Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases [StxI and StxII], an acetyl xylan esterase [StxIII], and an -L-arabinofuranosidase [StxIV] in the presence of xylan . Xylandegradation products [mainly xylobiose] produced by the actionof these enzymes entered the cell and were then degraded toxylose by an intracellular ß-xylosidase [BxlA] . Agene cluster involved in xylanolytic system of the strain wascloned and sequenced upstream of and including a BxlA-encoding gene [bxlA] . The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, andbxlA . Reverse transcriptase PCR analysis revealed that the genecluster is transcribed as polycistronic mRNA . The deduced gene products, comprising BxlE [a sugar-binding lipoprotein], BxlF [an integral membrane protein], and BxlG [an integral membrane protein], showed similarity to components of the bacterial ATP-binding cassette [ABC] transport system; however, the gene for the ATP binding protein was not linked to the bxl operon . The soluble recombinant BxlE protein was analyzed for its binding activityfor xylooligosaccharides . The protein showed high-level affinityfor xylobiose [K_d = 8.75 x 10^-9 M] and for xylotriose [K_d =8.42 x 10^-8 M] . Antibodies raised against the recombinant BxlErecognized the detergent-soluble BxlE isolated from S . thermoviolaceusmembranes . The deduced BxlF and BxlG proteins are predictedto be integral membrane proteins . These proteins contained theconserved EAA loop [between the fourth and the fifth membrane-spanningsegments] which is characteristic of membrane proteins frombinding-protein-dependent ABC transporters . In addition, thebxlR gene located upstream of the bxl operon was cloned andexpressed in Escherichia coli . The bxlR gene encoded a 343-residuepolypeptide that is highly homologous to members of the GalR/LacIfamily of bacterial transcriptional regulators . The purifiedBxlR protein specifically bound to a 4-bp inverted sequenceoverlapping the -10 region of the bxl operon . The binding ofBxlR to the site was inhibited specifically by low concentrationsof xylobiose . This site was also present in the region locatedbetween stxI and stxIV and in the upstream region of stxII.BxlR specifically bound to the regions containing the invertedsequence . These results suggest that BxlR might act as a repressorof the genes involved not only in the uptake system of xylandegradation products but also in xylan degradation of S . thermoviolaceusOPC-520.

 
Streptomyces bacteria are gram positive, soil inhabiting, and filamentous, with a high G+C content in their DNA . They producea number of secondary metabolites and extracellular proteins,including enzymes hydrolyzing different types of polysaccharidessuch as xylan, cellulose, and chitin [12] . Unlike celluloseand chitin, xylans have a relatively complex structure consistingof a ß-1,4-linked D-xylose polymer replaced with L-arabinofuranosyl,glucuronyl, 4-O-methlglucuronyl, and acetyl groups [37] . For complete hydrolysis of xylan, many xylanolytic microorganisms coordinately synthesize the multiple groups of xylanolytic enzymes, such as endo-ß-1,4-xylanases [EC 3.2.1.8], ß-xylosidase[EC 3.2.1.37], -L-arabinofuranosidases [EC 3.2.1.55], and acetylxylanesterases [EC 3.1.1.6] . The microbial hydrolysis of xylan iscentral to the recycling of photosynthetically fixed carbonand plays a pivotal role in the turnover of abundant organicmolecules in the biosphere [24].

Streptomyces thermoviolaceus OPC-520, a thermophilic actinomycete isolated from decayed wood, grows actively on xylan as a sole carbon source and does not have cellulase activity [29] . The biosynthesis of xylanolytic enzymes in S . thermoviolaceus OPC-520 was induced by xylan or xylobiose [which is the smallest molecule to induce the production of xylanolytic enzymes] and repressed by readily metabolized sugars such as glucose [31] . The bacteriumproduces four extracellular enzymes [designated StxI throughStxIV] in the presence of xylan [29-32] . StxI and StxII areendo-ß-1,4-xylanases, StxIII is an acetylxylan esterase,and StxIV is a -L-arabinofuranosidase . These enzymes effectivelyconvert xylan into xylooligosaccharides [mainly xylobiose, whichis a major product of xylan degradation] . The generated xylobioseand small amounts of xylooligosaccharides enter the cells andare further hydrolyzed to xylose by an intracellular ß-D-xylosidase[BxlA] [31] . We have cloned and sequenced the genes involvedin the xylan degradation of the strain [30-32] . Furthermore,they have been expressed in Streptomyces lividans or Escherichiacoli and the biochemical properties of each recombinant proteinhave been investigated [30-32] . A variety of enzymes that candegrade xylans have been identified, and the corresponding geneshave been cloned from saprophytic prokaryotes [5, 8, 10, 23]and eukaryotes [4, 11, 33] . However, little is known about theuptake system for xylan degradation products and the molecular mechanisms of the gene regulation in Streptomyces species.

