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Instituto de Microbiología Bioquímica/Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas (CSIC)/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain
Correspondence
Ramón I. Santamaría
santa{at}usal.es
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ABSTRACT |
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-p-tosyl-L-lysine chloromethyl ketone hydrochloride
These two authors contributed equally to the results obtained in this paper.
The GenBank accession number for the sequences determined in this work are AJ496191 and AJ496192.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Chater, 1989; Hodgson, 2000). Carbon source accessibility can repress or trigger the expression of many genes, most of them involved in the hydrolysis of polysaccharides, although others, such as those encoding proteases (Ellaiah & Srinivasulu, 1996; Sampath & Chandrakasan, 1998), and even the genes involved in antibiotic production, are also affected (Bhatnagar et al., 1988; Kojima et al., 1995).
Xylan, the main component of hemicellulose, is one of the most abundant polysaccharides in soil and is totally or partially degraded by a wide number of eukaryotic and prokaryotic organisms that need several types of enzymes to achieve total degradation. Endoxylanases (EC 3.2.1.8) have attracted industrial interest due to their potential applications in very different fields, such as biobleaching of kraft paper pulps and as an additive in animal feeding (Bajpai, 1999; Beg et al., 2000). Glycoside hydrolases have been classified into 90 families (see http://afmb.cnrs-mrs.fr/CAZY/index.html) and all the endoxylanases had been included in families 10 and 11 until the recent inclusion of a xylanase from Pseudoalteromonas haloplanktis in family 8 (Collins et al., 2002). Xylanases are most often multidomain enzymes containing one or more catalytic domains and a non-catalytic domain, usually a carbohydrate-binding domain, connected to the catalytic domain by a flexible linker region rich in proline, glycine and hydroxyamino acids. The role of these linker sequences is unclear and some authors have reported that, together with the carbohydrate-binding domain, they play an important role in the activity of xylanases against complex substrates (Black et al., 1997).
Proteolytic processing of actinomycete cellulases and xylanases has been reported (Gilkes et al., 1988; Lao & Wilson, 1996; Moorman et al., 1993; Ruiz-Arribas et al., 1997) and generally, the substrate-binding domain is removed from the original protein to release a functional catalytic monodomain enzyme (Gilkes et al., 1988; Ruiz-Arribas et al., 1997). A putative metalloprotease has been described as being involved in the processing of Streptomyces reticulii cellulase (Moorman et al., 1993) and a serine protease has been cloned and studied in Thermobifida fusca (formerly Thermomonospora fusca) as implicated in cellulase processing (Lao & Wilson, 1996). The latter is a member of the chymotrypsin family and is synthesized as a pre-pro-protein of 375 aa that is cleaved to produce the 194-residue mature protease. Although proteolysis is not necessary for the activation of cellulases or xylanases, the existence of a free form of the enzyme may facilitate the hydrolysis of soluble oligosaccharides.
The gene xysA from Streptomyces halstedii JM8 encodes a protein of 45 kDa (Xys1L) that is processed extracellularly, producing a protein of 33·7 kDa (Xys1S) after removal of a cellulose-binding domain at the carboxy terminus of Xys1L. However, proteins Xys1L and Xys1S have similar specific activities against xylan (Ruiz-Arribas et al., 1997). Here we show that this proteolysis, which occurs in all Streptomyces strains used as hosts, is mainly carried out by serine proteases. We cloned the genes corresponding to two of these serine proteases from Streptomyces lividans 66, a strain used in most of our expression experiments, and here demonstrate the role of both proteases in the processing of Xys1L and other extracellular proteins. We also demonstrate the in vivo role of serine proteases in protein processing by co-expression of a serine protease inhibitor and xysA genes.
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METHODS |
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was routinely used for subcloning and isolation of plasmids.
The xysA gene was obtained from Streptomyces halstedii JM8 (Ruiz-Arribas et al., 1997) and expressed in S. lividans 66 using plasmid pJM9 (Ruiz-Arribas et al., 1995). All plasmids used are indicated in Table 1.
