The phosphotransferase system of Streptomyces coelicolor IIA Crr exhibits properties that resemble transport and inducer exclusion function of enzyme IIA Glucose of Escherichia coli Annette Kamionka 1 , Stephan Parche 1 , Harald Nothaft 1 ,Jo¨ rg Siepelmeyer 2 , Knut Jahreis 2 and Fritz Titgemeyer 1 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 IIA Glucose of 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,thecrr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIA Crr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIA Crr is expressed in vivo. The functionality of IIA Crr was 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 IIA Glucose , was mutated. The capacity of IIA Crr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIA Crr and IIA Glucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIA Crr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIA Glucose -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 IIA Glucose (IIA Glc ). IIA Glc 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 IIA Glc 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 IIA Glc 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]. IIA Glc further 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 IIA Glc . Recently, another cellular function for IIA Glc has 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) Eur. J. Biochem. 269, 2143–2150 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02864.x 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. MATERIALS AND METHODS Bacterial strains, growth conditions, and plasmid construction S. coelicolor A3(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 Kan r (pLysS Cm r ) was used to produce native and hexa-histidine (His)-tagged S. coelicolor IIA Crr ,His- tagged S. coelicolor HPr, and His-tagged E. coli IIA Glc [16]. M15(pREP4, pAG3) was used to produce His-tagged Bacillus subtilis EI [16,20]. The glucose-negative E. coli crr mutant strain LM1 tonA galT nagE manAI kba ts rpsL xyl metB thi his mglA-C argG crr was 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 37 °C or in mineral medium supplemented with 0.1% casamino acids or 50 m M carbon source at 28 °C [17]. E. coli cultures were grown in Luria– Bertani medium at 37 °C. 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 NdeIandBamHI, respectively (Crr1, 5¢-GGAGGTTTCATATGACCACCGTTTCTTC CCCGC-3¢ and Crr2, 5¢-GACGGATCCGACGTCAC TTCCAGAGG-3¢, restriction sites are in italic type). The amplified DNA was digested with NdeIandBamHI 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. coelicolor M145 served as template together with oligo- nucleotide Crr3 (5¢-GCGTGCTGACC GCTCTCGG 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 IIA Glc of 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,thecrr 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 IIA Crr ) and pFT112 (IIA Crr )[24]. Protein overproduction and purification Recombinant His-tagged HPr from S. coelicolor,His- tagged IIA Crr from S. coelicolor, His-tagged IIA Glc from E. coli, and His-tagged EI from B. subtilis were overpro- duced and purified as described previously [16]. Purification of native IIA Crr was achieved in a single step by anion exchange chromatography (HQ-column; 1.6 mL bed vol- ume; Poros) in buffer (20 m M Tris/HCl pH 7.5, 3 m M dithiothreitol) with a linear gradient of 0–500 m M NaCl. Protein concentrations were determined with the Bio-Rad protein assay. Proteins were stored at )20 °Cor )70 °C. Phospho enol pyruvate-dependent phosphorylation Preparation of [ 32 P]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 IIA Crr activity was assayed by complementation of the glucose-specific PTS of E. coli measuring phosphoenolpyru- vate-dependent phosphorylation of methyl [a- 14 C]glucoside ([ 14 C]aMG; Amersham) in the presence of E. coli LM1 cell extract [16]. The assay was carried out at 30 °Cinareaction volume of 0.1 mL containing rate-limiting amounts of IIA protein (50 pmoles), 55 lgproteinofLM1extract,and a final concentration of 12 l M [ 14 C]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 37 °C in 100 mL Luria–Bertani medium supplemented with 25 m M maltose. At D 600 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 m M isopropyl 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 D 600 ¼ 1.0. All cells were harvested and washed twice in chilled transport buffer (50 m M Tris/HClpH7.5,50m M NaCl, 10 m M KCl). Cells were resuspended in transport buffer, adjusted to D 600 ¼ 1.0 and kept on ice. For transport analysis an aliquot of cells was preincubated for 5 min at 37 °C. Uptake was initiated by addition of [ 14 C]maltose to a final concentration of 20 l M (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 0.1 M LiCl. Radioactivity was determined by liquid scintillation counting. 2144 A. Kamionka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Western blot analysis Western blot analyses were carried out as described by Parche et al. [16]. Rabbit polyclonal antibodies were raised against His-tagged IIA Crr of S. coelicolor (Eurogentec). A dilution of 1 : 3000 yielded specific signals against 10 ng His-tagged IIA Crr and against 5 lgofS. 