Recently, sequence analysis of the upstream region of the bxlA gene revealed a gene cluster consisting of four complete open reading frames [ORFs] organized in the order bxlE, bxlF, bxlG, and bxlA . Upstream of the bxlE gene, furthermore, the bxlR gene[which had an opposite orientation] was found . In the presentwork, we show that the clustered genes encode an ATP-binding cassette [ABC]-type transporter for xylooligosaccharides andan intracellular ß-D-xylosidase required for hydrolysisof xylooligosaccharides . The substrate-binding protein [BxlE]of the ABC transporter was expressed in E . coli and showed thehighest level of affinity for xylobiose among xylooligosaccharidesfrom dimer to hexamer . Furthermore, we report that BxlR is acommon regulatory protein that specifically binds to the same4-bp inverted repeat located upstream of bxlE, stxI, stxII,and stxIV.

 
Bacterial strains, vectors, and culture conditions. S . thermoviolaceus OPC-520 was grown at 50°C in medium [1%glucose, 0.5% proteose peptone, 0.1% yeast extract, 0.1% K₂HPO₄, 0.02% MgSO₄^· • 7H₂O] and was used as the sourceof chromosomal DNA . To extract total RNA, 500-ml flasks containing100 ml of minimum medium [NNMP; 0.2% [NH₄]₂SO₄, 0.5% CasaminoAcids, 0.06% MgSO₄^· • 7H₂O, 5% polyethylene glycol6000, 0.001% each of ZnSO₄^· • 7H₂O, MnCl₂^· • 4H₂O, and CaCl₂, 0.01 M phosphate buffer [pH 6.8]] supplementedwith either oat spelt xylan [Sigma] or glucose were inoculatedwith 1 ml of mycelium grown for 24 h in YPG medium [29] . Theflasks were incubated at 50°C with vigorous shaking . TheE . coli strains employed in this study were JM109 and BL21[DE3]pLysS . E . coli cells were grown in Luria broth supplementedwith appropriate antibiotics at 37°C . The vectors used werepUC18, pUC19 [Takara Biochemicals, Shiga, Japan], pThioHis C[Invitrogen Co.], and pGEX-6P-1 [Amersham Biosciences].

General recombinant DNA techniques. S . thermoviolaceus chromosomal DNA was isolated by the methodof Hopwood et al . [6] . Restriction enzymes, T4 DNA ligase, andother modifying enzymes were purchased from Toyobo [Osaka, Japan].Agarose gel electrophoresis, plasmid DNA preparation, transformationof E . coli, and Southern hybridization were performed as describedby Sambrook and Russell [15] . Nucleotide sequencing was carried out by a dideoxy chain termination method [16] using a DYEnamicET terminator cycle sequencing premix kit [Amersham Biosciences]on a DNA sequencer [ABI Prism 310 genetic analyzer; AppliedBiosystems] . Sequence data were analyzed using a GENETYX-WIN program [Software Development Co., Ltd.].

Cloning of the 5' upstream region of bxlA. We carried out cloning of the 5' upstream region of the bxlAgene by the DNA-probing method with a 0.30-kb PstI-BamHI fragment of pBXL3 encoding an intracellular ß-D-xylosidase as a probe . The fragment was labeled with alkaline phosphatase [AlkPhos DIRECT; Amersham Biosciences] according to the manufacturer's instructions . Chromosomal DNA of S . thermoviolaceus was digested with various restriction enzymes and electrophoresed on a 0.6% agarose gel . Southern hybridization revealed that the probe hybridized with the 2.7-kb chromosomal fragment digested withBamHI . The DNA fragments corresponding to 2.7 kb were excisedfrom the gel and purified with a GenElute gel extraction kit[Sigma] . These were ligated into the dephosphorylated BamHIsite of pUC19, and the recombinant plasmids were introducedinto competent E . coli JM109 . The library was screened by colonyhybridization with the labeled probe as previously described[31] . The resulting plasmid was designated pBXL3.1 . To clonethe 5' upstream region of the 2.7-kb BamHI-BamHI fragment ofpBXL3.1, the second colony hybridization was performed usinga 0.18-kb BamHI-BglII fragment as a probe . The resulting plasmidwas designated pBXL3.2 . The 2.7-kb SphI-BamHI fragment of pBXL3.1and 4.4-kb BglII-SphI fragment of pBXL3.2 were ligated together,and the resulting 7.1-kb fragment was inserted into the corresponding sites of pUC19 . The resulting plasmid was named pBXL3.3.

