Báo cáo Y học: The phosphotransferase system of Streptomyces coelicolor IIACrr exhibits properties that resemble transport and inducer exclusion function of enzyme IIAGlucose of Escherichia coli pptx

The phosphotransferase system of Streptomyces coelicolorIIACrr exhibits properties that resemble transport and inducer exclusion function of enzyme IIAGlucose of Escherichia coli Annette

The phosphotransferase system of Streptomyces coelicolor

IIACrr exhibits properties that resemble transport and inducer exclusion function

of enzyme IIAGlucose of Escherichia coli

Annette Kamionka1, Stephan Parche1, Harald Nothaft1, Jo¨rg Siepelmeyer2, Knut Jahreis2

and Fritz Titgemeyer1

1 Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Lehrstuhl fu¨r Mikrobiologie, Erlangen, Germany; 2 Universita¨t Osnabru¨ck, Lehrstuhl fu¨r Genetik, Fachbereich Biologie/Chemie, Osnabru¨ck, Germany

We have investigated the crr gene of Streptomyces coelicolor

that encodes a homologue of enzyme IIAGlucoseof

Escheri-chia coli, which, as a component of the

phosphoenolpyru-vate-dependent sugar phosphotransferase system (PTS)

plays a key role in carbon regulation by triggering glucose

transport, carbon catabolite repression, and inducer

exclu-sion As in E coli, the crr gene of S coelicolor is genetically

associated with the ptsI gene that encodes the general

phosphotransferase enzyme I The gene product IIACrrwas

overproduced, purified, and polyclonal antibodies were

obtained Western blot analysis revealed that IIACrr is

expressed in vivo The functionality of IIACrrwas

demon-strated by phosphoenolpyruvate-dependent

phosphoryla-tion via enzyme I and the histidine-containing phosphoryl

carrier protein HPr Phosphorylation was abolished when

His72, which corresponds to the catalytic histidine of E coli IIAGlucose, was mutated The capacity of IIACrrto operate in sugar transport was shown by complementation of the

E coli glucose-PTS The striking functional resemblance between IIACrrand IIAGlucosewas further demonstrated by its ability to confer inducer exclusion of maltose to E coli

A specific interaction of IIACrrwith the maltose permease subunit MalK from Salmonella typhimurium was uncovered

by surface plasmon resonance These data suggest that this IIAGlucose-like protein may be involved in carbon meta-bolism in S coelicolor

Keywords: inducer exclusion; protein phosphorylation; protein–protein interaction; Streptomyces; surface plasmon resonance

Streptomycetes undergo global changes in gene expression

and enzyme activities in response to developmental stages,

secondary metabolite production (antibiotics), carbon

util-ization, and stress conditions [1–5] The focus of our

research is the regulation of carbon source utilization

(C-regulation) and how this influences the other

above-mentioned processes

Streptomyces coelicolor metabolizes a wide variety of

nutrients Their utilization is subject to C-regulation, in

which glucose kinase appears to be of significant importance

[6,7] However, the signal transduction pathways are poorly

understood In many other bacteria, components of the

phosphoenolpyruvate-dependent sugar phosphotransferase

system (PTS) trigger C-regulation by mechanisms known as

carbon catabolite repression and inducer exclusion [8,9] One key element in Escherichia coli is enzyme IIAGlucose

(IIAGlc) IIAGlc becomes phosphorylated by the general PTS proteins, which are histidine-containing phosphoryl carrier protein (HPr) and enzyme I (EI) In turn, it phosphorylates the sugar-specific PTS permeases that catalyse the uptake of glucose, trehalose, and sucrose [8,10,11] Mutations in the respective gene crr exhibit a pleiotropic catabolite repression resistant phenotype [12] The underlying mechanisms are that unphosphorylated IIAGlc inhibits a set of catabolic enzymes and sugar permeases including the MalK subunit of the maltose permease by protein–protein interaction (inducer exclu-sion) At the same time the cellular cAMP level is low, because dephosphorylated IIAGlc is unable to stimulate adenylate cyclase Under these conditions the cAMP-dependent catabolite activator protein CAP, which serves

as a global activator of many catabolite-controlled genes, remains in a switched off state [9] IIAGlcfurther appears to

be involved in carbon catabolite repression exerted by non-PTS substrates such as glucose 6-phosphate [13] This could

be correlated with the variation of the phosphorylation state

of IIAGlc Recently, another cellular function for IIAGlchas been proposed that suggests that it may be involved in the linkage between carbon metabolism and stress response [14]

