Báo cáo khoa học: Transcriptional regulation of the desferrioxamine gene cluster of Streptomyces coelicolor is mediated by binding of DmdR1 to an iron box in the promoter of the desA gene doc

cluster of Streptomyces coelicolor is mediated by bindingof DmdR1 to an iron box in the promoter of the desA gene Sedef Tunca1, Carlos Barreiro1, Alberto Sola-Landa1, Juan Jose´ R.. Prod

cluster of Streptomyces coelicolor is mediated by binding

of DmdR1 to an iron box in the promoter of the desA gene Sedef Tunca1, Carlos Barreiro1, Alberto Sola-Landa1, Juan Jose´ R Coque1,2and Juan F Martı´n1,2

1 Instituto de Biotecnologı´a, INBIOTEC, Leo´n, Spain

2 A ´ rea de Microbiologı´a, Facultad de CC Biolo´gicas y Ambientales, Universidad de Leo´n, Spain

Iron is an essential element required for many key

metabolic processes (including cytochrome and Fe-S

electron transporters) in almost all micro-organisms

The bioavailability of iron is very low because salts

of the oxidized ferric ion formed under normal oxic

conditions are largely insoluble [1] To solve the

problem of iron availability, many micro-organisms

synthesize different high-affinity iron chelators (sidero-phores), forming very stable complexes with ferric iron [2] Streptomyces species are soil-dwelling Gram-positive saprophytic bacteria that produce different types of siderophores [3,4] Desferrioxamines are nonpeptide hydroxamate siderophores composed of alternating dicarboxylic acid and diamine units

Keywords

desferrioxamine biosynthesis; gene

expression; iron regulation; lysine

decarboxylase gene; siderophores

Correspondence

J F Martı´n, Instituto de Biotecnologı´a,

INBIOTEC, Parque Cientı´fico de Leo´n,

Avenue del Real no 1, 24006 Leon, Spain

Fax: + 34 987 210 388

Tel: + 34 987 210 308

E-mail: jf.martin@unileon.es

(Received 11 October 2006, revised 19

December 2006, accepted 21 December

2006)

doi:10.1111/j.1742-4658.2007.05662.x

Streptomyces coelicolor and Streptomyces pilosus produce desferrioxamine siderophores which are encoded by the desABCD gene cluster S pilosus is used for the production of desferrioxamine B which is utilized in human medicine We report the deletion of the desA gene encoding a lysine decarboxylase in Streptomyces coelicolor A3(2) The DdesA mutant was able to grow on lysine as the only carbon and nitrogen source but its des-ferrioxamine production was blocked, confirming that the l-lysine decarb-oxylase encoded by desA is a dedicated enzyme committing l-lysine to desferrioxamine biosynthesis Production of desferrioxamine was restored

by complementation with the whole wild-type desABCD cluster, but not by desA alone, because of a polar effect of the desA gene replacement on expression of the downstream des genes The transcription pattern of the desABCD cluster in S coelicolor showed that all four genes were coordi-nately induced under conditions of iron deprivation The transcription start point of the desA gene was identified by primer extension analysis at a thy-mine located 62 nucleotides upstream of the translation start codon The )10 region of the desA promoter overlaps the 19-nucleotide palindromic iron box sequence known to be involved in iron regulation in Streptomyces Binding of DmdR1 divalent metal-dependent regulatory protein to the desApromoter region of both S coelicolor and S pilosus was shown using electrophoretic mobility-shift assays, validating the conclusion that iron regulation of the desABCD cluster is mediated by the regulatory protein DmdR1 We conclude that the genes involved in desferrioxamine produc-tion are under transcripproduc-tional control exerted by the DmdR1 regulator in the presence of iron and are expressed under conditions of iron limitation

Abbreviations

DmdR, divalent metal-dependent regulatory protein; ILMM, iron-limited minimal medium; YEME, yeast extract and malt extract culture medium.

linked by amide bonds They are produced by

many Streptomyces species, including Streptomyces

coelicolor [5], Streptomyces griseus [6] and

Streptomyces pilosus; the latter is used for industrial

production of desferrioxamine B for medical uses

[7,8]