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). Submerged cultures of Streptomyces were grown in YES medium (1 % yeast extract, 10·3 % sucrose, 5 mM MgCl2, pH 7; Ruiz-Arribas et al., 1995) supplemented with 1 % xylose. These cultures were grown in baffled flasks with 0·1 volume of medium. Thiostrepton (5–10 µg ml-1) or neomycin (15 µg ml-1) was added when necessary. Cells were grown at 28 °C and 250 r.p.m. in an orbital shaker (Adolf Kühner) for as long as required for each assay (2–5 days).
DNA manipulations and transformations.
Streptomyces total genomic and plasmid DNA was obtained as described by Kieser et al. (2000). S. lividans protoplasts were obtained from cells grown for 30 h in YEG (1 % yeast extract, 1 % glucose, 10·3 % sucrose, pH 7) supplemented with 5 mM MgCl2 and 0·5 % glycine. Manipulation of E. coli DNA was accomplished as indicated by Sambrook et al. (1989).
Cloning of the genes encoding the S. lividans proteases SpB and SpC.
The complete ORFs of both protease genes were amplified by PCR using oligonucleotides based on S. coelicolor sequences. For the equivalent gene to S. coelicolor protease SCF43A.19, oligonucleotides VR017 (5'-tttttttcatATGGTCGGCAGACACGCCGCC-3') and VR018 (5'-tattatcTCGAGGACCCGCTGCCACAGGGCCGG-3') were used. For the equivalent gene to S. coelicolor protease SCD40A.16c, oligonucleotides VR019 (5'-tttttttcatATGCCCCACCGACACCGACAC-3') and VR020 (5'-tattatcTCGAGCACTCGCTGCCACAGCGCCGG-3') were used. The underlined parts indicate the sequences for an NdeI site (oligonucleotides VR017 and VR019) and a XhoI site (oligonucleotides VR018 and VR020) that were added to facilitate the cloning steps. The capital letters represent the sequences complementary to SCF43A.19 (oligonucleotides VR017 and VR018) and SCD40A.16c (oligonucleotides VR019 and VR020). Amplification was carried out in an MJ Research thermocycler with Taq polymerase (Dynazyme), 1·5 mM MgCl2 and 100 ng genomic S. lividans as template. Cycling conditions were: 5 min at 94 °C, ‘Hot Start’ and 31 cycles of 1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C; final extension at 72 °C for 10 min. The PCR products (1·4 kb) were purified by agarose gel electrophoresis, digested with NdeI and XhoI, and cloned in pET22b(+) (under ampicillin selection and with E. coli DH5 as host).
The same amplicons were cloned under control of the xysA promoter and were flanked by mmrt and fdt transcriptional terminators using the E. coli/Streptomyces shuttle vector pN702GEM3. The plasmids obtained, pVR047 and pVR048 respectively (Table 1
), were transformed into S. lividans 66 and S. lividans 3.10 for protease expression.
DNA sequencing.
The DNA sequences were determined in both strands using a Perkin Elmer ABI Prism 377 DNA sequencer. Several oligonucleotides designed from the DNA sequences collected were used to obtain the definitive DNA sequence. Manipulation was done with the Gene Construction Kit (GCK) and analyses were made with DNA Strider (Marck, 1988). Comparisons of DNA or protein sequences were carried out online (http://www2.ebi.ac.uk/) with FASTA (Pearson & Lipman, 1988) and BLAST (Altschul et al., 1997). CLUSTAL W (Thompson et al., 1994) was used for sequence alignment.
The sequences of the genes of both proteases have been deposited in the EMBL/GenBank/DDBJ database with the accession numbers AJ496191 and AJ496192.
Isolation of strains deficient in serine protease inhibitor (SLPI-).
SLPI- strains were obtained from S. lividans 66 following the observations of Taguchi et al. (1995a) in the case of S. albogriseolus. These authors reported that SLPI- strains were obtained at high frequency after protoplast regeneration due to the gene encoding this protein being located close to the end of the linear chromosome which undergoes frequent deletions (Kuramoto et al., 1996). After S. lividans protoplast cell wall regeneration, colonies defective in differentiation were selected and protease overproduction was studied in plates containing 0·5 % skimmed milk. The putative SLPI- strains were later corroborated by immunodetection, using anti-SLPI polyclonal antibodies. One of the strains obtained was denominated S. lividans 3.10.