We have described that the PTS is operative in strepto-mycetes [15,16] Analysis of the S coelicolor genome revealed the presence of nine genes that may encode four sugar-specific permeases, as well as the genes ptsH and ptsI

Correspondence to F Titgemeyer, Friedrich-Alexander-Universita¨t

Erlangen-Nu¨rnberg, Lehrstuhl fu¨r Mikrobiologie, Staudtstrasse 5,

91058 Erlangen, Germany Fax: + 49 91318528082,

Tel.: + 49 91318528095, E-mail: ftitgem@biologie.uni-erlangen.de

Abbreviations: aMG, methyl a-glucoside; EI, enzyme I; HPr, histidine

containing phosphoryl carrier protein; II(ABC) sugar , enzyme II(ABC)

transporter protein; PTS, phosphoenolpyruvate-dependent sugar

phosphotransferase system; isopropyl, thio-b- D -galactose (IPTG);

Enzymes: enzyme I of the phosphoenolpyruvate-dependent sugar

phosphotransferase system (EC 2.7.3.9); enzyme II of the

phos-phoenolpyruvate-dependent sugar phosphotransferase system

(EC 2.7.1.69).

(Received 22 October 2001, revised 25 February 2002, accepted

4 March 2002)

encoding HPr and EI [17] Beside this, a crr-like gene was

found upstream of ptsI In this communication we provide

evidence that this putative crr gene is expressed in vivo and

that it constitutes a functional equivalent of its homologue

in E coli

M A T E R I A L S A N D M E T H O D S

Bacterial strains, growth conditions, and plasmid

construction

S coelicolorA3(2) M145 (SCP1-, SCP2-, prototroph) was

used as wild-type strain [18] E coli DH5a was the host

strain for subcloning experiments [19] E coli FT1

DptsHIcrr Kanr(pLysS Cmr) was used to produce native

and hexa-histidine (His)-tagged S coelicolor IIACrr,

His-tagged S coelicolor HPr, and His-His-tagged E coli IIAGlc[16]

M15(pREP4, pAG3) was used to produce His-tagged

Bacillus subtilisEI [16,20] The glucose-negative E coli crr

mutant strain LM1 tonA galT nagE manAI kbatsrpsL xyl

metB thi his mglA-C argG crrwas used for heterologous

complementation experiments [21]

S coelicolor cultures were grown for 30–72 h with

vigorous shaking in complex medium (tryptic soy medium

without dextrose; Difco) at 37C or in mineral medium

supplemented with 0.1% casamino acids or 50 mMcarbon

source at 28C [17] E coli cultures were grown in Luria–

Bertani medium at 37C

Total DNA from S coelicolor M145 was isolated as

described [16] Cloning of the crr gene of S coelicolor was

performed as follows A DNA fragment of 475 bp

compri-sing crr was amplified by PCR with Pfu DNA polymerase

using S coelicolor M145 wild-type chromosomal DNA as

template together with oligonucleotides engineered to

introduce the restriction sites NdeI and BamHI, respectively

(Crr1, 5¢-GGAGGTTTCATATGACCACCGTTTCTTC

CCCGC-3¢ and Crr2, 5¢-GACGGATCCGACGTCAC

TTCCAGAGG-3¢, restriction sites are in italic type) The

amplified DNA was digested with NdeI and BamHI and

cloned into plasmids pET15b and pET3c (Novagen)

resulting in crr expression plasmids pFT41 and pFT42,

respectively [22] A two-step PCR mutagenesis procedure as

described by Landt et al was used to change the codon for

His72 to an alanine codon [23] Chromosomal DNA of

S coelicolorM145 served as template together with

oligo-nucleotide Crr3 (5¢-GCGTGCTGACCGCTCTCGG

GATCGAC-3¢; altered positions are underlined) and the

two flanking primers as described above The NdeI–BamHI

digested PCR fragment was cloned into pET3c digested

with the same enzymes giving pFT44 The expression

plasmid pCRL13 for the production of a C-terminal

His-tagged IIAGlcof E coli was derived by cloning of an NdeI–

HindIII fragment into plasmid pET23a(+) (Novagen) [22]