In Streptomyces species, as in other Gram-positive

bacteria, the expression of genes involved in iron

meta-bolism is under the control of a divalent

metal-depend-ent regulatory protein (DmdR) analogous to the

diphtheria toxin repressor of Corynebacterium

diphthe-riae [9,10] S coelicolor has two similar genes, dmdR1

and dmdR2, encoding regulatory proteins of this family

[11] In a previous study, Flores et al [12] reported

that S coelicolor DmdR1 binds specific sequences

(iron boxes) in the upstream region of the diphtheria

toxin (tox) gene of C diphtheriae and the desA gene of

S pilosus

Several putative iron boxes were found by

bioinfor-matics analysis upstream of 10 different ORFs in the

genome of S coelicolor [12] One of the putative iron

boxes is located in the promoter of the desABCD gene

cluster, which was assumed to be responsible for

des-ferrioxamine biosynthesis [13] Barona-Go´mez et al

[14] proposed a possible pathway for desferrioxamine

biosynthesis from l-lysine and reported that desD is

essential for desferrioxamine formation (Fig 1) [15]

The first step in the desferrioxamine pathway is the

conversion of l-lysine into cadaverine catalyzed by the

enzyme lysine decarboxylase [7,8] which, in S

coeli-color, appears to be encoded by desA, although no

conclusive genetic evidence was available until now, as

other putative lysine decarboxylase-encoding genes

occur in the S coelicolor genome (e.g SCO2017)

As one of the iron boxes was located in the

upstream region of the desABCD cluster, it was of

interest to perform a transcriptional analysis of this

cluster and also to characterize the promoter region

(transcription start point and regulatory sequences) in

order to analyze the role of iron and the DmdR1

regu-lator in the transcriptional control of the

desferrioxam-ine cluster

In this study, we report the deletion of the first gene

of the desABCD gene cluster (desA) in S coelicolor

A3(2), which caused cessation of desferrioxamine E

and B biosynthesis Transcriptional analysis of this

region showed that the genes involved in

desferrioxam-ine production are under iron control The

transcrip-tion start point of the desA gene was shown to overlap

with the palindromic iron box Binding of purified

DmdR1 protein to the desA promoter region of both

S coelicolorand S pilosus, as shown by

electrophoret-ic mobility-shift assay, proved that iron control of the

expression of the des cluster is mediated by the DmdR1 regulator

Results

Deletion of the desA gene of S coelicolor blocks desferrioxamine biosynthesis

The organization of the putative desferrioxamine gene cluster, as deduced from the S coelicolor genome, is shown in Fig 1B To clarify the role of the l-lysine decarboxylase encoded by desA and its possible involvement in desferrioxamine biosynthesis, two apra-mycin-resistant and kanamycin-sensitive transformants were isolated among S coelicolor transformants with the desA gene replacement construction (see Experi-mental procedures), and the DdesA mutation was veri-fied in one of the mutants by PCR and Southern blot analysis A 1462-bp PCR band corresponding to the extended resistance cassette was found only in the mutant strain, and a 1372-bp PCR band corresponding

to the desA gene was present only in the wild-type strain but not in the DdesA mutant These results were confirmed by Southern blot hybridization of ScaI-digested DNA A hybridization band of  4200 bp was obtained for the wild-type with a desA fragment (1372 bp) as probe, and a band of about 4220 bp was found for the mutant with aac(3)IV fragment (935 bp)

as probe, as expected Hybridization and PCR analysis results indicate that the desA gene has been deleted and replaced by the apramycin resistance gene

HPLC analysis showed that no desferrioxamines could be detected in the culture supernatants of the DdesA mutant, whereas desferrioxamines B and E were produced in the parental strain (Fig 2)

The DdesA mutant was able to grow on Streptomy-ces minimal medium containing l-lysine as the only carbon and nitrogen source, indicating that the desA gene is not involved in the catabolism of lysine

Complementation of the S coelicolor desA mutation with cosmid Stc105 restored desferrioxamine biosynthesis

Complementation of the DdesA mutant was tested by conjugation with Escherichia coli containing either (a) a plasmid construct pRAdesAKn (see Experimental pro-cedures) carrying a 4204-bp fragment containing the desA coding region or (b) cosmid Stc105 In the plas-mid-mediated complementation, one of the KnR conju-gants was selected for further analysis A 1372-bp PCR fragment corresponding to the desA gene was present

in the complemented and in the wild-type strains but

B

Fig 1 Proposed pathway for desferrioxamine biosynthesis indicating the conversions catalyzed by the enzymes encoded by desA, desB, desC and desD (A) and organization of the S coelicolor des cluster and the upstream SCO2780 and SCO2781 genes (B) The iron box located upstream of desA is indicated by an open box RBS, Ribosome-binding site The hairpin structure corresponds to a stem and loop structure (putative transcriptional terminator) found downstream of desD Solid bars indicate the DNA fragments amplified by RT-PCR in the gene expression studies (see Fig 4).