Antibody generation and immunodetection.
The synthetic peptide RAVTLTCAPTASGTHP derived from the S. lividans protease inhibitor sequence (SLPI) (GenBank accession no. M80576) was obtained from Chiron Technologies and was inoculated into two rabbits to obtain polyclonal antibodies. Immunodetection was performed on proteins transferred to Immobilon-P membranes (Millipore) with anti-rabbit alkaline-phosphatase-conjugated antibodies (Promega). A 1/25 000 dilution of the antipeptide antiserum was used.
Protein purification.
S. lividans 66 transformed with pVR052 (Table 1) was used for S. lividans protease inhibitor (SLPI) production. S. lividans 3.10, a SLPI- strain transformed with pVR047 or pVR048 (Table 1), was used to obtain the proteases SpB and SpC, respectively. Two litres of YES supplemented with 1 % xylose and 15 µg neomycin ml-1 was inoculated with 200 ml of a culture of the corresponding strain, which had been grown in the same medium for 36 h and subsequently divided into 500 ml aliquots in 2-litre baffled flasks. Incubation on a rotary shaker was continued at 28 °C for 5 days. The supernatants were filtered through 0·45 µm membranes, concentrated 30 times in a Minitan cell (Millipore) equipped with a 10 kDa cut-off polysulfone membrane, and diafiltered with 20 mM Tris/HCl, pH 7·2, or with MilliQ water.
Chromatographic procedures were performed with an ÄKTA Purifier system controlled by Unicorn 3.1 software (Amersham Pharmacia Biotech). Absorbance was monitored at 280 nm and 205 nm.
For SLPI inhibitor purification, the concentrated supernatant was equilibrated with 20 mM Tris/HCl buffer, pH 7·2, loaded into a MonoQ (HR 5/5 anion-exchange) column (Pharmacia Fine Chemicals) equilibrated with the same buffer, and eluted with a 0–0·4 M NaCl gradient in the same buffer at a flow rate of 1 ml min-1. The purified inhibitor eluted as a major peak at 0·11 M NaCl.
For SpB protease purification, the concentrated sample was equilibrated with 10 mM sodium acetate buffer, pH 5·0, and loaded into a MonoQ column equilibrated with the same buffer. The protein was eluted with a 0–0·84 M sodium acetate gradient in the same buffer at a flow rate of 1 ml min-1. Fractions from the anion-exchange column showing biological activity (eluting between 0·52 and 0·73 M sodium acetate) were pooled, concentrated in Centricon Plus-20 devices equipped with 10 kDa molecular mass cut-off membranes, and diafiltered with 20 mM phosphate buffer, pH 7·2. Ammonium sulphate (1·7 M final concentration) was added prior to loading into a Phenyl-Superose column (HR 5/5 hydrophobic interaction column; Pharmacia Fine Chemicals) equilibrated with the same buffer. Elution was accomplished at a flow rate of 1 ml min-1, with a decreasing-step gradient from 1·7 to 0·85 M ammonium sulphate in 2 ml, followed by 0·85–0·68 M ammonium sulphate in 15 ml. The xylanase-processing fractions (eluting between 0·80 and 0·73 M) were pooled and concentrated 15-fold in Centricon Plus-20 devices equipped with 10 kDa molecular mass cut-off membranes. Aliquots of 500 µl were loaded, at a flow rate of 0·1 ml min-1, into a Superdex-75B column (HR 10/30 gel filtration column; Pharmacia Fine Chemicals), equilibrated in PBS, pH 6·8. The active fractions (eluting at 11·687 ml; approx. molecular mass 29 kDa) were pooled to afford homogeneous preparations of a single protein.
For protease SpC, the concentrated supernatant was equilibrated with 20 mM Tris/HCl buffer, pH 7·7, and loaded into a MonoQ column equilibrated with the same buffer. The protease was eluted with a 0–0·3 M NaCl gradient in the same buffer. Fractions from the anion-exchange column showing biological activity (eluting between 0·15 and 0·22 M NaCl) were pooled, concentrated in Centricon Plus-20 devices equipped with 10 kDa molecular mass cut-off membranes, and diafiltered with 20 mM phosphate buffer pH 7·2.