Construction of expression plasmids. The expression plasmid pThioHis-BxlE, coding for BxlE, was constructedas follows . Two oligonucleotide primers, P1 and P2 [Table 1],were synthesized and were modified to contain XhoI and PstI recognition sites to facilitate cloning in frame into pThioHisC . The bxlE gene was amplified by PCR with the primers, withplasmid pBXL3.3 as the template . PCR was performed for 30 cyclesconsisting of 97°C for 15 s, 63°C for 30 s, and 68°Cfor 80 s . The amplified DNA was digested by XhoI and PstI, andthe resulting fragment was inserted into the corresponding sitesof pThioHis C . On the other hand, a DNA fragment of bxlR, codingfor BxlR, was prepared by PCR with plasmid pBXL3.3 as the templateand the primers P3 and P4 [Table 1] . PCR was performed for 30 cycles consisting of 97°C for 15 s, 55°C for 30 s, and68°C for 80 s . The amplified DNA was digested by BglII andEcoRI, and the resulting fragment was inserted in frame intothe glutathione S-transferase [GST] fusion protein expressionvector pGEX-6P-1 . The resulting expression plasmid was designatedpGEX-BxlR . The nucleotide sequences of the junctions betweenvectors and inserts and the whole amplified DNA were confirmedwith a DYEnamic ET terminator cycle sequencing premix kit withsynthesized primers.

 
Purification of recombinant BxlE and BxlR. E . coli TOP10 cells harboring pThioHis-BxlE were induced with1 mM IPTG [isopropyl-ß-D-thiogalactopyranoside] atthe mid-exponential growth phase and further incubated for 3h at 37°C . The cells were harvested by centrifugation, washed,and resuspended with 20 mM phosphate buffer [pH 7.4] . The cellswere disrupted by sonication, and the lysate were centrifugedat 10,000 x g for 30 min . The fusion protein was purified fromthe supernatant by affinity chromatography with a nickel-chargedSepharose resin [ProBond resin; Invitrogen Co.] . The purifiedfusion protein was treated with enterokinase [Invitrogen Co.]for 16 h at 37°C to obtain BxlE . The recombinant BxlE included the extra six amino acid residues in the N-terminal portion.On the other hand, E . coli BL21[DE3] pLysS cells harboring pGEX-BxlR were induced with 1 mM IPTG at the mid-exponential growth phase and further incubated for 2 h at 37°C . The lysate was preparedin the same manner as the E . coli TOP10 cells harboring pThioHis-BxlE. The GST-fusion protein was purified from the lysate by affinity chromatography with glutathione-Sepharose 4B [Amersham Biosciences]. The purified fusion protein was treated with PreScission protease [Amersham Biosciences] for 4 h at 4°C to obtain BxlR . TheN-terminal amino acid sequences of BxlE and BxlR were confirmedby protein sequencing [Procise 491 HT protein sequencer; AppliedBiosystems].

RT-PCR. S . thermoviolaceus OPC-520 was grown for 24 h at 50°C inNMMP containing 1% xylan, 1% xylobiose, or 1% glucose . TotalRNA was extracted from 1.0-ml suspensions of S . thermoviolaceusOPC-520 cells with an SV total RNA isolation system [Promega]in accordance with the manufacturer's instructions . Total RNA[2 µg] and primer P5 were used to reverse the bxl transcripts.The reaction was carried out at 42°C for 60 min with Moloneymurine leukemia virus reverse transcriptase [RT] [RNase H minus;Promega] and terminated by heating at 70°C for 15 min . The RT products were used as a template for PCR, and the following primer pairs were designed: primers P6 and P7, primers P8 andP9, primers P10 and P11, and primers P12 and P13 [Table 1]. PCR was performed for 30 cycles consisting of 97°C for 15s, 55°C for 30 s, and 68°C for 80 s . As negative controls,the reactions were performed in the absence of RT or RNA template.

Preparation of membrane proteins. S . thermoviolaceus OPC-520 was grown on NNMP [10 ml] supplementedwith 1% xylan or 1% glucose at 50°C for 12 h . Mycelia wereharvested by centrifugation [5,000 x g for 15 min], washed twice in 50 mM Tris-HCl buffer [pH 7.5] containing 0.2 M NaCl, and suspended in 10 ml of the same buffer . The mycelia were disruptedby sonication, and membranes were isolated by centrifugation[10,000 x g for 1 h at 4°C] . Membrane proteins were extractedfor 30 min with 1% N-lauroylsarcosine in 50 mM Tris-HCl buffer[pH 7.5] containing 0.2 M NaCl and centrifuged at 10,000 x g for 20 min at 4°C . The supernatant was used for Westernblotting.

Western blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]was done by the method of Laemmli [9] . Proteins on the gel weretransferred to a Sequi-Blot polyvinylidene difluoride membrane.The membrane was incubated for 1 h at room temperature withanti-BxlE polyclonal mouse serum diluted to 1:1,000 in phosphate-bufferedsaline containing 2.0% skim milk [Difco] . Bound antibody wasdetected as described previously [14].

