Functional analysis of two divalent metal-dependent
regulatory genes dmdR1 and dmdR2 in Streptomyces
coelicolor and proteome changes in deletion mutants
Francisco J. Flores
1
, Carlos Barreiro
2
, Juan Jose
´
R. Coque
1,2
and Juan F. Martı
´
n
1,2
1A
´
rea de Microbiologı
´
a, Facultad de Ciencias Biolo
´
gicas y Ambientales, Universidad de Leo
´
n, Spain
2 Institute of Biotechnology of Leo
´
n, INBIOTEC, Parque Cientı
´
fico de Leo
´
n, Spain

a, Facultad
de Ciencias Biolo
´
gicas y Ambientales,
Universidad de Leo
´
n, 24071 Leo
´
n, 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 medi-
ated 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 ana-
lysis of dmdR1 and dmdR2 was performed by deletion of each copy. Dele-
tion 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

responded to the expected gene, they were cloned in
pBluescriptKS+ and sequenced. Both PCR products
showed high nucleotide sequence identity with a dtxR-
like 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
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 differ-
ent 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 differ-
ent from the others and was later renamed 2 ⁄ 10A7
[12,14]. The two dtxR homologous genes that we iso-
lated were named dmdR1 and dmdR2, respectively, as
they belong to the family of divalent metal-dependent
regulatory proteins (see below).

77% identity with the S. lividans desR gene.
A characteristic common to both DmdR1 and
DmdR2 proteins is the high conservation of the N-ter-
minal region, particularly domains 1 and domain 2,
when compared with other DtxR-like proteins
(Fig. 2A). The high conservation of these domains
agrees with the important role of domain 1 on
DNAÆprotein 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)
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.
F. J. Flores et al. Two iron-dependent regulators in S. coelicolor
FEBS Journal 272 (2005) 725–735 ª 2005 FEBS 727
containing dmdR1, as indicated in Fig. 3. In this con-
struction, 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.

-dependent metallo-
enzymes, indicating that the formation of these
enzymes is under control of the divalent metal regula-
tor, DmdR1. One interesting example is the Zn
2+
-
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 regu-
lation in response to DmdR1 (see below). In other
micro-organisms this DNA-binding haemoprotein con-
fers resistance to peroxide damage during periods of oxi-
dative stress and long-term nutrient limitation [15,16].
One of the more interesting dmdR1-regulated pro-
teins is a hypothetical phosphatidylethanolamine-bind-
ing 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 tran-
script, 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.

-or
Mn
2+
-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, indica-
ting that a single recombination, resulting in chromo-
somal integration of the plasmid, had occurred. When
this transformant was allowed to sporulate, a clone
was selected that was resistant to kanamycin and sensi-
tive to thiostrepton. In subsequent replicas, 100% of
the clones obtained from spores were kanamycin-resist-
ant and thiostrepton-sensitive, confirming that a dou-
ble recombination with deletion of the dmdR2 gene
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.

sco4018 Phosphatidylethanolamine-
binding protein
533 nt
17 926 Da
Phosphatidylethanolamine-binding
proteins in other bacteria
Gene located upstream of the iron regulator dmdR2
Both P1 and DmdR2-encoding genes appear
to form an operon
P2
Medium increase
sco0596 DNA-binding protein
of the ferritin family
563 nt
20 052 Da
Contains domains typical of the ferritin-like
superfamily, such as: DNA-binding ferritin-
like protein, ferritins and bacterioferritins
Putative dpsA gene
The Dps protein family includes DNA-binding hemoproteins
in several bacteria
P3
Medium increase
sco6501 Gas-vesicle protein 773 nt
26 914 Da
Best match in S. avermitilis and
M. tuberculosis with a gas vesicle protein
This gene appears to form part of a large operon encoding
at least 10 proteins
P4

(Zn
2+
-dependent)
1031 nt
36 926 Da
High homology with several fructose
1,6-biphosphate aldolases
Enzymes with zinc-binding ability
P7
Large increase
sco0604 Hydrolase activity of the
SGHN superfamily
1175 nt
41 719 Da
Best matches in S. avermitilis and
M. tuberculosis to hydrolases
Enzymes of the SGHN hydrolase superfamily
P8
Medium increase
sco5501 Glu-tRNA Gln amidotransferase
(subunit B)
1514 nt
54 485 Da
Homologous to subunit B of
Glu-tRNAGln amidotransferases
High score with GatB
Probable Glu-tRNA Gln amidotransferase
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.

(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
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 Size Homology Remarks
P9
Large decrease
(disappeared)
sco0999 Superoxide dismutase
(Fe
2+
or Mn
2+
-dependent)
647 nt
23 599 Da
High homology with other
superoxide dismutases
Fe
2+
or Mn
2+
-dependent superoxide
dimutase Putative scdF2 gene
P10

tion with a dmdR2 probe (XhoI-SacII frag-
ment) and (C) hybridization of ApaI-digested
total DNA with an aphII (XbaI-HindIII
fragment) probe. Lane 1, Streptomyces coeli-
color 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.
F. J. Flores et al. Two iron-dependent regulators in S. coelicolor
FEBS Journal 272 (2005) 725–735 ª 2005 FEBS 731
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 pro-
teins P1 to P11 are probably controlled by transcrip-
tional 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

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 pro-
tein 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
AB
Fig. 6. Western blot analysis of DmdR1 and DmdR2 levels in the parental Streptomyces coelicolor strain and in the dmdR1-ordmdR2-dis-
rupted 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.
Two iron-dependent regulators in S. coelicolor F. J. Flores et al.
732 FEBS Journal 272 (2005) 725–735 ª 2005 FEBS
the same amino acid sequence as protein P10 but differ-
ent pI). Most proteins that respond to dmdR1 disrup-
tion are (a) metallo-enzymes that require Fe

mation procedures and PCR DNA amplification were per-
formed by standard methods [27]. Disruption of genes and
gene replacement were performed following the usual pro-
cedures for S. coelicolor [26].
Cell-free extracts and SDS ⁄ PAGE
Crude extracts of S. coelicolor were obtained by cell disrup-
tion 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 micro-
scopic observation. Cell debris was removed by centrifuga-
tion at 18 000 g. SDS ⁄ PAGE was performed by standard
methods.
2D electrophoresis
2D electrophoresis was performed using the procedure des-
cribed by Go
¨
rg et al. [28]. A total of 350 mg of crude protein
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 recommen-
ded 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 Genotype ⁄ gene Source ⁄ reference
Bacterial strain Genotype

FRBGL3: 5¢-GAAGATCTCAGCACGCCGCCCGCCGACTC-3¢
F. J. Flores et al. Two iron-dependent regulators in S. coelicolor
FEBS Journal 272 (2005) 725–735 ª 2005 FEBS 733
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 soft-
ware [29].
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].
Acknowledgements
This work was supported by a grant (Generic Project
10-2 ⁄ 98 ⁄ LE ⁄ 0003) from the ADE of Castilla and Leo
´
n
(Valladolid, Spain). F. J. Flores received a fellowship
of the Fundacio
´
n Ramo
´
n Areces (Madrid, Spain). We

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