The purity of all different proteins was checked by SDS-PAGE followed by silver staining (Morrisey, 1981). The amino-terminal sequence of all of the proteins was determined using an Applied Biosystems Protein Sequenator.
Protein analysis and enzyme assays.
Protein concentrations were determined by the method of Peterson (1977). Electrophoresis in denaturing polyacrylamide gels (SDS-PAGE) was performed as described elsewhere (Ruiz-Arribas et al., 1995). Detection of proteolytic activity (zymogram analysis) was performed after electrophoresis in 15 % acrylamide SDS-PAGE containing 0·2 % gelatin (Sigma) or 0·2 % skimmed milk (Sveltesse, Nestlé) included in the separating gel. The samples were mixed with the loading buffer and heated for 15 min at 50 °C. After electrophoresis, the gel was rinsed several times in MilliQ water and washed for 30 min in 2·5 % Triton X-100 followed by a 30 min wash in 0·2 M sodium phosphate, pH 7. The gel was incubated for 2–4 h at 45 °C in the same buffer and then stained with 0·5 % Coomassie brilliant blue R. Clear bands corresponding to proteolytic activity were observed after destaining.
Protease activity on Xys1L was studied in an in vitro experiment in which Xys1L (supernatants containing either Xys1L or pure protein) was mixed with the filtered supernatant containing the protease or with pure protease solution and incubated for different periods of time at 37 °C. Typically, 4 µg pure Xys1L and 0·25–1 µg pure proteases were tested in each assay at a final volume of 30 µl. When total supernatants were used as the source of protease, 6 µg total protein was used. The effect of chemical protease inhibitors was tested in this type of assay at the concentrations and under the conditions described in the literature (Díaz-Rodríguez et al., 1999). The inhibitors assayed were: 50–150 µM N-acetyl-Leu-Leu-Met-al (ALLM); 50–150 µM N-acetyl-Leu-Leu-Norleucinal (ALLN); 10–30 µg aprotinin ml-1; 1–30 mM EDTA; 1–30 mM EGTA; 1–5 mM 1,10-phenanthroline; 10–50 µM fosforamidon; 10–50 µg leupeptin ml-1; 2–10 µM pepstatin; 1–2 mM PMSF; 10–30 µg soybean trypsin inhibitor (STI) ml-1; 5-15 mM N-p-tosyl-L-arginine methyl ester hydrochloride (tame); 10-100 µm n-p-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK); 25–100 µM iodoacetic acid.
The effect of the pure S. lividans proteinaceous serine protease inhibitor (subtilisin type) SLPI was also studied at concentrations varying between 0·02 and 1 µg.
Xylanase activity was measured in culture supernatants by the dinitrosalicylic acid (DNS) method, using xylose as standard (Bernfeld, 1951; Biely et al., 1985). One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol reducing sugars (expressed as xylose equivalents) over 1 min.
Enzymes and reagents were purchased from Boehringer Mannheim, Promega, Bethesda Research Laboratories, Pharmacia, Sigma, Merck, Panreac, Bio-Rad, Santa Cruz and Ambion, and were used following the manufacturers' guidelines.
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RESULTS |
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). An in vitro experiment was developed as follows: the filtered supernatant from a 48 h culture of S. lividans 66 bearing plasmid pJM9, which permits overexpression of xylanase Xys1L, was incubated at 37 °C for 1–3 h in the presence of S. halstedii JM8 filtered supernatant. The processing of Xys1L to originate Xys1S increased with the incubation time and non-specific degradation of Xys1S was also observed after longer incubations (3 h, Fig. 1a). These processing-degradation events were not observed when the S. halstedii JM8 supernatant had been previously boiled for 5 min (Fig. 1a). The results of this experiment demonstrated the presence of extracellular thermolabile protease(s) responsible for the processing-degradation observed. A similar effect was observed with all supernatants from all the Streptomyces strains tested (data not shown).
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). Inhibitors for other types of proteases, such as metalloproteases (EDTA, EGTA), aspartyl proteases (pepstatin) and cysteine proteases (iodoacetic acid), did not show any effect. From these results, we concluded that the main proteases involved in this process belong to the serine protease group.