Surface plasmon resonance analyses. BIAcore X system and carboxymethylated dextran [CM5] sensorchips were purchased from Pharmacia Biosensor . To immobilizeBxlE to the CM5 sensor chip surface, carboxyl groups along thecarboxymethylate-dextran chains of the sensor chip surface wereactivated by exposure [35 µl at 5 µl/min] to a mixtureof 0.1 M N-hydroxysuccinimide and 0.1 M N'-[3-diethylaminopropyl]carbodiimide hydrochloride [1:1[vol/vol]] . BxlE was injectedover the surface at 50 µg/ml in coupling buffer [10 mMsodium acetate buffer, pH 3.0] . After coupling, unreacted surfaceester groups were blocked by exposure to 1 M ethanolamine [pH8.5] . Bovine serum albumin was used as the control protein.The interaction of BxlE with xylooligosaccharides [at concentrationsranging from 3 x 10^-9 to 2 x 10^-4 M] was analyzed at a flowrate of 10 µl/min in 10 mM HEPES buffer [pH 7.4] containing150 mM NaCl and 3 mM EDTA . The association time was 3.5 min,and the dissociation time was 5 min . The kinetic parameters were determined with a BIA evaluation program [version 3.0].

Gel retardation assay. The upstream region of bxlE [corresponding to positions -114to -61 [taking A of the initiation codon of bxlE as position+1]] was amplified by PCR using P14 and P15 . The oligonucleotide-directedmutagenesis of the region was performed by PCR using primerpairs P16 and P17, P16 and P18, and P16 and P19 . The upstreamregions of stxI [corresponding to positions -418 to -246 [takingthe initiation codon of stxI as position +1]], stxII [correspondingto positions -149 to -38 [taking the initiation codon of stxIIas position +1]], stxIII [corresponding to positions -40 to+94 [taking the initiation codon of stxIII as position +1]],and stxIV [corresponding to positions -78 to +21 [taking theinitiation codon of stxIV as position +1]] were amplified byPCR using primer pairs P20 and P21, P22 and P23, P24 and P25,and P26 and P27 [Table 1], respectively . PCR was performed for30 cycles consisting of 97°C for 15 s, 53°C for 30 s,and 68°C for 80 s . The amplified fluorescein isothiocyanate[FITC]-labeled DNA fragments were used in the gel retardationassay . Binding reactions contained 10 ng of FITC-labeled DNAs,10 to 100 ng of purified BxlR, and 10 ng of poly[dI-dC] in afinal volume of 20 µl of buffer consisting of 10 mM HEPES-KOHbuffer [pH 7.5], 1 mM MgCl₂, 0.5 mM dithiothreitol, and 50 mMKCl . Binding reaction mixtures were allowed to equilibrate for30 min at 37°C and immediately loaded onto 5% polyacrylamidegels . The gels were electrophoresed at 200 V for 20 min by usinga Mini-Protean II apparatus [Bio-Rad] in Tris-acetate-EDTA buffer.The gels were visualized by a fluoro image analyzer [FLA-2000;Fujifilm, Tokyo, Japan].

Primer extension. Total RNA was extracted from 1.5 ml of cell suspensions of S.thermoviolaceus OPC-520 grown in NNMP supplemented with 1% xylanby using an SV total RNA isolation system [Promega] . About 10.0µg of RNA was used to map the 5' end of the bxlE transcript.Reverse transcription was initiated from P28 [the FITC-labeledprimer] complementary to the 5' end of the bxlE coding region.The reaction was carried out at 50°C for 60 min with Moloneymurine leukemia virus RT [RNase H minus; Promega] . The primer extension and the sequencing reaction products were analyzedon a 6.0% denaturing polyacrylamide gel by a DNA sequencer [Hitachi SQ3000] . The sequence reaction was performed with the same primer.

Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper will appearin the DDBJ, EMBL, and GenBank nucleotide sequence databasesunder accession numbers AB110643 [stxI and stxIV], AB110644[stxII and stxIII], and AB110645 [bxlR, bxlE, bxlF, bxlG, and bxlA].

 
Cloning and sequence analysis of the gene cluster. Previous study of an intracellular ß-D-xylosidase [BxlA] in S . thermoviolaceus OPC-520 led to the identification and characterization of bxlE, bxlF, bxlG, and bxlR genes [31].A 0.3-kb PstI-BamHI fragment of pBXL3 [Fig . 1] coding for the COOH-terminal end of BxlG [last hydrophobic membrane-spanningregion] was used as a probe to screen a S . thermoviolaceus BamHIgene library constructed with pUC19 . Among 800 transformants,only one clone [pBXL3.1] [which hybridized with the probe] wasisolated by colony hybridization [Fig . 1] . Analyses by restriction enzyme digestion and sequencing of the fragment revealed that the insert of pBXL3.1 and that of pBXL3 shared a 0.3-kb PstI-BamHI region . Analysis of the entire nucleotide sequence of pBXL3.1 led to the prediction of two complete ORFs [bxlF and bxlG] and one truncated frame [bxlE] [Fig . 1] . Then the 5' upstream regionof the insert of pBXL3.1 [designated pBXL3.2] was further clonedin the second colony hybridization, using the 0.18-kb BamHI-BglIIfragment as a probe . The 2.7-kb SphI-BamHI fragment of pBXL3.1and 4.4-kb BglII-SphI fragment of pBXL3.2 were ligated together,and the resulting 7.1-kb fragment was inserted into the correspondingsites of pUC19 . The resulting plasmid was named pBXL3.3 [Fig.1].