The crr fragment was generated by PCR (primers: Crr4,

5¢-GGAGAAGCATATGGGTTTGTTCG-3¢ and Crr5,

5¢-TTAAAGCTTGATGCGGATAACCGG-3¢; restriction

sites are in italic type) All PCR-based constructs were

confirmed by DNA sequencing For constitutive expression

of crr, the crr alleles from plasmids pFT41 and pFT42 were

prepared by sequential treatment with XbaI, T4 DNA

polymerase, and HindIII The fragments were cloned

into the pSU2718 derivative pFT76 (K Mahr, unpublished

data) that was sequentially treated with KpnI, T4

DNA-polymerase, and HindIII giving plasmids pFT111 (his-tagged IIACrr) and pFT112 (IIACrr) [24]

Protein overproduction and purification Recombinant tagged HPr from S coelicolor, His-tagged IIACrr from S coelicolor, His-tagged IIAGlc from

E coli, and His-tagged EI from B subtilis were overpro-duced and purified as described previously [16] Purification

of native IIACrr was achieved in a single step by anion exchange chromatography (HQ-column; 1.6 mL bed vol-ume; Poros) in buffer (20 mM Tris/HCl pH 7.5, 3 mM

dithiothreitol) with a linear gradient of 0–500 mM

NaCl Protein concentrations were determined with the Bio-Rad protein assay Proteins were stored at)20 C or )70 C

Phosphoenol pyruvate-dependent phosphorylation Preparation of [32P]phosphoenolpyruvate and protein phos-phorylation assays were carried out as described previously [16] Radiolabelled proteins were detected by radiolumi-nography on a phosphoimager (Fuji)

Enzyme assays IIACrr activity was assayed by complementation of the glucose-specific PTS of E coli measuring phosphoenolpyru-vate-dependent phosphorylation of methyl [a-14C]glucoside ([14C]aMG; Amersham) in the presence of E coli LM1 cell extract [16] The assay was carried out at 30C in a reaction volume of 0.1 mL containing rate-limiting amounts of IIA protein (50 pmoles), 55 lg protein of LM1 extract, and

a final concentration of 12 lM[14C]aMG (1.4 mCiÆmmol)1) Phosphorylation of aMG was linear within the first minute The initial phosphorylation rates were calculated from triplicates by subtraction of the blank value (LM1 extract without IIA protein) of 140 ± 8 nmol aMG-PÆmin)1

Transport assays Cells of E coli FT1 bearing either plasmid pET23a(+), pCRL13(crr+E coli), pET3c, or pFT42(crr+S coelicol-or) were grown at 37C in 100 mL Luria–Bertani medium supplemented with 25 mM maltose At

D600 ¼ 0.8, 50 mL of FT1(pCRL13) or FT1(pFT42) culture were harvested The remaining 50 mL of the cultures were supplemented with 1 mMisopropyl thio-b-D -galactose (IPTG) to induce crr expression Incubation was continued for 45 min FT1(pET23a(+)) and FT1(pET3c) were grown to a final D600 ¼ 1.0 All cells were harvested and washed twice in chilled transport buffer (50 mM T ris/HCl pH 7.5, 50 mM NaCl, 10 mM KCl) Cells were resuspended in transport buffer, adjusted to

D600 ¼ 1.0 and kept on ice For transport analysis an aliquot of cells was preincubated for 5 min at 37C Uptake was initiated by addition of [14C]maltose to a final concentration of 20 lM (5 mCiÆmmol)1) Samples of 0.5 mL were taken between 0.5 and 5 min, rapidly filtered (1 mLÆs)1) through nitrocellulose filters (NC45), and washed three times with 2 mL ice-cold 0.1M LiCl Radioactivity was determined by liquid scintillation counting