Effect of proteinaceous serine protease inhibitor (subtilisin type) on the processing of Xys1L
Since S. lividans 66 is a commonly used laboratory host strain for xylanase gene (xysA) overexpression, we studied Xys1L processing when it was produced in this host. Xylanase processing in S. lividans occurs at the same position (after D362) (Ruiz-Arribas et al., 1997) as in the original S. halstedii strain, suggesting the presence of at least a number of similar proteases in both strains. In vitro experiments confirmed that the 4-day-old filtered culture of S. lividans 66 was also able to process Xys1L. Nevertheless, a longer incubation time was required (as compared to S. halstedii JM8 supernatants) and only long incubation times (6 h) afforded total processing of Xys1L. PMSF-induced inhibition of processing was also observed, again suggesting that extracellular serine proteases were involved (data not shown).
Since most members of the genus Streptomyces are a good source of proteinaceous serine protease inhibitors (Taguchi et al., 1993, 1997), we decided to study the effect of the subtilisin-type inhibitor (SLPI) from S. lividans on the processing of Xys1L. First, 20 putative S. lividans SLPI-defective strains were obtained following the method of Taguchi et al. (1995a) (see Methods); it was observed that most of them were also subject to growth problems in complex media. Two of these strains (named 3.1 and 3.10) were selected and the absence of SLPI was corroborated by Western blot analysis with anti-SLPI antiserum (Fig. 2a). In vitro experiments of Xys1L processing confirmed that the supernatant from these strains processed Xys1L more efficiently than the S. lividans wild-type strain, suggesting that the activity of the proteases responsible was somehow blocked by the inhibitor (Fig. 2b).
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) in order to obtain plasmid pVR052 (Table 1). This promoter is induced by xylose or xylan and is repressed by glucose, and has proved to be a very efficient promoter for homologous and heterologous gene expression in Streptomyces (M. Díaz et al., unpublished). The production of SLPI was accomplished as indicated in Methods and the protein was partially purified (up to 99 %). The effect of SLPI was studied in in vitro experiments in which pure Xys1L (Ruiz-Arribas et al., 1997) was incubated with the filtered supernatant from S. lividans 66 and different amounts (1–10 µg) of the purified protease inhibitor. The presence of inhibitor partially reduced the processing of Xys1L (data not shown). Therefore, proteinaceous subtilisin serine protease inhibitors partially inhibit the serine proteases responsible for Xys1L processing.
Coexpression of xylanase and protease inhibitor genes increases xylanase production
The effect of co-expressing the xylanase gene (xysA) and the gene that encodes the protease inhibitor SLPI (slpI) was also studied. A transcriptional fusion in which the xysA promoter controls the expression of both genes – xysA and slpI – was constructed. This fusion was cloned into the bifunctional plasmid pN702GEM3 (Table 1) flanked by transcriptional terminators to obtain plasmid pVR011 (Table 1, Fig. 2c). As a control, the same vector containing only the xysA gene (plasmid pNAMP7) was used. The supernatants from several independent clones were analysed by SDS-PAGE and their xylanase activity was measured.
The positive effect of co-expressing both genes as compared to the results corresponding to cultures expressing only the xysA gene was observable at two levels: (1) a reduction in xylanase Xys1L processing, and (2) an increase in the amount of xylanase produced (Fig. 2d). Xylanase activity measurements revealed that the supernatants co-expressing xysA and slpI displayed threefold more xylanase activity (480 U ml-1) than those expressing xysA alone (150 U ml-1). Therefore, this inhibitor has two positive effects that should yield a higher total amount of xylanase, first by blocking Xys1L processing to produce Xys1S, and then by reducing the proteolysis-degradation undergone by this protein.
