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1 Graduate School of Advanced Integration Science, Chiba University, Matsudo 648, Matsudo City, Chiba 271-8510, Japan
2 National Institute of Agro-Environmental Sciences, Kannondai 3-1-1, Tsukuba, Ibaraki 305-8604, Japan
3 Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan
Correspondence
Akihiro Saito
takosaito{at}faculty.chiba-u.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; the enzymes hydrolyse chitin into (GlcNAc)2 as the predominant final product (Blaak et al., 1993; Ohno et al., 1996). We previously reported that the dasABC gene cluster encodes subunits of an ATP-binding cassette (ABC) transporter for the uptake of (GlcNAc)2 in Streptomyces coelicolor A3(2) (Saito et al., 2007). The genes encode the (GlcNAc)2-binding protein DasA and two putative integral membrane proteins, DasB and DasC, respectively. The dasA null mutant ASC2 shows a lower but significant rate (25 % of that of the parental strain M145) of decline of the (GlcNAc)2 concentration in the culture supernatant, suggesting the presence of (GlcNAc)2-uptake systems other than DasABC (Saito et al., 2007), although the types of the remaining transporters are unknown. Interestingly, the dasA mutant ASC2 showed a longer duration of chitinase production in the presence of (GlcNAc)2 (Saito et al., 2007). Another dasA mutant, SAF3, which was independently generated, exhibits a chitin-degradation activity apparently stronger than that of the parental strain M145 (Colson et al., 2008). The data imply that (GlcNAc)2 uptake is important in controlling chitinase production in S. coelicolor A3(2).
Various genes encoding other oligosaccharide-uptake systems have been identified in streptomycetes: cebEFG for cellobiose and cellotriose in Streptomyces reticuli (Schlösser et al., 1999); malEFG for maltose in S. coelicolor (van Wezel et al., 1997a); ngcEFG for GlcNAc and (GlcNAc)2 in Streptomyces olivaceoviridis (Xiao et al., 2002); and bxlEFG for xylobiose in Streptomyces thermoviolaceus (Tsujibo et al., 2004). Interestingly, all of these gene systems encode subunits of ABC transporters, with the E and FG genes of these uptake systems encoding sugar-binding proteins (SBPs) (CebE, MalE, NgcE and BxlE) and two putative integral membrane proteins (CebFG, MalFG, NgcFG and BxlFG), respectively. It is noteworthy that in streptomycetes, gene clusters that encode ABC transporters for oligosaccharides lack genes for ABC proteins, which provide the necessary energy for the corresponding transporters by hydrolysing ATP to ADP (Bertram et al., 2004). The MsiK protein is known to be the ABC protein that assists the cellobiose-, xylobiose- and maltose-uptake systems in Streptomyces lividans (Hurtubise et al., 1995; Schlösser et al., 1997), and the trehalose-uptake system in S. reticuli (Schlösser, 2000). It is therefore assumed that the msiK gene is globally involved in oligosaccharide-uptake systems in Streptomyces species. Hurtubise et al. (1995) have indicated that the uptake of cellobiose and xylobiose assisted by the msiK product is essential for induction of cellulase and xylanase production, respectively, in S. lividans.
S. coelicolor A3(2) possesses the ORF SCO4240, which encodes an MsiK homologue sharing 93 and 91 % amino acid identities with those of S. reticuli and S. lividans, respectively. In this report, by constructing and analysing an msiK null mutant, we confirmed that the product of the msiK gene is required for (GlcNAc)2 uptake in S. coelicolor A3(2). Unexpectedly, it was found that (GlcNAc)2-uptake systems other than DasABC are also ABC transporters, in contrast to Gram-negative Escherichia coli and Serratia marsescens, in which uptake of the disaccharides is mediated by the phosphotransferase system (PTS) (Keyhani et al., 2000; Uchiyama et al., 2003). The msiK mutant also allowed us to demonstrate that (GlcNAc)2 uptake is necessary for the induction of chitinase production.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). E. coli JM109 (Yanisch-Perron et al., 1985) was used as the host for gene manipulation. E. coli ET12567 (dam dcm hsdS) (MacNeil et al., 1992) was used to prepare plasmids for S. coelicolor A3(2) transformation, to avoid the methylation-specific restriction system of the bacterium. Plasmid vectors used are listed in Table 1. Luria–Bertani (LB) medium (Sambrook & Russell, 2001) was used for culture of S. coelicolor A3(2); E. coli transformants were grown in LB medium supplemented with 50 µg ampicillin ml–1 or 100 µg hygromycin B ml–1. A minimal medium (10 mM K2HPO4, 10 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, supplemented with 0.1 %, v/v, trace element solution) (Schlochtermeier et al., 1992) was used to investigate the responses of S. coelicolor A3(2) cells to various carbon sources. Soya flour–mannitol (SFM) agar medium (van Wezel et al., 1997b) was used to prepare spores of S. coelicolor A3(2) strains.