Western blot analysis

Western blot analyses were carried out as described by

Parche et al [16] Rabbit polyclonal antibodies were raised

against His-tagged IIACrrof S coelicolor (Eurogentec) A

dilution of 1 : 3000 yielded specific signals against 10 ng

His-tagged IIACrr and against 5 lg of S coelicolor cell

extract that corresponded to a molecular size of 19 kDa and

17 kDa, respectively

Surface plasmon resonance analysis

Interactions of proteins were detected by surface plasmon

resonance analysis using a BIAcore X optical biosensor

(Biacore AB) Three micrograms of S coelicolor

His-tagged IIACrr (180 pmoles), of E coli His-tagged IIAGlc

(150 pmoles), and of E coli His-tagged tetracycline

repres-sor TetR (125 pmoles; control for nonspecific binding) were

applied for the immobilization on an NTA sensor chip The

efficiency for the coupling reaction was 1200 resonance units

(71 fmoles) for His-tagged IIACrr, 450 resonance units

(24 fmoles) for his-tagged IIAGlc, and 1500 resonance units

(60 fmoles) for His-tagged TetR For binding analysis, 5 lg

(120 pmoles) of purified MalK protein of Salmonella

typhimurium was dialysed twice against eluent buffer

(10 mM Hepes pH 7.4, 150 mM NaCl, 50 lM EDTA,

0.005% (v/v) polysorbate 20) and introduced at a flow rate

of 5 lLÆmin)1for 10 min Three micrograms of purified glucose kinase protein (91 pmoles) from S coelicolor were used as a control for unspecific ligand binding

Computer analyses The program DNA STRIDERTM 1.2 and the Lasergene workstation software (DNASTAR) were used to process DNA sequence data [25] DNA databank and protein databank searches were performed using theBLASTserver of the National Center for Biotechnology Information at the National Institutes of Health Bethesda, MD, USA (http:// www.ncbi.nlm.nih.gov) Binary sequence comparisons were computed with theFASTAsoftware [26]

R E S U L T S

Identification of thecrr gene Figure 1A depicts a detailed genetic map of the crr ptsI genes that we had identified previously by in silico analysis [17] Both genes encode putative PTS phosphotransferase components that constitute homologues of E coli enzyme IIAGlcand EI, respectively They are flanked upstream by rrnC, which encodes ribosomal RNA and downstream by

an ORF of unknown function The sequence of the crr region contains two possible start codons Analysis of the

Fig 1 Genetic organization of the S coelicolor crr gene and protein alignment (A) T he genetic arrangement of the crr and ptsI gene is shown Arrows indicate transcriptional orientation of genes Numbers of base pairs and length of proteins (aa, amino acids) are denoted below coding regions Numbers in square brackets show the lengths of intergenic regions in bp (B) The IIA Crr sequence (above) is shown together with the consensus sequence (below) derived from an alignment with 11 amino acid sequences of IIACrrand IIACrr-like proteins found in the current databank These are: S coelicolor (AL353861), Bacillus stearothermophilus (P42015), Haemophilus influenzae (P45338), Corynebacterium glutam-icum (Q45298), Bacillus subtilis (P39816), Escherichia coli (P08837), Klebsiella pneumonieae (P45604), S mutans (P12655), Corynebacterium ammoniagenes (3098512), Lactobacillus delbrueckii (P22733), S thermophilus (P23926) Residues conserved in > 80% of all proteins are displayed

in upper case letters while residues conserved in 50–80% of all proteins are shown in lower case letters Insertions/deletions are indicated by a dash Two conserved histidines are highlighted as putative phosphorylation (*) and active centre sites (!).

3

codon usage, of potential ribosome binding sites, and

sequence alignments favoured an ORF of 449 bp beginning

at the second start codon that encodes a gene product with a

calculated mass of Mr 15 236 The protein sequence of

IIACrr was aligned with 11 homologues (Fig 1B) The

derived consensus sequence revealed two well-conserved

histidines in IIACrr (57 and 72) that matched the active

centre residue histidine 75 and the experimentally proven

phosphorylation site histidine 90 of E coli IIAGlc[27,28]

Overexpression and purification ofS coelicolor IIACrr

and IIACrr(H72A)

To study the function of IIACrr, we overexpressed three crr

alleles in E coli encoding His-tagged IIACrr, native IIACrr,

and native IIACrr(H72A) Therefore, plasmids pFT41,

pFT42, and pFT44 were transformed into the

deletion mutant FT1(pLysS) Recombinant proteins were

produced and purified as outlined in Materials and

meth-ods As depicted in Fig 2, His-tagged IIACrr, IIACrr, and

IIACrr(H72A) showed overexpression characteristics

reveal-ing prominent protein bands that migrated correspondreveal-ing

to a size of 19 kDa for the His-tagged protein and 17 kDa

for the native protein (Fig 2, lanes 2, 4, 6) His-tagged

IIACrr was purified yielding  20 mg proteinÆL)1 E coli

culture, and IIACrrand IIACrr(H72A) were purified yielding

 9 mg and  10 mg proteinÆL)1 E coli culture,

respect-ively (Fig 2, lanes 3, 5, 7) His-tagged IIACrrwas used to

raise polyclonal antibodies

Is the putativecrr gene expressed in S coelicolor?