Cloning of two serine proteases from S. lividans involved in Xys1L processing
The role of a serine protease in the processing of cellulases has been described in Thermomonospora fusca (now Thermobifida fusca), where the protease encoded by the tfpA gene cleaves its cellulases into many different isoenzymes (Calza et al., 1985; Lao & Wilson, 1996). A BLAST search (Altschul et al., 1997) with the sequence of this protease (GenBank accession number O86984) against the S. coelicolor database (http://www.sanger.ac.uk/Projects/S_coelicolor/) detected two different ORFs with clear similarity: ORF SCF43A.19 (GenBank accession number Q9XA96) shows 45 % identity (58 % similarity), and ORF SCD40A.16c (GenBank accession number Q9L0J5), which has 46 % identity (59 % similarity). Two oligonucleotides were designed for each gene (see Methods) and both ORFs were amplified by PCR from S. lividans 66 genome and cloned in pET22b(+). The complete DNA sequences of these ORFs were obtained and compared with the S. coelicolor database. The proteases encoded by the S. lividans genes were denominated SpB (similar to SCF43A.19) and SpC (similar to SCD40A.16c). The SpB ORF is 458 aa long and is almost identical (99 %) to SCF43A.19 from S. coelicolor, which is also 458 aa long. Two amino acid changes were observed: one located in residue 75 (L in SCF43A.19 and P in SpB) and another in residue 341 (E in SCF43A.19 and G in SpB). Only this latter change is present in the mature protein (see below). SpC is also 458 aa long and its homologue (98 %) in S. coelicolor is SCD40A.16c (463 aa). The changes observed between both ORFs are: residue 66, L in SCD40A.16c and I in SpC; residue 122, I in SCD40A.16c and V in SpC; residue 244, G in SCD40A.16c and D in SpC; and residue 377, E in SCD40A.16c and G in SpC. The most significant change is a 5 aa deletion between residues 409 and 413, corresponding to the amino acid repeat GDDGG that is present three times in S. coelicolor SCD40A.16c and only twice in SpC from S. lividans. These amino acids are located at a linker region that connects the protease catalytic domain with a chitin-binding domain present at the C terminus of all these proteins. That domain is 47 aa long and shows similarity to chitin-binding domains present in several chitinases. Chitin-binding domains in proteases have been previously described for protease C from Streptomyces griseus (Sidhu et al., 1994). The complete ORF of this protease shares 55 % identity with proteases SpB and SpC.
In order to express both ORFs in S. lividans, they were cloned under control of the xysA promoter in the bifunctional plasmid pN702GEM3 to afford plasmids pVR047 and pVR048 (Table 1), which were transformed into S. lividans 66 (wild-type) and S. lividans 3.10 (SLPI-). Protease production in both strains was studied by SDS-PAGE in supernatants from several independent clones. No differential bands were detected by Coomassie blue staining when compared to transformants carrying an empty vector. However, several active protease bands were detected in zymograms carried out in renatured SDS-PAGE containing gelatin or skimmed milk, in which the samples were not boiled. This diversity of active bands was identical in both host strains (wild-type and SLPI-) (data not shown).
In vitro experiments using filtered supernatants from S. lividans 3.10 transformants expressing SpB or SpC confirmed that Xys1L was more efficiently processed by them than by the supernatant from the same strain transformed with the empty vector (Fig. 3a).
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). The N-terminus determined for both of them starts in the EDLV sequence in SpB and in the YDLR sequence in SpC. In both proteins, the first residue obtained corresponded to residue 207. SDS-PAGE of both pure proteins revealed a single band of 31–33 kDa for protease SpB and a band of about 23–25 kDa for protease SpC. Zymogram analyses of both pure proteases, in which the samples were not boiled, detected several active bands for both of them (Fig. 3c). Zymograms with boiled samples did not reveal protease activity (data not shown).
The observation of in vitro Xys1L processing with SpB or with SpC pure proteases demonstrated that both were able to process Xys1L efficiently. However, SpB was more active since 0·25 µg protease sufficed to process 4 µg Xys1L almost totally after 3 h of incubation, while 0·50 µg SpC was required to obtain a similar extent of processing (Fig. 3d, lane 0). Partial biochemical characterization of both proteases was accomplished using Xys1L as substrate. Both were more active at 50 °C than at 37 °C (the temperature normally used in our in vitro experiments) and the presence of 10 mM CaCl2 or 10 mM MgCl2 in the reaction mixture failed to affect their activity (data not shown). The inhibitory effect of pure serine protease inhibitor SLPI (from 0·02 to 1 µg) increased with the amount of SLPI added in the experiment and 0·5 µg was sufficient to block the processing of Xys1L (Fig. 3d).