 
The nucleotide sequence of the gene cluster of pBXL3.3 was determined. The overall G+C content of the sequenced fragment was 74% . This value is in agreement with the G+C content of Streptomyces [38]. Upstream of the bxlA gene, three ORFs [bxlE, bxlF, and bxlG]were found which were carried on the same strand and had thesame directions of transcription . On the other hand, the bxlRgene was located in an opposite orientation 171 bp upstream of the bxlE gene . There are only 44 nucleotides between the TGA termination codon of bxlG and the ATG initiation codon of bxlA, 4 nucleotides between bxlF and bxlG, and 17 nucleotidesbetween bxlE and bxlF.

The bxlG gene consists of 906 nucleotides encoding a protein of 301 amino acids with a predicted molecular mass of 32,428 Da . The deduced amino acid sequence of the encoded protein [BxlG]was compared with those of the other proteins . A search of theBLAST database found that BxlG had similarity with a putativebinding protein-dependent transport protein from Streptomycescoelicolor A3 [2] [accession no. CAB88163] [54% identity], BxlGfrom S . lividans [accession no. AAC99627] [52% identity], ahypothetical protein from Thermobifida fusca [accession no. ZP_00056972] [43% identity], and a putative sugar ABC transporter integral membrane protein from S . coelicolor A3 [2] [accessionno. CAD55434] [41% identity] . The bxlF gene consists of 876 nucleotides encoding a protein of 291 amino acids with a predicted molecular mass of 31,789 Da . The deduced BxlF was closely relatedto BxlF from S . lividans [accession no. AAC99626] [53% identity],a putative binding protein-dependent transport protein fromS . coelicolor A3 [2] [accession no. CAB88162] [49% identity],and a hypothetical protein from Thermobifida fusca [accessionno. ZP_00056971] [45% identity] . When their hydrophobicity profiles were analyzed, both BxlG and BxlF were predicted to span themembrane six times [data not shown] . Furthermore, they containthe consensus sequence EAAX₂DGAX₈IXLP between the fourth and the fifth membrane-spanning segments [which is characteristicof membrane proteins from binding protein-dependent ABC transporters][17] [Fig . 2A].

 
The bxlE gene consists of 1,311 nucleotides encoding a protein of 436 amino acids with a predicted molecular mass of 46,661Da . The deduced amino acid sequence of BxlE showed sequencehomology with several sugar-binding proteins, such as a putativesugar-binding lipoprotein from S . coelicolor A3 [2] [accession no. CAB88161] [43% identity], BxlE from S . lividans [accession no. AAC99625] [43% identity], and a hypothetical protein from Thermobifida fusca [accession no. ZP_00056970] [38% identity].The deduced N-terminal portion of BxlE [MQSYSRRWFLGAGATTLISAAGLTACG]includes positively charged residues and the consensus sequence[L[S,A][A,G]C[S,G]] [which corresponds to the sites cleavedby lipoprotein-specific signal peptidases in gram-positive bacteria][27] . These results suggest that Cys-26 is at the amino terminusof the mature form and is covalently modified by the typicalester-linked and amide-linked acylation of lipoproteins . BxlEhas the signature sequence [cluster 1] of binding proteins specificfor multiple sugars and glycerol phosphate [28] [Fig . 2B] . The highly conserved lysine residue of the signature sequence is also conserved in BxlE . These results suggest that BxlE servesas a substrate-binding protein of the components comprisingan ABC transporter system.

The bxlR gene consists of 1,032 nucleotides encoding a protein of 343 amino acids with a predicted molecular mass of 36,706 Da . The deduced amino acid sequence of BxlR showed sequencehomology with transcriptional repressors classified into theGalR/LacI family . The N-terminal helix-turn-helix DNA-bindingmotif [TLAEIAREAGVSAPTVSKVLNG] [located between amino acid 13and amino acid 34] was found at the amino-terminal portion ofBxlR . BxlR showed sequence homology with a putative transcriptionalregulator from S . coelicolor A3 [2] [accession no. CAA20410][86% identity], a probable LacI-family transcriptional regulatorfrom S . avermitilis [accession no. BAC72695] [58% identity],and BxlR from S . lividans [accession no. AAC99624] [55% identity].The amino acid sequence [residues 200 to 204, 251 to 254, and277 to 281] of BxlR also shares some homology with that of thesugar-binding sites of the GalR/LacI proteins.

The bxlEFG and bxlA genes are polycistronically transcribed. We performed RT-PCR to examine transcription of the gene clusterby using the primers indicated in Fig . 3 . When total RNA isolatedfrom S . thermoviolaceus mycelia grown in the presence of xylanor xylobiose was used, the expected sizes of DNA fragments [655bp [lanes 1 and 2], 388 bp [lanes 4 and 5], and 566 bp [lanes7 and 8]] were amplified . However, when total RNA from glucose-grownmycelia was used, DNA fragments were not amplified [lanes 3,6, and 9] . These results indicate that the gene cluster comprisingbxlEFG and bxlA is induced in the presence of xylan or xylobioseand is polycistronically transcribed . On the other hand, theDNA fragments between P12 and P13 were not amplified [lanes10, 11, and 12] when total RNAs prepared from the medium containingxylan, xylobiose, or glucose were used.