To address this question, we monitored the presence of

IIACrr protein in S coelicolor A Western blot analysis

showed IIACrr-specific immunosignals in extracts of

wild-type mycelia (Fig 3) IIACrrprotein was detectable under all

conditions tested and showed the highest levels in

glucose-grown mycelia, intermediate levels when fructose and glycerol served as the carbon source, and lower levels in mycelia grown on casamino acids or glutamate

Is IIACrrphosphorylated by HPr?

In vitrophosphorylation assays were performed to demon-strate phosphoenolpyruvate-dependent phosphorylation of IIACrr in the presence of the general PTS phosphotrans-ferases EI and HPr (Fig 4) As shown in lane 1 of Fig 4, IIACrrof S coelicolor became phosphorylated upon incu-bation with radiolabelled phosphoenolpyruvate, EI of

B subtilis, and HPr of S coelicolor, while IIACrrincubated

Fig 2 Overexpression and purification of IIACrr proteins An

SDS/12% polyacrylamide gel stained with Coomassie brilliant blue is

shown Lane 1, protein marker; lane 2, 30 lg crude cell extract of

FT1(pFT41); lane 3, 8 lg purified His-tagged IIACrr; lane 4, 30 lg cell

extract of FT1(pFT42); lane 5, 5 lg purified IIA Crr ; lane 6, 30 lg cell

extract of FT1(pFT44); lane 7, 5 lg purified IIA Crr (H72A).

Fig 3 Western Blot analysis A Western Blot of an SDS/12% poly-acrylamide gel shows the immunoreactive signal of IIACrr In each lane

10 lg protein of crude cell extract were subjected to gel electrophoresis Extracts were prepared from cells grown in mineral medium contain-ing 0.1% casamino acids (CAA; lane 1), or in minimal medium con-taining 50 m M of either fructose (lane 2), glucose (lane 3), glycerol (lane 4), or glutamate (lane 5) The figure is representative for several simi-larly performed Western blot experiments.

Fig 4 Phosphorylation of EI, HPr, and IIACrr The phospholumino-gram of an SDS/12% polyacrylamide gel shows [32 P]phos-phoenolpyruvate-dependent phosphorylation of purified B subtilis His-tagged EI (16 pmol), S coelicolor His-tagged HPr (67 pmol), and

of S coelicolor IIA Crr and IIA Crr (H72A) (235 pmol) The following combinations were examined: lane 1: EI, HPr, and IIACrr; lane 2: EI and HPr; lane 3: EI and IIA Crr ; lane 4: HPr; lane 5: EI, HPr, and IIA Crr boiled for 10 min prior to protein gel loading; lane 6: EI, HPr, and IIACrr(H72A); lane 7: EI and IIACrr(H72A) The migration of proteins

is indicated Note that phosphorylated EI is not or barely visible due to the low protein amounts used.

only with EI was not phosphorylated (lane 3) After boiling,

IIACrr-phosphate became dephosphorylated indicating a

heat-labile aminoacyl phosphorylation of IIACrr(lane 5) as

occurs by histidine phosphorylation When histidine 72 was

replaced by an alanine, the resulting product IIACrr(H72A)

could not be phosphorylated (lane 6)

Can IIACrr function in sugar transport?

After it was shown that IIACrris phosphorylated by HPr,

we investigated whether it could interact with an enzyme II

permease As no such enzyme II has been characterized so

far in S coelicolor, we asked whether IIACrr can replace

IIAGlc of E coli with respect to glucose transport We

constructed plasmids pFT111 (His-tagged IIACrr) and

pFT112 (IIACrr), in which the crr genes should be expressed

constitutively When pFT111 and pFT112 were

trans-formed into the crr mutant LM1, fermentation of glucose

was restored as indicated by red glucose-fermenting colonies

on MacConkey agar supplemented with glucose (Fig 5)

This showed that IIACrr could interact with the E coli

components of the glucose-specific PTS, HPr, and enzyme

IIBCGlc The complementation was quantified by a

glucose-PTS assay, in which cell extracts of LM1 were combined

with rate-limiting amounts of purified His-tagged IIACrrof

S coelicolor and His-tagged IIAGlcof E coli The initial

phosphorylation rates of methyl a-glucoside were

152 ± 22 nmol aMG-PÆmin)1 when IIACrr was added

and 429 ± 39 nmol aMG-PÆmin)1when IIAGlcwas added

This indicated that under these conditions the heterologous

IIACrrprotein could compensate to about 35% the function

of E coli IIAGlc

Can IIACrrfunction in inducer exclusion?