The effect of other inhibitors such as 2 mM PMSF, 30 µg aprotinin ml-1, 30 µg STI ml-1, 100 µM TLCK, 30 µg leupeptin ml-1, 10 µM pepstatin, 4·5 mM 1,10-phenanthroline and 30 mM EDTA was also studied. As expected, both proteases were totally inhibited by PMSF, although SpB was almost totally inhibited by aprotinin while SpC was not. Among the other inhibitors studied only leupeptin showed partial inhibition of SpB and did not have any clear effect on SpC activity (at the concentration assayed) (Fig. 3e).
All these experiments demonstrated that both serine proteases are involved in the processing of Xys1L and that both respond similarly to the presence of the inhibitors SLPI and PMSF but not to the presence of other inhibitors such as aprotinin and leupeptin. These small differences in their inhibition profiles suggest that SpB and SpC are similar but not absolutely equivalent.
Other serine proteases from Streptomyces also process Xys1L
Now that the S. coelicolor genome has become available, at least 12 proteins have been identified as serine proteases by sequence similarity. Some homologues of these proteases have been cloned from different Streptomyces strains. Among them, SAM-P20 (Taguchi et al., 1995b), SAM-P26 (Taguchi et al., 1998) and SAM-P45 (Suzuki et al., 1997) from Streptomyces albogriseolus have been studied. Pure enzymes (a SAM-P20 homologue isolated from S. coelicolor Müller, SAM-P26 and SAM-P45 from S. albogriseolus), provided by Dr S. Taguchi, were used to study the processing effects on Xys1L in in vitro experiments. All of them were able to process the xylanase in a similar manner to SpB and SpC (Fig. 4a). Because these three serine proteases interact with the subtilisin inhibitor of S. albogriseolus, homologous to the SLPI inhibitor from S. lividans, we conducted studies aimed at determining whether pure SLPI might block their activity on Xys1L, observing that the proteolytic activity was blocked by the presence of this inhibitor.
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These results suggest that Xys1L processing is not carried out by a single enzyme but it is, instead, susceptible to the action of a variety of Streptomyces serine proteases, which are probably produced differentially, depending on the culture conditions.
Effects of SpB and SpC on the processing of other extracellular proteins
Our group is also studying the production of other proteins in S. lividans and some of them have also been found to undergo post-translational modifications. We therefore tested the effects of S. halstedii JM8 supernatant and pure SpB and SpC proteases on the following proteins: p40, a protein with no known catalytic activity and with a cellulose-binding domain at the carboxy terminus, isolated from S. halstedii JM8 (Garda et al., 1997), that is structurally similar to the xylanase Xys1L; Amy, an -amylase from S. griseus IMRU3570 (Vigal et al., 1991), and Xyl30, a xylanase from Streptomyces avermitilis (NCBI AAD32560). In vitro experiments were carried out mixing S. lividans supernatants in which each protein of interest had been overexpressed with the S. halstedii JM8 supernatant or with pure SpB or SpC proteases. The S. halstedii JM8 supernatant was able to process and degrade all the proteins studied. Both proteases were able to process p40, producing a protein of 35 kDa that corresponded to the formerly named p35 (Garda et al., 1997). This protein had previously been observed in supernatants of aged cultures (older than 48 h) overexpressing the p40 gene and corresponds to a truncated form in which the cellulose-binding domain has been removed. A weak partial processing of the amylase from S. griseus was observed but no effect on the xylanase Xyl30 from S. avermitilis was observed when pure SpB or SpC proteases were used (Fig. 4b). These results clearly indicate that the serine proteases studied are active in the processing and/or degradation of extracellular proteins and that their role should be considered when studying the overexpression of proteins of interest in Streptomyces hosts.
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DISCUSSION |
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; Lao & Wilson, 1996; Moorman et al., 1993). In cellulases and xylanases, this process normally occurs at the linker region that joins the catalytic and the substrate-binding domains and originates free forms of the enzyme able to the hydrolyse soluble oligosaccharides generated after a first attack on the substrate.