 
Expression and purification of BxlE and BxlR. The sequence analysis of the gene cluster suggests that BxlE,BxlF, and BxlG are components of an ABC transporter system andthat BxlR is a transcriptional repressor of bxlEFGA . To clarifythe roles of these proteins in the xylanolytic system of S.thermoviolaceus OPC-520, BxlE and BxlR were expressed in E.coli with the procedure described in Materials and Methods.The six-His-tagged BxlE was purified by HisTrap column chromatography.On the other hand, the fusion protein [GST-BxlR] was purifiedby affinity chromatography with glutathione-Sepharose 4B . Thepurified GST-BxlR was treated with PreScission protease, andthen BxlR was purified with glutathione-Sepharose 4B . The molecularmasses of BxlE and BxlR calculated from each of the amino acidsequences were in reasonable agreement with those estimatedby SDS-PAGE [Fig . 4] . On the other hand, gel filtration chromatography[Superdex 200; Amersham Biosciences] showed that BxlR had anapparent molecular mass of 75 kDa [data not shown] . This resultsuggests that BxlR forms a dimer.

 
BxlE is a sugar-binding protein. To clarify the role of BxlE in an ABC transporter system, recombinantBxlE was immobilized on a BIAcore sensor chip . Xylose or eachof xylooligosaccharides [from dimer to hexamer] was passed overthe sensor chip, and the binding to BxlE was monitored directlyby surface plasmon resonance detection . Sensorgrams for theinteraction of various concentrations of ligands with BxlE wereexamined [data not shown] . The equilibrium dissociation constants[K_d] were calculated according to the ratio [K_d = the dissociationrate constant [k_off]]/[the association rate constant [k_on]]. Among the sugars tested, xylobiose showed the highest affinity towards BxlE [K_d = 8.75 x 10^-9 M] and xylotriose showed thesecond highest affinity [K_d = 8.42 x 10^-8M], as shown in Table2 . The affinity of xylooligosaccharides towards BxlE showeda tendency to decrease with increases in the degree of polymerization.However, xylose and glucose showed weak affinity towards BxlE.

 
In gram-positive bacteria, solute-binding proteins are locatedat the surface of the cytoplasmic membrane by a lipid anchor[27] . To examine the distribution of native BxlE in S . thermoviolaceus OPC-520, Western blotting analysis was performed . Membrane vesicles from S . thermoviolaceus OPC-520 were prepared from mycelia grown in NNMP supplemented with xylan or glucose . Native BxlE was detected only in membrane fraction from mycelia grown in thepresence of xylan and not in the culture supernatant and cytoplasm[Fig. 5] . Taken together, these findings show that BxlE located at the cytoplasmic membrane is a sugar-binding protein which serves as one of the components of an ABC transporter for xylobiose and larger oligosaccharides.

 
BxlR binds to the inverted repeat sequence. To investigate whether recombinant BxlR binds specifically tothe region located between the bxlR and bxlE genes, gel retardationassays were performed . The perfect inverted repeat sequence [5'-CGAA-Nx-TTCG-3'] was found in the intergenic region . Toidentify the transcriptional start site of the bxl operon, primer extension analysis was carried out for total RNA prepared fromthe cells of S . thermoviolaceus OPC-520 . The primer extension revealed that the inverted repeat sequence and the -10 regionoverlap each other [Fig . 6] . Then, a FITC-labeled 54-bp DNA fragment containing the inverted repeat sequence was amplifiedby PCR using P14 and P15 . As shown in Fig . 7, the purified BxlR was found to bind to the amplified intergenic bxlR-bxlE . As the amount of BxlR increased, the amounts of DNA-protein complex increased . We reported that the smallest molecule to inducethe production of both xylanase and ß-xylosidase inS . thermoviolaceus OPC-520 was xylobiose [31] . Then, we examinedthe effect of the presence of xylobiose on BxlR protein-DNAinteraction . The binding of BxlR to the FITC-labeled DNA fragmentwas weakened by xylobiose at concentrations of 1 to 250 mM andwas lost by the addition of high concentrations [500 mM] ofxylobiose [Fig. 7] . However, the presence of xylose and glucosehad no effect on binding even at a concentration of 500 mM [datanot shown] . These results indicate that BxlR is a transcriptional repressor of the bxl operon.

 
To investigate whether BxlR specifically binds a 4-bp inverted sequence, oligonucleotide-directed mutagenesis was performed.An A-to-T transversion [5'-CGAA-Nx-TTCG-3'; the A is indicatedby underlining] resulted in loss of the binding of BxlR to theamplified DNA fragment containing the inverted sequence . Twoadditional base changes [a T-to-G transversion [5'-CGAA-Nx-TTCG-3']and a T-to-G transversion [5'-CGAA-Nx-TTCG-3']; the T is indicated by underlining] also resulted in loss of BxlR-DNA interaction[data not shown] . These data suggest that BxlR specificallybinds to a 4-bp inverted repeat sequence.