not in the DdesA mutant, as expected (Fig 3A) In this

conjugant, the Southern blot hybridization pattern

agreed with the integration of the intact wild-type desA

gene (Fig 3B,C) When the desA fragment (1372 bp) was used as probe, a band of 3700 bp was found only

in the wild-type and complemented mutant (Fig 3B)

A 1520-bp positive band was obtained only in the com-plemented strain when a kan (kanamycin resistance) fragment (1519 bp) was used as probe, as expected (Fig 3C) Complementation of the desA deletion in the mutant strain with the wild-type gene failed to restore desferrioxamine production under iron-deficient condi-tions (data not shown) However, functional comple-mentation of the DdesA mutant was achieved with cosmid Stc105, which includes the entire siderophore biosynthetic gene cluster (Fig 2C)

The failure of the 4204-bp fragment containing a wild-type copy of the desA gene to complement the mutation in trans suggests that the DdesA mutation affects expression of the downstream genes desBCD in the des cluster and that the presence of wild-type desA gene product was not sufficient to restore the ability to produce these siderophores The complementation with cosmid Stc105 indicates that the four genes in the des cluster are probably transcribed as one polycistronic mRNA (see below), allowing complementation of the desAmutant, even if expression of the endogenous des-BCDgenes is disturbed in the DdesA mutant

Fig 2 Lack of desferrioxamine production in the S coelicolor

DdesA mutant and restoration by complementation with the des

cluster HPLC analysis of siderophore production in S coelicolor

A3(2) parental and the DdesA mutant strain before and after

com-plementation with the Stc105 cosmid Desferrioxamine E (retention

time 15.3 min) is the major desferrioxamine produced by S

coeli-color Desferrioxamine B showed a retention time of 13.6 min.

Fig 3 Verification of the complementation of desA deletion by PCR using primers for the desA gene (A) and by Southern blot hybridization (B, C) (A) 1-kb Plus DNA ladder (Invitrogen) (lane 1); S coelicolor A3(2) wild-type strain (lane 2); DdesA mutant (lane 3); complemented strain (lane 4) (B, C) Southern blot analysis of PvuII-digested genomic DNA probed with a 1372-bp desA fragment (B) and of BamHI + SacI-digested genomic DNA probed with a kan (kanamycin resistance) fragment (1519 bp) (C) Size markers (kDNA-HindIII-digested) (lane 1); S coelicolor A3(2) wild-type strain (lane 2); desA-deleted strain (lane 3); complemented strain (lane 4).

Production of desferrioxamines B and E

is regulated by iron

The desferrioxamines Tris–hydroxamate–Fe3+

com-plexes were determined in the supernatants of cultures

grown in (a) iron-limited minimal medium (ILMM),

(b) ILMM with 2,2¢-dipyridyl, and (c) ILMM

supple-mented with 35 lm iron S coelicolor A3(2) grown in

iron-deficient medium produces desferrioxamine B and

E Addition of iron to the culture medium completely

suppressed desferrioxamine production, indicating that

the biosynthesis of these siderophores is strictly

regula-ted by iron (not shown)

Expression of the desABCD cluster is

coordinately derepressed after iron deprivation

Four different genes (desA to desD) have been reported

to be involved in the biosynthesis of desferrioxamine

[14] Upstream of the desA gene, two other genes

enco-ding siderophore-related proteins are located (Fig 1B)