In actinomycetes, Moormann et al. (1993) described the purification of a 36 kDa putative metalloprotease involved in the processing of an 82 kDa avicelase from S. reticuli (Cel1). The protease cleavage site is located at the beginning of the catalytic domain of the enzyme and generates two fragments: an amino-terminal inactive fragment of 40 kDa that contains the cellulose-binding domain, and a carboxy-terminal fragment of 45 kDa that retains the catalytic activity and even exerts increased activity against p-nitrophenyl 
-D-cellobioside (Moorman et al., 1993). Serine proteases have been reported to be involved in the processing of glucanases in two different actinomycetes. Thus, processing of the endoglucanase CenA and the exoglucanase Cex from Cellulomonas fimi is carried out by the same serine protease that cuts at the linker region of both enzymes (Gilkes et al., 1988). No further investigations concerning this protease have been published and it is therefore not possible to compare it with those studied here. The role of serine proteases in the processing of cellulases has been documented in T. fusca, for which Lao & Wilson (1996) described the cloning and expression of a protease gene (tfpA) in S. lividans. This 368 aa protease, which might be included in the chymotrypsin family, is secreted as a pre-pro-protein with 194 residues in its mature form. The two genes cloned here encode proteases sharing about 45 % identity with the catalytic domain of the T. fusca protease and with the S. griseus protease C (Sidhu et al., 1994).
Proteases SpB and SpC are encoded as two proteins of 458 aa that have putative signal peptides at the N-terminus of 31 aa for SpB and 32 aa for SpC, followed by a propeptide up to amino acid 207, which is eliminated in both cases to produce the mature proteins. The sequences around the residue 207 PRVL
EDLV for SpB and PRTFYDLR for SpC are similar to the processing sequences of S. griseus serine proteases A, B, C and D. The putative catalytic triad corresponds to amino acids H242, D270 and S351, which are included in conserved regions, but further investigations would be necessary to confirm their role as part of the active centre. Both S. lividans proteases (SpB and SpC) and their homologues in S. coelicolor have a carboxy-terminal domain of 47 aa, similar to those of chitin-binding domains present in several chitinases. The presence of this type of domain has previously been described in protease C from S. griseus and its putative role was explained on the basis of the coexistence of chitin and proteins in insect cuticles. It is possible that the efficient degradation of chitin would require the collaboration of proteases recruited through their chitin-binding domain (Sidhu et al., 1994).
The processing of Xys1L is not exclusive to the proteases SpB and SpC or their homologues, depending on the host. Here we observed that another three serine proteases isolated from a different strain of Streptomyces, SAM-P20, SAM-P26 and SAM-P45, also process Xys1L. All five proteases have homologues in the S. coelicolor genome, whose sequence has recently been completely determined (Bentley et al., 2002). Proteases SpB and SpC have almost identical proteins encoded by ORFs SCF43A.19 and SCD40A.16c respectively. Protease SAM-P20 is 85 % identical to ORF SCI11.35c; SAM-P26 is 85 % identical to SCI11.30c, and SAM-P45 shares 43 % identity with the proteins encoded in ORFs SCF51A.10 and SCC24.17c, which have been described as putative secreted peptidases. Another eight ORFs in the S. coelicolor genome have been described as putative serine proteases, namely 2SCG61.37c, SCI35.29c, SC8E4A.07, SCH17.01c, SCH5.30, SC3C3.08, SC3C3.17c and SC10G8.13c. The multiplicity of enzymes with overlapping activities could be explained in terms of a different expression pattern that permits the expression of one or another gene, depending on the growth conditions. In the case of proteases, the effect of carbon or nitrogen sources on the differential expression of the corresponding genes has been described (reviewed by Hodgson, 2000). Study of the expression patterns of these ORFs and their role in the processing and degradation of overproduced extracellular proteins will be one of our objectives in future work. Meanwhile, co-expression of serine protease inhibitors together with target proteins might be a useful approach in biotechnological applications where a target protein is processed or degraded by endogenous proteases of the serine protease family.
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Received 8 November 2002;
revised 21 February 2003;
accepted 24 March 2003.
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