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

In Gram-negative bacteria the mechan-ism of control is mediated by the global regulatory protein Fur [2,3], whereas in Gram-positive bacteria the expression of iron-regulated genes is me

regulatory genes dmdR1 and dmdR2 in Streptomyces

coelicolor and proteome changes in deletion mutants

Francisco J Flores1, Carlos Barreiro2, Juan Jose´ R Coque1,2and Juan F Martı´n1,2

2 Institute of Biotechnology of Leo´n, INBIOTEC, Parque Cientı´fico de Leo´n, Spain

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

mechan-ism 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 Rhodococ-cus fascians [9], and the IdeR protein of Mycobacte-rium smegmatisand Mycobacterium tuberculosis [10] Taking into account the industrial interest in several Streptomyces strains for the production of secondary

Keywords

iron metabolism; proteome changes;

regulatory proteins; Streptomyces

Correspondence

de Ciencias Biolo´gicas y Ambientales,

Universidad de Leo´n, 24071 Leo´n, Spain

Fax: +34 987 291506

Tel: +34 987 291505

E-mail: degjmm@unileon.es

(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 contrast, deletion of dmdR2 did not affect significantly the protein profile

of S coelicolor One of the proteins (P1, a phosphatidylethanolamine-bind-ing 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 con-firmed 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

Abbreviations

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

metabolites [11], and the genetic knowledge on

Strep-tomyces 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

func-tional 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

con-served 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

cor-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)

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 coelicolorgenome

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 Streptomy-ces coelicolor DNA regions in cosmids D10

and dmdR2 genes The arrows indicate the location of the ORFs and the orientation in each DNA fragment The ApaI fragments of

figure, were subcloned in plasmids pD10a and pA7a, respectively.

extensive homology with the DtxR protein of C

diph-theriaeand with the homologous proteins of C

lacto-fermentum, R fascians, M tuberculosis, M leprae,

M smegmatis, R erythropolis, R equi, S pilosus and

S lividans The cloned dmdR1 gene showed 99%

iden-tity at the nucleotide level to the known S lividans

desR gene, confirming that it corresponds to the

S coelicolorhomologous 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-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.

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

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

coe-licolor (Fig 3B, lane 6), indicating that the dmdR1 has

been partially deleted and replaced with the

apramy-cin-resistance gene (Fig 3) One of the disrupted

trans-formants (all of which showed identical hybridization

patterns) was randomly selected and named S

coeli-color 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 MALDI-TOF peptide mass fingerprinting and identified with full confidence (Table 1 and 2) Several of these pro-teins correspond to Fe2+- or Zn2+-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 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 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

A

Fig 3 Disruption of dmdR1 (A) Strategy for disruption Plasmid pHZD10HAM was con-structed 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) Hybridiza-tion with an aac(3)IV probe (1.5 kb PstI– EcoRI fragment) Lane 6, control Streptomy-ces 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 cross-hybridization.

In addition to P10 (putative Zn2+-dependent

fruc-tose 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,

indica-ting that a single recombination, resulindica-ting 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

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 ori-gin [17], to obtain pHZBH9 This plasmid was used to transform the S coelicolor dmdR1 and one ant was selected at random Cultures of this transform-ant 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

A

D

Fig 4 Two-dimensional protein resolution of the wild-type (A), the dmdR1 mutant (B) and the complemented strain (dmdR1 mutant comple-mented 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).

GenBank accession no.

P1 Large

Phosphatidylethanolamine- binding

Phosphatidylethanolamine-binding proteins

P2 Medium

P3 Medium

P4 Small

P5 Medium

F 1,6-biphosphate

P7 Large

P8 Medium

One of the 1350 clones tested had a double

recombina-tion and was sensitive to both apramycin and

thio-strepton 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

phe-notype 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

(phosphatidylethanol-amine-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

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

P9

Large decrease

(disappeared)

647 nt

23 599 Da

High homology with other superoxide dismutases

dimutase Putative scdF2 gene

Large decrease

(disappeared)

P11

Medium decrease

(almost disappeared)

dehydrogenase

1385 nt

49 480 Da

High homology with other glutamate dehydrogenases.

Putative gdhA gene

Contains a GLFV dehydrogenase active site, similar to that of GdhA 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.

A

Fig 5 Disruption of dmdR2 (A) Strategy for

disruption Plasmid pHZA7AKM was

con-structed by inserting the

kanamycin-resist-ance (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)

Hybridiza-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 coelicoeli-color

transformants Note the endogenous dmdR2

band in S coelicolor (arrow) and the change

of the hybridizing band in different disrupted

clones.

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

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)

reg-ulatory proteins is common in Gram-positive bacteria

[13] A related protein family, SirR, occurs in

Staphy-lococcus 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

conser-vation of motifs in domains 1 and 2 ( 70% identical

residues), particularly in the DNA-binding region

(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-regula-ted promoters in S coelicolor and other Streptomyces species [24] Both DmdR proteins recognize the consen-sus 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 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

Fig 6 Western blot analysis of DmdR1 and DmdR2 levels in the parental Streptomyces coelicolor strain and in the dmdR1- or dmdR2-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.

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 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

regula-tor, 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 transfor-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

EDTA, pH 8.0) and the disruption was followed by micro-scopic observation Cell debris was removed by

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

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.

D(lacZYA-argF) U169

BRL (Bethesda Research Laboratory), MD, USA

pHZD10HAM

(a derivative of pHZ1351)

pHZA7AKM

(a derivative of pHZ1351)

pHZBH 9

(a derivative of pHZ1351)

This work; [1]

Oligonucleotides used as primers

FRBGL1: 5¢-GAAGATCTGGCGGACCGGCATCTGGA-3¢

FRBGL2: 5¢-GAAGATCTACGACGTCTTGCCCTCCTG-3¢

FRBGL3: 5¢-GAAGATCTCAGCACGCCGCCCGCCGACTC-3¢

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

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

acknowledge the help of J A Oguiza and the technical

support of M Corrales and M Mediavilla

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