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 IIA Crr (180 pmoles), of E. coli His-tagged IIA Glc (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 IIA Crr , 450 resonance units (24 fmoles) for his-tagged IIA Glc , 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 m M Hepes pH 7.4, 150 m M NaCl, 50 l M EDTA, 0.005% (v/v) polysorbate 20) and introduced at a flow rate of 5 lLÆmin )1 for 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 STRIDER TM 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 the BLAST server 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 the FASTA software [26]. RESULTS Identification of the crr 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 IIA Glc and 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)Thegeneticarrangementofthecrr 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 IIA Crr and IIA Crr -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 Ó FEBS 2002 IIACrr of Streptomyces coelicolor (Eur. J. Biochem. 269) 2145 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 M r 15 236. The protein sequence of IIA Crr was aligned with 11 homologues (Fig. 1B). The derived consensus sequence revealed two well-conserved histidines in IIA Crr (57 and 72) that matched the active centre residue histidine 75 and the experimentally proven phosphorylation site histidine 90 of E. coli IIA Glc [27,28]. Overexpression and purification of S. coelicolor IIA Crr and IIA Crr (H72A) To study the function of IIA Crr , we overexpressed three crr alleles in E. coli encoding His-tagged IIA Crr ,nativeIIA Crr , and native IIA Crr (H72A). Therefore, plasmids pFT41, pFT42, and pFT44 were transformed into the 1 DptsHIcrr deletion mutant FT1(pLysS). Recombinant proteins were produced and purified as outlined in Materials and meth- ods. As depicted in Fig. 2, His-tagged IIA Crr , IIA Crr ,and IIA Crr (H72A) showed overexpression characteristics reveal- ing prominent protein bands that migrated corresponding 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 IIA Crr was purified yielding  20 mg proteinÆL )1 E. coli culture, and IIA Crr and IIA Crr (H72A) were purified yielding  9mgand 10 mg proteinÆL )1 E. coli culture, respect- ively (Fig. 2, lanes 3, 5, 7). His-tagged IIA Crr was used to raise polyclonal antibodies. Is the putative crr gene expressed in S. coelicolor ? To address this question, we monitored the presence of IIA Crr protein in S. coelicolor. A Western blot analysis showed IIA Crr -specific immunosignals in extracts of wild- type mycelia (Fig. 3). IIA Crr protein 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 IIA Crr phosphorylated by HPr? In vitro phosphorylation assays were performed to demon- strate phosphoenolpyruvate-dependent phosphorylation of IIA Crr in the presence of the general PTS phosphotrans- ferases EI and HPr (Fig. 4). As shown in lane 1 of Fig. 4, IIA Crr of S. coelicolor became phosphorylated upon incu- bation with radiolabelled phosphoenolpyruvate, EI of B. subtilis,andHProfS. coelicolor, while IIA Crr incubated Fig. 2. Overexpression and purification of IIA Crr proteins. An SDS/12% polyacrylamide gel stained with Coomassie brilliant blue is shown. Lane 1, protein marker; lane 2, 30 lgcrudecellextractof FT1(pFT41); lane 3, 8 lg purified His-tagged IIA Crr ;lane4,30lgcell extract of FT1(pFT42); lane 5, 5 lg purified IIA Crr ;lane6,30lgcell 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 IIA Crr .Ineachlane 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 IIA Crr . 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 IIA Crr ;lane2:EI and HPr; lane 3: EI and IIA Crr ;lane4:HPr;lane5:EI,HPr,andIIA Crr boiled for 10 min prior to protein gel loading; lane 6: EI, HPr, and IIA Crr (H72A); lane 7: EI and IIA Crr (H72A). The migration of proteins is indicated. Note that phosphorylated EI is not or barely visible due to the low protein amounts used. 2146 A. Kamionka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 only with EI was not phosphorylated (lane 3). After boiling, IIA Crr -phosphate became dephosphorylated indicating a heat-labile aminoacyl phosphorylation of IIA Crr (lane 5) as occurs by histidine phosphorylation. When histidine 72 was replaced by an alanine, the resulting product IIA Crr (H72A) could not be phosphorylated (lane 6). Can IIA Crr function in sugar transport? AfteritwasshownthatIIA Crr is 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 IIA Crr can replace IIA Glc of E. coli with respect to glucose transport. We constructed plasmids pFT111 (His-tagged IIA Crr )and pFT112 (IIA Crr ), 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 IIA Crr could interact with the E. coli components of the glucose-specific PTS, HPr, and enzyme IIBC Glc . 