Alignment of the upstream regions of xylanase [stxI and stxII], acetylxylan esterase [stxIII], and -L-arabinofuranosidase [stxIV]genes in S . thermoviolaceus OPC-520 revealed that [like bxlE]stxI, stxII, and stxIV had the sequence 5'-CGAA-Nx-TTCG-3' inthe putative promoter regions [Fig . 8] . To clarify whether BxlRregulates transcription of these genes, DNA fragments containingthe inverted repeat were amplified by PCR and interactions ofBxlR with each of the amplified DNA fragments were examinedby gel retardation assays . As shown in Fig . 9, BxlR specificallybinds to DNA fragments containing the perfect inverted repeatsequence but not to DNA fragments from stxIII . The tandem organizationof stxII and stxIII genes forms an operon, as in the case ofthe gene cluster bxlE, bxlF, bxlG, and bxlA [unpublished data].These results suggest that BxlR is a common transcriptional regulator not only of the bxl operon but also of the stxI, stxII,stxIII, and stxIV genes dispersed throughout the genome of S.thermoviolaceus OPC-520.

 
The presented data suggest that xylooligosaccharides are specifically transported to the cytoplasm through an ABC transporter systemand that xylooligosaccharides are then degraded to xylose byan intracellular ß-xylosidase . In addition to thesefindings, we clarified that BxlR is a regulator not only ofthe bxl operon for xylooligosaccharide uptake and degradationand but also of the genes involved in xylan degradation in S.thermoviolaceus OPC-520.

The bxl operon is composed of four genes encoding xylooligosaccharide binding protein [bxlE], two permeases [bxlF and G], and an intracellularß-xylosidase [bxlA] . Analysis of the deduced aminoacid sequence of BxlE showed that the protein is a membrane-associatedlipoprotein probably serving as a solute-binding protein inan ABC transport system . Solute-binding proteins have been classifiedinto eight clusters [28] . The sequence from Ala-61 to Asp-81of BxlE shows similarity to the signature sequence characteristicof cluster 1 binding proteins . These include MalE [essentialfor import of maltose in S . coelicolor A3] [2, 35], CebE [essentialfor import of cellobiose and cellotriose in Streptomyces reticuli][20], and NgcE [essential for import of N-acetylglucosamineand N,N'-diacetylchitobiose in S . olivaceoviridis] [40].

To investigate the substrate specificity of the identified ABC transporter system, BxlE was expressed in E . coli and analyzed [using surface plasmon resonance] for the kinetics of sugarbinding . The association [k_on] and dissociation [k_off] rateconstants of various sugars were determined for BxlE, and the equilibrium dissociation constant [K_d] was calculated . Amongthe sugars tested, xylobiose showed the highest affinity [K_d = 8.75 x 10^-9 M] followed by xylotriose [K_d = 8.42 x 10^-8 M].The lowest value was measured for xylohexaose [K_d = 1.16 x 10^-6 M] . Kinetic parameters for bacterial sugar-binding proteinsso far reported have been determined by several methods, suchas equilibrium dialysis [19], sugar uptake by whole cells [39],and stopped-flow techniques [13] . A sugar-binding protein ofS . reticuli showed the highest affinity [K_d = 1.5 x 10^-6 M]for cellobiose and cellotriose [19] . The dissociation constant of recombinant trehalose/maltose-binding protein from Thermococcus litoralis was determined to be 1.6 x 10^-7 M [7] . Recently, theequilibrium dissociation constants [K_d] of NgcE purified from S . olivaceoviridis were ascertained [using surface plasmon resonance] for N-acetylglucosamine [K_d = 8.28 x 10^-9 M] and chitobiose [K_d = 2.87 x 10^-8 M] [40] . These values were very close to theK_d values of xylobiose and xylotriose, although the recombinantBxlE includes the extra amino acid residues [VPGMLSS] in theN-terminal portion [CGSGSGS] . In the case of trehalose/maltose-bindingprotein of T . litoralis, the N-terminally truncated proteinwas expressed in E . coli, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein whose N-terminal cysteine is covalently modified by lipid [3] . Therefore,it seems likely that the lipid anchor of BxlE does not influencethe binding affinity [since binding is brought about by the movement of the two soluble lobes forming the binding site between them] [26] . These results indicate that BxlE is a sugar-bindingprotein involved in xylan metabolism of S . thermoviolaceus OPC-520[which shows a high specificity for xylobiose and larger oligosaccharides].