We then studied whether IIACrr could replace its E coli counterpart in a C-regulatory capacity The DptsHIcrr deletion strain FT1 provided the possibility to monitor inducer exclusion of maltose uptake If crr is expressed in such a genetic background, the product should not be phosphorylated due to the lack of EI and HPr Non-phosphorylated IIAGlcwill block the activity of the maltose-specific ABC transport complex by interaction with MalK This effect is shown in Fig 6A, where maltose uptake was severely reduced when IIAGlc of E coli was expressed in strain FT1(pCRL13) The same result, although less pronounced, was observed when IIACrr was expressed in strain FT1(pFT42) (Fig 6B) It should be noted that both strains overproduced similar amounts of IIA protein as judged by comparison of protein band intensities of a CBB-stained SDS/polyacrylamide gel (data not shown)

To corroborate this finding, we performed protein– protein interaction analysis of IIACrrwith purified MalK from Salmonella typhimurium, which exhibits 95% amino

Fig 5 Complementation of an E coli crr mutant The figure shows a

MacConkey agar plate supplemented with 25 m M glucose While

E coli LM1 crr bearing plasmid pSU2718 (control) formed white

colonies (no glucose fermentation), LM1(pFT111) producing

His-tagged IIACrr of S coelicolor or LM1(pFT112) producing native

IIA Crr of S coelicolor yielded red (dark grey) colonies indicating

aci-dification of the medium as a result of glucose fermentation.

Fig 6 Time-course of maltose uptake (A) Maltose uptake of E coli FT1 bearing either pET23a(+) (control, d), pCRL13 (E coli His-tagged IIA Glc ) after induction with IPTG (.) (B) Maltose uptake of

E coli FT1 bearing either pET3c (control, d) or pFT 42 (S coelicolor his-tagged IIACrr) after induction with IPTG (.) Values were deter-mined in triplicate and experiments were performed at least three times Standard deviations are displayed by error bars.

acid identity with MalK of E coli (Fig 7) Therefore,

his-tagged IIACrr was coupled to an NTA-sensor chip and a

solution of MalK was allowed to flow over the immobilized

protein A binding signal of 400 resonance units was

detected, while no interaction was observed when MalK

solution was passed over immobilized His-tagged TetR

protein (negative control) Immobilized His-tagged IIAGlc

yielded a response of 500 resonance units with the MalK

protein (positive control) Therefore, the observed reduction

of maltose uptake in E coli by IIACrrcould be confirmed by

the demonstration of its interaction with the MalK subunit

of the maltose permease complex

D I S C U S S I O N

In this study, we report on the analysis of an S coelicolor

ORF that encodes a protein, IIACrr, with significant

similarity to enzyme IIAGlcof E coli, a global-acting factor

of carbon metabolism We provided evidence that the gene

is expressed in vivo and that IIACrris phosphorylated in vitro

by the general PTS phosphotransferases EI and HPr IIACrr

could replace the functions of E coli IIAGlc in glucose

transport and inducer exclusion These findings suggest that

IIACrr might be involved in carbohydrate transport and

C-regulation in S coelicolor

The crr gene of S coelicolor shares the highest similarity

to a putative crr gene of S griseus (accession AB030569),

which indicated that crr is also present in other

strepto-mycetes crr genes are further found in Gram-negative

bacteria such as E coli and Haemophilus influenzae, and in

some mycoplasma species [8,29] In contrast, many other

microorganisms including the actinomycetes

Corynebacte-rium diphtheriae and Mycobacterium smegmatis, some

mycoplasmae, and low-GC Gram-positive bacteria such

as Bacillus subtilis possess no crr gene These have crr

homologues as part of sugar-specific enzyme IIABC permeases that solely appear to fulfil transport function (F Titgemeyer, unpublished data; [30–32]) For Gram-negative species a multiple role of IIAGlc has been documented and proposed [9,13,14,29]