The first is a siderophore-interacting protein (viuB

gene), whereas the second encodes a putative secreted

protein (SCO2780) annotated as a hypothetical

sidero-phore-binding lipoprotein [13] To elucidate if these

two genes are expressed and to study their possible

involvement in desferrioxamine biosynthesis, the

tran-scriptional pattern of the entire region was analyzed

by RT-PCR under iron-deprivation conditions

Because of the lack of growth after iron deprivation,

the cultures were initially grown in complex medium

[yeast extract and malt extract culture medium

(YEME)] for 36 h and then starved of iron (see

Experimental procedures) After iron deprivation, five

samples (taken at 2, 6, 8, 24 and 48 h) were analyzed,

and the RNA from one nonstarved culture was used

as control A small increase in dry weight until 6 h

was observed, but no further growth occurred

there-after

The RT-PCR analysis revealed induction of the

tes-ted desA and desD genes (locates-ted at the beginning and

end of the cluster) under iron-limiting conditions,

indi-cating a coordinated transcription (Fig 4) This result

supports the existence of the desABCD operon

sugges-ted by Barona-Gomez and coworkers [14] that is

tran-scribed as a polycistronic mRNA and confirms the

need of the entire des cluster (as in cosmid Stc105) to

complement the DdesA mutant described above

Maximum induction of desA and desD was found

6–8 h after iron limitation, and a significant decrease

in expression was observed after 48 h of iron

depriva-tion, indicating that the culture was unable to

main-tain expression of the cluster for prolonged periods,

probably because of the lack of iron-dependent respir-atory metabolism after extended iron deprivation

An upstream gene encoding a putative siderophore-binding protein is also derepressed after iron deprivation

The transcription pattern of the genes located upstream of the desA gene was also analyzed The viuB gene did not show RT-PCR amplification, suggesting that it is not expressed, or very poorly so, under the experimental conditions used On the other hand, a transcription pattern similar to that of the desABCD operon was found for the SCO2780 gene located upstream of viuB (Fig 4) encoding a putative sidero-phore-binding lipoprotein (see Discussion) Our results confirm the regulation by iron of the expression of this gene In contrast with the desABCD operon, the gene encoding this putative siderophore-binding lipoprotein (SCO2780) does not show an obvious consensus iron box in its promoter region, suggesting that SCO2780 is controlled by indirect iron regulation, probably medi-ated by a cascade mechanism

The desA promoter of S pilosus showed higher expression ability than the same promoter from

S coelicolor Streptomyces pilosus is used industrially for desferriox-amine production and it produces higher levels of those siderophores than S coelicolor [16,17] To com-pare the efficiency of expression of the des cluster from

Fig 4 Expression of the desA–D genes and the upstream genes

at 2, 6, 8, 24 and 48 h after iron starvation (t ¼ 0) Controls without RNA (lane –) and with DNA instead of RNA (lane +) were per-formed simultaneously The hrdB gene was used as control of RNA amounts.

these two Streptomyces species, the desA promoter

region (511-bp PCR product) of both S coelicolor and

S pilosus were cloned in BamHI–EcoRI-digested

pIJ4083 (7.6 kb) carrying the promoterless xylE

repor-ter gene encoding catechol dioxygenase (constructions

named pCoedesAP and pPildesAP, respectively) The

511-bp fragment of either S coelicolor or S pilosus

showed iron-regulated promoter activity when

intro-duced in both S coelicolor and Streptomyces lividans

(Fig 5) Catechol oxygenase activity was observed

only under iron-limited conditions in both strains The

S pilosus desApromoter clearly showed higher

expres-sion ability than the equivalent S coelicolor promoter

region when introduced in either S lividans or S

coeli-color, suggesting that the S pilosus promoter is

recog-nized more efficiently by the transcribing RNA

polymerase complex

Transcription start point: the)10 region overlaps

the iron box

Primer extension experiments with increasing S

coeli-color RNA concentrations (50–150 lg RNA) using a

fluorescein-labelled 17-bp oligonucleotide [18] as

primer (O6, Table 2) allowed clear identification of the desA transcription start point at a thymine located 62 nucleotides upstream of the ATG translation initiation codon of desA This transcription start point is located immediately downstream of the iron box (boxed in Fig 6) and allowed us to identify the )10 Pribnow box as TAGGCT in agreement with the proposed con-sensus sequence for Streptomyces promoters TAgPu-PuT [19] It is interesting that the )10 sequence is located inside the iron box of desA (nucleotides 7–12

of the iron box), explaining the regulation of desA expression by binding of DmdR1 to the iron box The same overlapping was found in S pilosus

DmdR1 binds to the promoter region of desABCD

in both S coelicolor and S pilosus Binding of purified DmdR1 to the desA promoter region of S coelicolor was studied using a 511-bp PCR fragment of this region in the DNA-protein binding reaction DmdR1 showed a high affinity for the desA promoter region of both S coelicolor and S pilosus, resulting in retardation of the digoxigenin-labelled fragment which was prevented by competition with excess unlabelled probe (Fig 7) The mobility shift was clearly higher at increasing protein concentrations, giving two DNA–protein complexes of different size This is in agreement with our previous finding on the binding of two (or four) DmdR1 molecules to the iron boxes of either Corynebacterium glutamicum or