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 IIA Crr of S. coelicolor and His-tagged IIA Glc of E. coli. The initial phosphorylation rates of methyl a-glucoside were 152 ± 22 nmol aMG-PÆmin )1 when IIA Crr was added and 429 ± 39 nmol aMG-PÆmin )1 when IIA Glc was added. This indicated that under these conditions the heterologous IIA Crr protein could compensate to about 35% the function of E. coli IIA Glc . Can IIA Crr function in inducer exclusion? We then studied whether IIA Crr 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 IIA Glc will 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 IIA Glc of E. coli was expressed in strain FT1(pCRL13). The same result, although less pronounced, was observed when IIA Crr 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 IIA Crr with 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 IIA Crr 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)orpFT42(S. coelicolor his-tagged IIA Crr ) 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. Ó FEBS 2002 IIACrr of Streptomyces coelicolor (Eur. J. Biochem. 269) 2147 acid identity with MalK of E. coli (Fig. 7). Therefore, his- tagged IIA Crr 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 IIA Glc yielded a response of 500 resonance units with the MalK protein (positive control). Therefore, the observed reduction of maltose uptake in E. coli by IIA Crr could be confirmed by the demonstration of its interaction with the MalK subunit of the maltose permease complex. DISCUSSION In this study, we report on the analysis of an S. coelicolor ORF that encodes a protein, IIA Crr , with significant similarity to enzyme IIA Glc of E. coli, a global-acting factor of carbon metabolism. We provided evidence that the gene is expressed in vivo and that IIA Crr is phosphorylated in vitro by the general PTS phosphotransferases EI and HPr. IIA Crr could replace the functions of E. coli IIA Glc in glucose transport and inducer exclusion. These findings suggest that IIA Crr 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,andin 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 IIA Glc has been documented and proposed [9,13,14,29]. The reported data demonstrate that IIA Crr could effi- ciently cross-communicate with the proteins HPr, enzyme IIBC Glc , and MalK from enteric bacteria. This striking functional resemblance to E. coli IIA Glc and the observation that S. coelicolor IIA Crr is present under all nutritional conditions tested may provide good indications that IIA Crr functions in a similar way in S. coelicolor. The amount of IIA Crr washigherwhenS. 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 IIA Crr ? We could demonstrate that IIA Crr is 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 IIA Crr is not an enzyme IIA Glc as streptomycetes appear to lack the glucose-specific PTS [15,17]. An analysis of the S. coelicolor genome 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 IIA Crr 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 IIA Crr could replace the inducer exclusion function of E. coli IIA Glc by inhibition of maltose uptake might be a good indication for this hypothesis. The demonstration of the IIA Crr –MalK interaction suggests that IIA Crr 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% identicalaminoacidsisMsiK,whichservesastheATPase subunit for ABC transporters specific for maltose, cellobi- ose, xylobiose, and trehalose [34–36]. MsiK could therefore be a potential candidate for regulation by IIA Crr .Initial attempts to demonstrate IIA Crr -MsiK binding by surface plasmon resonance failed probably because overproduced MsiK forms inclusion bodies yielding incorrectly folded protein (unpublished data) [37]. IIA Crr 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 IIA Glc 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 IIA Crr of S. coelicolor operatesinasimilarway. Finally, another hint as to a possible function of IIA Crr should be mentioned here. Ueguchi and coworkers have reported that E. coli IIA Glc controls 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, IIA Crr could be involved in controlling some of the many sigma factors that S. coelicolor possesses [4,41]. Fig. 7. Surface plasmon resonance analysis. A real-time interaction analysis of his-tagged IIA Crr (broken line) and His-tagged IIA Glc (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) 4 as a function 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). 2148 A. Kamionka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 IIA Crr in relation to the nutritional state of the streptomycetes mycelium. ACKNOWLEDGEMENTS These studies were carried out in the laboratories of W. Hillen. 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(2001) A connection between stress and development in the multicellular prokaryote Streptomyces coeli- color A3(2). Mol. Microbiol. 40, 804–814. 2150 A. Kamionka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . The phosphotransferase system of Streptomyces coelicolor IIA Crr exhibits properties that resemble transport and inducer exclusion function of enzyme. 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