The reported data demonstrate that IIACrr could effi-ciently cross-communicate with the proteins HPr, enzyme IIBCGlc, and MalK from enteric bacteria This striking functional resemblance to E coli IIAGlcand the observation that S coelicolor IIACrr is present under all nutritional conditions tested may provide good indications that IIACrr functions in a similar way in S coelicolor The amount of IIACrr was higher when S coelicolor was grown on carbohydrates than it was when the organism was grown

on amino acids Thus, further investigation should be carried out to determine in more detail which carbon sources induce expression of crr

What are the targets of IIACrr? We could demonstrate that IIACrris phosphorylated by S coelicolor HPr There-fore, it should act as a PTS phosphotransferase With respect to carbon source transport, it seems to be clear that IIACrris not an enzyme IIAGlcas streptomycetes appear to lack the glucose-specific PTS [15,17] An analysis of the

S coelicolorgenome revealed two loci, malX2-nagE1-nagE2 and malX1

2 , that encode PTS permeases of the glucose/ sucrose family [17,33] The fact that all lack a IIA domain may support the speculation that IIACrr serves as the corresponding phosphotransferase

A fascinating issue to investigate is whether the mechan-ism of inducer exclusion is realized in S coelicolor Our observation that IIACrrcould replace the inducer exclusion function of E coli IIAGlcby inhibition of maltose uptake might be a good indication for this hypothesis The demonstration of the IIACrr–MalK interaction suggests that IIACrr may regulate the function of some of the

> 140 MalK homologues found in the S coelicolor genome The one with the highest similarity of 46% identical amino acids is MsiK, which serves as the AT Pase subunit for ABC transporters specific for maltose, cellobi-ose, xylobicellobi-ose, and trehalose [34–36] MsiK could therefore

be a potential candidate for regulation by IIACrr Initial attempts to demonstrate IIACrr-MsiK binding by surface plasmon resonance failed probably because overproduced MsiK forms inclusion bodies yielding incorrectly folded protein (unpublished data) [37]

IIACrr could also play a role in carbon catabolite repression The mechanism of this phenomenon is not solved in streptomycetes [6,7,38–40] It appears that glucose kinase serves a global regulatory function, but how it senses and transmits carbon source signals is unclear It has been demonstrated that IIAGlc of E coli senses C-regulatory signals from both PTS and non-PTS carbon sources and responds via its phosphorylation state [9,13] It would be of great interest to examine whether IIACrr of S coelicolor operates in a similar way

Finally, another hint as to a possible function of IIACrr

should be mentioned here Ueguchi and coworkers have reported that E coli IIAGlccontrols the sigma factor of the general stress response RpoS [14] They suggested that this could be a linkage between carbon metabolism and stress response upon nutrient starvation Thus, IIACrr could be involved in controlling some of the many sigma factors that

S coelicolorpossesses [4,41]

Fig 7 Surface plasmon resonance analysis A real-time interaction

analysis of his-tagged IIACrr (broken line) and His-tagged IIAGlc

(dotted line) with MalK is shown The control with tetracycline

repressor (TetR) is depicted by a solid line The sensorgram represents

the binding responses of MalK in resonance units (RU)

of time MalK solution was passed for 10 min over immobilized

protein resulting in an increase of RU caused by buffer components

and protein binding Removal of MalK by application of washing

buffer revealed an RU-increase of the baseline (dotted line) indicating

solely the binding of MalK to immobilized IIA protein (arrows).

The experiment was repeated three times with almost identical results.

When purified glucose kinase from S coelicolor was applied as a

ligand, no binding was observed (negative control).

Further analyses are required to address the ideas

mentioned above These should cover phenotype analysis

of a crr mutant, protein–protein interaction studies with

candidate proteins, and the determination of the levels of

nonphosphorylated/phosphorylated IIACrr in relation to

the nutritional state of the streptomycetes mycelium

A C K N O W L E D G E M E N T S

These studies were carried out in the laboratories of W Hillen His

support is greatly appreciated We thank E Schneider for providing

MalK protein and O Scholz for a gift of TetR protein We are grateful

to K Mahr for critical reading of the manuscript The work was

funded by SFB171 and SFB473 of the Deutsche

Forschungsgemein-schaft J S was supported through SFB431 grant given to J W.

Lengeler.

R E F E R E N C E S

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