S pilosus[12]

Discussion

Several desferrioxamines are produced by different Streptomyces species [5,6] Desferrioxamine B is used clinically for the treatment of iron overload during metabolic alterations in humans Initial work on the biosynthesis of desferrioxamine B in S pilosus indica-ted that the first step in the desferrioxamine biosynthe-sis is the decarboxylation of lysine by a lysine decarboxylase encoded by the desA gene [8,9] Lysine decarboxylases occur in different Streptomyces species and are involved in the utilization of l-lysine as a nitrogen source [20], but the DesA decarboxylase might be specific for desferrioxamine biosynthesis Unfortunately, the complete desferrioxamine gene clus-ter in S pilosus is not known On the basis of the sequence of the S coelicolor genome, Barona-Go´mez

et al [14] proposed a biosynthetic pathway in which cadaverine formed by lysine decarboxylation is subsequently hydroxylated to N-hydroxycadaverine by the protein encoded by desB (Fig 1) which is later

Fig 5 S coelicolor A3(2) and S pilosus desA promoter activity in

S lividans and in S coelicolor A3(2) in ILMM cultures as measured

by determining the catechol oxygenase of the coupled reporter

gene.

acylated with succinyl-CoA (or alternative acyl-CoA

esters to form succinyl-N-hydroxycadaverine), which is

finally oligomerized by the action of DesD [15]

A separate putative lysine decarboxylase (SCO2017)

showing 38% end to end identity (53% functionally

conserved residues) with DesA occurs in the S

coeli-color genome We have shown in this study that the

desA gene is essential for desferrioxamine biosynthesis

but not for growth on lysine as the only carbon and

nitrogen source, indicating that the encoded lysine

decarboxylase is a dedicated enzyme committing

l-lysine to the desferrioxamine pathway, as occurs with

p-aminobenzoic acid synthase in the biosynthesis of

candicidin [21,22] and a few other examples of

‘com-mitting’ enzymes for secondary metabolites that have

evolved as variants of enzymes involved in primary

metabolism [23]

All the evidence from this work indicates that the

desABCD cluster is expressed as a polycistronic

transcript Expression of the four genes is coordinately regulated by iron limitation, as shown by the RT-PCR analysis, and there is overlapping of the desB transla-tion terminatransla-tion triplet with the ATG of desC and also

of desC and desD (so-called translational coupling); moreover, there are no intergenic regions between any

of the four genes Downstream of desD, we have located a putative transcriptional terminator [calcula-ted DG )30.7 kcalÆmol)1(128.5 kJÆmol)1)] (Fig 1B) In the four genes, there is strong overexpression, which is maximal 8 h after iron deprivation and decreases at

24 h The coordinated regulation by iron of the expres-sion of the entire cluster ensures simple and efficient up-regulation of desferrioxamine biosynthesis after iron limitation

The two upstream genes (ORFs SCO2780 and SCO2781) have been annotated to encode proteins related to siderophore uptake and metabolism [13] (SCO database, http://streptomyces.org.uk), but there

Fig 6 Primer extension analysis of the transcription start (TS) point of the S coelicolor desA promoter Comparison of the reaction sequences of the promoter region (T, G, C, A) with that of the primer extension reaction product (inclined arrow) using 50, 100 and 150 lg RNA The )10 region is shown, and the transcriptional start point is indicated as +1 (bent arrow) The19-bp palindromic region, which con-tains the repressor-binding site, is boxed, and the ribosome-binding site is underlined The ATG is shown in bold letters The reported tran-scription start site of the S pilosus desA promoter is indicated by three asterisks Note the strict conservation of the 19-nucleotide iron box and the )10 sequence in both Streptomyces species.

is very little evidence for or against this claim SCO2780

encoding a putative secreted siderophore-binding

lipoprotein with a conserved Fhu (Fe2+ siderophore

binding) domain showed an iron-limitation response

similar to that of the desABCD cluster; it was clearly

induced at 2 h and reached maximal expression at 6–8 h

after iron deprivation There is no consensus iron box

in the upstream region of SCO2780, and its regulation

is probably mediated by a cascade mechanism, rather

than by direct interaction of DmdR1

On the other hand, the viuB gene (SCO2781) was

not transcribed under the conditions tested (Fig 4),

and its role in iron metabolism remains obscure This

putative siderophore-interacting protein is similar to

the Vibrio fischeri ViuB vibriobactin utilization protein

[24]