Both BxlG and BxlF contain the conserved EAA cytoplasmic loopthat is found in all other integral membrane components of binding protein-dependent transport systems [27] and that may interactwith the membrane-associated ATP-hydrolyzing subunit . However,we could not discover the identity of the gene encoding an ATP-hydrolyzingsubunit in the vicinity of the bxl operon . That the gene encodingan ATP-hydrolyzing subunit is absent also holds for the cellobioseoperon from S . reticuli [18], the maltose operons from S . lividans[22] and S . coelicolor [35], and the N-acetylglucosamine operonfrom S . olivaceoviridis [40] . Schlösser has shown thatthe Streptomyces ATP-binding component MsiK assists in cellobioseand maltose transport systems as a general ATP-hydrolyzing subunit[18] . Thus, MsiK [or an MsiK-like protein which is homologousto the ATP-hydrolyzing subunit Malk in the maltose transportsystem in E . coli] [1, 2] seems to be encoded elsewhere on thechromosome of S . thermoviolaceus OPC-520.

The bxlR genes occur upstream of the bxl operon organized in the order bxlE, bxlF, bxlG, and bxlA . The gene organization[bxlR-bxlE-bxlF-bxlG-bxlA] is the same as those for the maloperon [malR-malE-malF-malG-aglA] from S . coelicolor A3 [2][35] and the ceb operon [cebR-cebE-cebF-cebG-bglC] from S . reticuli [20] . The mal operon and the ceb operon correspond to the maltoseand cellobiose-cellotriose import systems, respectively . Theseoperons are regulated by MalR and CebR belonging to GalR/LacIfamily [21, 34, 35] . The deduced protein BxlR is related to members of the GalR/LacI regulatory family . Thus, the bxlR gene was expressed in E . coli to investigate whether BxlR is the transcriptional regulator of the bxl operon . The purified BxlR was found to bind specifically to the 61-bp region located between the bxlR and bxlE . The GalR/LacI regulators bind to their targetDNA sites as homodimers, and their operator sequences are invertedrepeats [25] . Since the BxlR protein shares a number of commonfeatures with other members of GalR/LacI family, we investigatedwhether there are the inverted repeats within the 61-bp regionlocated between the bxlR and bxlE . Computer analysis revealedthat the sequence required for recognition by BxlR appearedto be a 4-bp inverted repeat [5'-CGAA-Nx-TTCG-3'] located inthe -10 region of the bxl operon . Several base changes withinthis sequence resulted in loss of the binding activity of BxlR,indicating that BxlR recognizes a 5'-CGAA-Nx-TTCG-3' sequenceas an operator . Most proteins of the GalR/LacI family bind carbohydrateor nucleoside effectors [36] . Our results showed that BxlR-DNAinteraction was weakened in the presence of low concentrationsof xylobiose [1 to 10 mM] and was not affected by the high concentrationsof xylose or glucose [500 mM] . The synthesis of ß-xylosidase[BxlA] and sugar-binding protein [BxlE] in S . thermoviolaceusOPC-520 was induced by the presence of xylobiose but not thatof xylose . Therefore, it is presumed that xylobiose is the trueinducer of the bxl operon which leads to release of the BxlRfrom the operator upstream of the bxlE gene . In the absenceof xylobiose, BxlR presumably binds to the inverted repeat andblocks the transcription of the bxl operon.

Xylanase production [StxI and StxII] in S . thermoviolaceus OPC-520 is also induced in the presence of xylobiose . These results suggest that xylanase genes and the bxl operon might be coordinately controlled by the same regulatory system . As expected, the inverted repeat sequences corresponding to the BxlR binding motif were identified in the regions located between stxI and stxIV and in the upstream region of stxII . Indeed, BxlR specifically bound to the regions containing a 4-bp inverted repeat [5'-CGAA-Nx-TTCG-3']. Our results resemble those of investigations of the regulatory system of the cellulase genes in Thermomonospora fusca thatis controlled by CebR, a member of GalR/LacI family [25] . The 14-bp inverted repeat is present in the regions upstream ofall six cellulase genes in T . fusca . CebR specifically bindsto the inverted repeat as a repressor and is released from thebinding site through a direct interaction of CelR with cellobiose.These results indicate that the expression of genes encodingenzymes involved in xylan degradation and of an ABC transporterrequired for the uptake of xylan degradation products is controlledby BxlR . This simple regulation may allow quick adaptation ofthe strain to various environments and avoidance of the unnecessaryproduction of proteins involved in the xylan degradation system.Furthermore, the 5'-CGAA-Nx-TTCG-3' sequence was found in theupstream regions of the genes involved in xylan degradationsystems from different Streptomyces species [such as S . coelicolorA3 [2] and S . lividans] containing ORFs very similar to thatof S . thermoviolaceus OPC-520 . These findings suggest that thegenes involved in the xylan degradation systems of different Streptomyces species might be also regulated in the same manner as those of the system regulating S . thermoviolaceus OPC-520. Our aim is to investigate our major remaining question: whether bxlR gene disruption leads to the loss of catabolite repression by glucose of many genes involved in xylan metabolism of S. thermoviolaceus OPC-520 [although the strain is not amenableto general recombinant DNA techniques].

 
* Corresponding author . Mailing address: Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Osaka 569-1094, Japan . Phone and fax: [81-726] 90-1057 . E-mail: tsujibo@gly.oups.ac.jp.

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