in the
other DmdR proteins.

The DmdR regulatory proteins control iron-regulated promoters in S. coelicolor and other Streptomyces
species [24]. Both DmdR proteins recognize the consensus iron box sequence TTAGGTTAGGCTCACC
TAA [1]. Neither dmdR1 nor dmdR2 contain an iron
box in their upstream region, indicating that expression
of these genes is not directly self-regulated. The same
observation was made in the C. lactofermentum gene [8]
and all other reported dmdR-like genes. However, the
finding that protein P1 encoded by ORF3, located
immediately upstream of dmdR2, increases in response
to dmdR1 disruption suggests that the ORF3–dmdR2
cluster is negatively regulated by the DmdR1 regulator.
Indeed, Western blot analysis confirmed that DmdR2
is only formed in the dmdR1-disrupted mutant.
The second dmdR copy is silent when dmdR1 is
expressed normally. This second dmdR copy may serve
as a backup regulator to control the large number of
important siderophores produced by soil-dwelling
Streptomyces. Removal of the dmdR1 gene by targeted
gene replacement in S. coelicolor resulted in a change in
the protein profile of the disrupted mutant. Eight protein spots clearly increased their level, whereas at least
three others decreased their concentration in the dmdR1
mutant, as compared to that of the parental strain. One
of the proteins (P10) decreased in the mutant, but a
modified form was accumulated as protein P6 (having
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Two iron-dependent regulators in S. coelicolor

the same amino acid sequence as protein P10 but different pI). Most proteins that respond to dmdR1 disruption are (a) metallo-enzymes that require Fe2+ or other
divalent ions, (b) members of the ferritin family, or
(c) superoxide dismutase proteins. Ferritin is known to
be differentially regulated by iron and manganese in
staphylococci [25], but there is no information available
regarding ferritin regulation in Streptomyces species.
In addition to the 11 proteins listed in Tables 1 and
2, minor changes in other proteins were observed.
These proteins may be involved in other reactions of
iron metabolism (e.g. siderophore biosynthesis) or may
be regulatory proteins that respond to DmdR1.
In summary, the important role of the DmdR1 regulator, but not of the DmdR2 regulator in the control of
gene expression in S. coelicolor has been confirmed by
changes in the proteome of S. coelicolor detected by
using 2D protein gel analysis. This is consistent with the
finding that dmdR2 is very poorly expressed in wild-type
S. coelicolor.

Experimental procedures
Microbial strains, plasmids and culture
conditions
The bacterial strains, plasmids and oligonucleotides used in
this work are listed in Table 3. S. coelicolor cultures were
grown in YEME or MEY media [26]. Escherichia coli
cultures were grown in LB (Luria–Bertani) or TB (terrific
broth), following standard procedures [27].

Recombinant DNA techniques and
DNA sequencing

Plasmid DNA isolation, Southern blotting, E. coli transformation procedures and PCR DNA amplification were performed by standard methods [27]. Disruption of genes and
gene replacement were performed following the usual procedures for S. coelicolor [26].

Cell-free extracts and SDS ⁄ PAGE
Crude extracts of S. coelicolor were obtained by cell disruption using a Branson sonicator (Sonifier B12, Danbury,
CT, USA). Cells were sonicated for 10 s, with 1.5 min
intervals, in TE buffer (10 mm Tris ⁄ HCl, pH 8.0, 1 mm
EDTA, pH 8.0) and the disruption was followed by microscopic observation. Cell debris was removed by centrifugation at 18 000 g. SDS ⁄ PAGE was performed by standard
methods.

2D electrophoresis
2D electrophoresis was performed using the procedure described by Gorg et al. [28]. A total of 350 mg of crude protein
ă
extract was used for IEF in 18 cm precast immobilized pH
gradient (IPG) strips with a linear pH gradient of 4.0–7.0
using an IPGphor IEF unit (Amersham Pharmacia Biotech,
Uppsala, Sweden). The second dimension was run in
SDS ⁄ polyacrylamide gels, of 12.5% (w ⁄ v) acrylamide, in an
Ettan Dalt apparatus (Amersham Biosciences), as recommended by the manufacturer, and the gels were subsequently
stained with Coomassie Brilliant Blue [27]. Precision Plus

Table 3. Bacterial strains, plasmids and oligonucleotides used in this work.
Bacteral strains ⁄ plasmids ⁄
oligonucleotides
Bacterial strain
E. coli DH5a

S. coelicolor A3(2)
Plasmid
pD10

pA7a
pHZD10HAM
(a derivative of pHZ1351)
pHZA7AKM
(a derivative of pHZ1351)
pHZBH 9
(a derivative of pHZ1351)

Genotype ⁄ gene

Source ⁄ reference

Genotype
F– recA1 endA2 gryA96 thi-1 hsdR17
(rk–mk+) sup44relA1 k– (/80 dLacZDM15)
D(lacZYA-argF) U169
Wild type

John Innes Institute, Norwich, UK

Gene
dmdR1
dmdR2
dmdR1::aac(3)IV

This work
This work
This work; [1]

dmdR2::aphII

This work; [1]

BRL (Bethesda Research Laboratory), MD, USA

This work; [1]

Oligonucleotides used as primers
FRBGL1: 5¢-GAAGATCTGGCGGACCGGCATCTGGA-3¢
FRBGL2: 5¢-GAAGATCTACGACGTCTTGCCCTCCTG-3¢
FRBGL3: 5¢-GAAGATCTCAGCACGCCGCCCGCCGACTC-3¢

FEBS Journal 272 (2005) 725–735 ª 2005 FEBS

733


Two iron-dependent regulators in S. coelicolor

protein Standards (Bio-Rad, Hercules, CA, USA) were used
as markers.
Protein spots were excised from gels and digested with
modified trypsin (Promega, Madison, WI, USA). Peptide
mass fingerprints were analyzed by using the mascot software [29].

F. J. Flores et al.

7

8

Immunodetection analysis of DmdR1 and DmdR2
Western blot analysis of DmdR1 and DmdR2, after
SDS ⁄ PAGE resolution of the proteins, was performed as
described previously [1]. Polyclonal rabbit antibodies
against pure DmdR1 or DmdR2 were raised and purified
by ammonium sulphate precipitation and FPLC using a
protein A–sepharose column (Amersham Biosciences), as
described in detail by Flores & Martı´ n [1].

9

10

Acknowledgements

11

This work was supported by a grant (Generic Project
´
10-2 ⁄ 98 ⁄ LE ⁄ 0003) from the ADE of Castilla and Leon
(Valladolid, Spain). F. J. Flores received a fellowship
´
´
of the Fundacion Ramon Areces (Madrid, Spain). We
acknowledge the help of J. A. Oguiza and the technical
support of M. Corrales and M. Mediavilla.

12

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13

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