Primer extension analysis of the promoter region of

the desABCD cluster identified the transcription start

point, which allowed us to deduce the )10 Pribnow

box as TAGGCT, in good agreement with the

consen-sus (TAgPuPuT))10 sequence of Streptomyces species

[19] It is very interesting that this )10 region is

located inside the 19-nucleotide iron box identified

pre-viously [12] Therefore, binding of the iron regulator

DmdR1 will interfere with RNA polymerase

interac-tion and expression of the desferrioxamine cluster

Indeed, binding of the pure DmdR1 protein to the

S coelicolor desA promoter region was shown for the

first time in this work As described previously,

bind-ing of DmdR1 to the iron box requires a bivalent

metal (Fe2+, Mn2+, or other bivalent metals) [12], and therefore when iron is depleted, DmdR1 is unable to bind to the cognate iron box, and transcription is enhanced leading to siderophore biosynthesis

It is interesting that the promoter of S pilosus desA showed higher transcription ability than the S

coelicol-or homologous promoter (both of 511 nucleotides, amplified with the same primers) when coupled to the reporter xylE gene in either S coelicolor or S lividans Although the )10 region of desA in both S coelicolor and S pilosus [16,17] was almost identical and in both species it is located within the iron box palindrome (Fig 6), the )35 and upstream regions are different These regions were found to be relevant for optimal expression from the desA promoter, as short promoter regions gave very poor expression of the reporter xylE gene

In summary, we provide evidence that the desA gene encoding a l-lysine decarboxylase is essential for des-ferrioxamine biosynthesis in S coelicolor and appears

to be a desferrioxamine-dedicated enzyme, in contrast with another putative lysine decarboxylase (SCO2017) that might be involved in lysine utilization [20] Expression of the desABCD cluster is coordinately regulated by iron concentrations in the culture med-ium, and this regulation is mediated by binding of the regulatory protein DmdR1 to the iron box located in the promoter region of the desABCD cluster Elec-trophoretic mobility-shift assays of the desA promoters

of both S coelicolor and S pilosus revealed that two different complexes of different size are formed in each case, supporting earlier suggestions that binding takes place in the form of dimers or tetramers [12]

Experimental procedures

Bacterial strains, plasmids and culture conditions

Bacterial strains and plasmids used in this work are listed

in Table 1 Streptomyces species were routinely grown in YEME medium [25,26] at 30C For siderophore produc-tion and promoter activity experiments Minimal Medium [3] was used Escherichia coli strains were grown in Luria– Bertani broth or Luria–Bertani broth supplemented with

20 mm glucose at 30C or 37 C E coli BW25113 [27] was used to propagate the recombination plasmid pIJ790 and

S coelicolor cosmid Stc105 [28] E coli DH5a (Stratagene,

La Jolla, CA, USA) was used as a host for plasmid con-structions E coli ET12567⁄ pUZ8002 [29] was used as the nonmethylating plasmid donor for intergeneric conjugation with S coelicolor A3(2) Ampicillin (100 lgÆmL)1), apramy-cin (50 lgÆmL)1), chloramphenicol (25 lgÆmL)1), and kana-mycin (50 lgÆmL)1) were added to growth media when

Fig 7 Binding of the DmdR1 protein to the desA promoter region

of either S pilosus (left) or S coelicolor (right) at increasing protein

concentrations The electrophoretic mobility-shift assays were

per-formed as indicated in Experimental procedures Lanes 1 and 7,

control probes without protein (dashed arrow) Lanes 2–5 and 8–11

contain 1, 2, 4 or 8 l M DmdR1 in the binding reactions,

respect-ively Note the formation of two DNA–protein complexes (arrows)

at high protein concentrations Lanes 6 and 12 contain 8 l M

DmdR1 with an excess of cold probe as control The DmdR1

pro-tein was purified as described by Flores et al [12].

necessary S lividans 1326 was used as a host for

Strepto-mycesplasmid constructions

DNA methods

Isolation of plasmid and bacterial chromosomal DNA,

restriction enzyme digestions, agarose gel electrophoresis and

Southern blot analysis were performed according to standard

molecular biology techniques [30] Plasmids were

trans-formed into E coli strains by standard chemical methods

or by electroporation Electroporation-competent cells (50

or 100 lL; 109colony-forming unitsÆmL)1) were mixed with

1–5 lL DNA solution in an ice-cold microcentrifuge tube

and electroporated at 2.5 kV with 25 lF and a resistance of

200 ohms or at 2.5 kV with 10 lF and resistance of

500 ohms DNA fragments used as probes were labelled with

digoxigenin using a random priming kit (DIG DNA labelling

Mix; Roche Diagnostics GmbH, Penzberg, Germany)

Isolation of a S coelicolor DdesA mutant

Deletion of the desA gene of S coelicolor A3(2) was

per-formed by replacing the wild-type gene with a cassette

con-taining the apramycin resistance gene as selectable marker

using a PCR-based system [31] The plasmid pIJ773 which

has a disruption cassette containing the apramycin

resist-ance gene [aac(3)IV] and oriT was used as template The

mutant was constructed using the oligonucleotides 5¢-acccc

tctcggaccgtccccaccggaggacccccccatgATTCCGGGGATC

CGTCGACC-3¢ and 5¢-aggccgatgcccacgaagtcgtacggggcgctggctt caTGTAGGCTGGAGCTGCTTC-3¢ as the forward and reverse primers, respectively (the sequence identical with the upstream region of the desA gene is underlined and in low-ercase; the sequence identical with the downstream region

of the desA gene is shown in italics and in lowercase) These two long PCR primers (59 and 58 nucleotides) were designed to produce a deletion of desA just after its start codon, leaving only its stop codon behind The 3¢ sequence

of each primer matches the right or left end of the disrup-tion cassette (the sequence is shown uppercase in both primers) The extended apramycin resistance cassette was amplified by PCR, and E coli BW25113⁄ pIJ790 bearing cosmid Stc105 was electro-transformed with this cassette The isolated mutant cosmid was introduced into nonmethy-lating E coli ET12567 containing the RP4 derivative pUZ8002; then the mutant cosmid was transferred to

S coelicolor by intergeneric conjugation [32,33] Double cross-over exconjugants were screened for their kanamycin sensitivity and apramycin resistance

S coelicolor DdesA mutant complementation

A 4204-bp ScaI fragment containing the desA coding region was cloned into the pBluescript SK EcoRV site As the DdesA mutant is apramycin resistant, the kanamycin resistance marker was cloned into an XbaI site of the new construct (pSKdesA) A 5723-bp XhoI + NotI fragment containing the desA gene and kanamycin gene (from

Table 1 Strains and plasmids used in this study.

Plasmids

pIJ4083 High copy number promoter-probe vector carrying the promoterless xylE

gene as reporter

Kieser et al [25]

E coli strains

DH5a F – recA1, endA1, gyrA96, thi-1, hsdR17 (rK– , mK+ ), sup44, relA1k-,

(r80 dLacZAM15), D(lacZYA-argF)U169

Hanahan [39]

Streptomyces strains

pSKdesAKn) was cloned into the EcoRV site of pRA

(5769 bp) The new construct (pRAdesAKn) having a

size of 11492 bp was used to transform E coli ET12567⁄

pUZ8002 After conjugation with the S coelicolor desA

mutant, KnRcolonies were selected

As complementation of the DdesA mutant was not

obtained with the above construct (see Results), the mutation

was complemented using the cosmid Stc105 A nonessential

gene (SCO2788) downstream of the desferrioxamine gene

cluster was replaced by the cassette from pIJ773 to allow

RP4 oriT-assisted conjugation by the method described

above to obtain the desA mutant After intergeneric

conjuga-tion between E coli ET12567⁄ pUZ8002 bearing the cosmid

with oriT and the S coelicolor DdesA mutant, single

cross-over exconjugants were screened for kanamycin resistance

Kanamycin-resistant colonies were isolated and analysed by

HPLC for their ability to produce desferrioxamines

Siderophore plate bioassays

Siderophore production assays by colonies were carried out

on chrome azurol S blue plates prepared by the protocol of

Schwyn & Neilands [34]

HPLC analysis of desferrioxamines

Bacteria were grown in ILMM [3] and distributed into

500-mL flasks (washed with 10% nitric acid and autoclaved)

CaCl2Æ2H2O (0.01% final concentration) and glucose

(2.5 gÆL)1final concentration), autoclaved separately, and

fil-ter-sterilized yeast extract (0.05 gÆL)1 final concentration)

were then added to the culture flasks The same medium

sup-plemented with FeSO4Æ7H2O (35 lm final concentration) was

used as control Cultures were grown on a rotary shaker

(250 r.p.m.) at 30C Biomass was removed by filtration,

and 50 mL culture supernatant was freeze-dried The solid

residue was redissolved in 1 ml distilled water, and, after

removal of the insoluble particles by centrifugation, 10 lL

1 m FeCl3was added to form Tris–hydroxamate–Fe3+

com-plexes Insoluble particles were removed by centrifugation,

and the solution was filtered through a Vivaspin concentrator

before HPLC analysis

HPLC separation of desferrioxamines was performed on a

reverse-phase column (Nucleosil C18, 5 lm, 4.6 by 150 mm)

with 150 lL injection volume and 1 mLÆmin)1flow rate for

25 min A solution of 0.1% aqueous formic acid⁄ methanol

was used as the solvent system The Tris–hydroxamate–Fe3+

complexes were detected at a wavelength of 435 nm

Determination of desA promoter activity

The desA promoter fragments of S coelicolor A3(2)

and S pilosus were amplified by PCR using primers Pf

(5¢-GGAATTCCGCGCGCGGGTCTGGCTTCA-3¢) and

CGTT-3¢) containing cleavage sites for EcoRI and BamHI

at their ends (in bold) The PCR products were extracted from gels and digested with EcoRI and BamHI The pro-moter fragments were introduced upstream of the xylE gene (catechol dioxygenase reporter) in the pIJ4083 vector

to generate pCoedesAP and pPildesAP The correct orienta-tion was confirmed by sequence analysis S coelicolor and

S lividanscells harbouring pCoedesAP and pPildesAP were grown in ILMM [3] containing 50 lgÆmL)1 thiostrepton Then 1 mL of the cells was withdrawn at 24, 48, 60 and

72 h and, after being washed with physiological saline, they were frozen and kept at)20 C Crude extracts of the cells were obtained by disruption using an ultrasonicator at

4C Cells were sonicated (4 · 15 s with 20 s intervals) in sample buffer [100 mm phosphate buffer (pH 7.5), 20 mm EDTA (pH 8.0), 10% (v⁄ v) acetone] Triton X-100 was added to a final concentration of 0.1%, and the mixture was incubated for 15 min on ice After clearing of the mix-ture by centrifugation (10 000 g, Eppendorf 5415R centri-fuge; Eppendorf, Hamburg, Germany) at 4C, the clear supernatant was assayed for catechol 2,3-dioxygenase activ-ity as described by Hopwood et al [32]

Primer extension analysis

RNA was isolated from a 48-h culture of S coelicolor har-bouring pCoedesAP plasmid under conditions of iron limi-tation by the procedure of Kieser et al [25] Primer extension analysis was performed as described by Patek

et al [35] and Barreiro et al [36] The fluorescein-labelled primer was hybridized to RNA in a solution containing 0.4 m NaCl, 40 mm Pipes (pH 6.4), 1 mm EDTA (pH 8.0), and 80% formamide at 45C for 12 h The precipitated RNA was dissolved in 20 lL reaction mixture: 4 lL Super-Script buffer (Invitrogen, Carlsbad, CA, USA), 5 lL dNTP (2 mm), 2 lL dithiothreitol (0.1 m), 2 lL actinomycin D (500 lgÆmL)1), 1 lL RNase inhibitor (40 U) and 5 lL

H2O After the addition of SuperScript II RT (Invitrogen), the reaction was run at 42C for 1 h and stopped by heat inactivation of the enzyme RNA was removed by RNase treatment and the protected RNA–DNA sample was preci-pitated with ethanol Then, the sample was dissolved in

6 lL TE buffer (10 mm Tris/HCl, 1 mm EDTA, pH 8.0) and 6 lL stop buffer (Thermo Sequenase Primer Cycle Sequencing Kit, Amersham Biosciences, Piscataway, NJ, USA) After heat denaturation, the sample was run in the ALFexpress DNA sequencer to identify the end of the pro-tected fragment

Transcriptional analysis

Culture conditions under iron limitation were as follows

S coelicolor inoculum cultures were grown for 36 h in YEME broth [25] The cell pellet was harvested by centrifugation and washed twice with distilled water Equal