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), with some modifications. Spores of S. coelicolor A3(2) strains that formed on SFM agar medium were inoculated into 30 ml LB medium in a 100 ml flask with a spring (Kieser et al., 2000) and grown for 19 h at 30 °C on a rotary shaker at 150 r.p.m. Mycelia were harvested by centrifugation (3000 r.p.m., 3 min), washed with minimal medium without carbon sources, suspended in 60 ml minimal medium, and divided into several aliquots. Each aliquot was supplemented with a different carbon source: 250 µM of glucose, maltose, cellobiose, xylobiose, GlcNAc or (GlcNAc)2, and 0.1 % (w/v) powdered chitin. For S. coelicolor transformants carrying pWHM3 or its derivative, 25 µg thiostrepton ml–1 was added to each culture. After sugar supplementation, cultures were again grown at 30 °C on a rotary shaker at 150 r.p.m. The culture fluids were sampled periodically, centrifuged to separate the supernatant and mycelia, and stored at –80 °C. The sugar concentration and chitinase activity of the supernatants were measured, whereas the mycelia were used for total RNA preparation.
Gene manipulation.
Plasmid preparation and restriction enzyme digestion were done as described by Sambrook & Russell (2001). DNA fragments were ligated by using a DNA ligation kit (Takara Bio) according to the manufacturer's instructions.
Disruption of msiK.
Regions (
1 kb) upstream and downstream of the msiK (SCO4240) gene were amplified by PCR using specific primers that we designed (Table 2). The products were cloned into pGEM-T Easy (Promega) and the sequences of the cloned fragments were confirmed to be identical to those registered in the genome database (http://www.sanger.ac.uk/Projects/S_coelicolor/). The fragment corresponding to the msiK downstream region was isolated with EcoRI and HindIII, and cloned into the corresponding sites of pBlueScript SK+ (Table 1
) to obtain the plasmid pDMK03. The HindIII–XhoI fragment of the msiK upstream region was then inserted into pDMK03 to obtain pDMK04. The HindIII fragment of the hyg cassette (Blondelet-Rouault et al., 1997) was integrated into the corresponding site on pDMK04. A plasmid clone in which the hyg gene was oriented opposite to the residual msiK gene (Fig. 1) was selected and named pDMK05. pDMK05 was digested with XhoI and PstI, and the fragment containing the upstream and downstream fragments of msiK and the hyg gene cassette was inserted into the SalI/PstI-digested pIJ2925 (Janssen & Bibb, 1993) to obtain pDMK06. The BglII fragment of pDMK06, which includes the entire SalI/XhoI–PstI fragment, was inserted into the BamHI site of the temperature-sensitive vector pAS100 (Table 1) to obtain the msiK-disruption plasmid pDMK07. S. coelicolor A3(2) M145 was transformed with pDMK07, which was prepared from E. coli ET12567 according to the method described by Kieser et al. (2000). After obtaining thiostrepton-resistant transformants at 30 °C, we selected strains that grew at 39 °C on SFM agar medium supplemented with 50 µg hygromycin B ml–1. After streaking the obtained colonies on SFM agar medium containing hygromycin and culturing at 30 °C, we obtained strains that were resistant to hygromycin B but sensitive to thiostrepton. Disruption of msiK was verified by Southern blot analysis, using the labelled msiK and hyg genes as probes.
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) by using high-performance anion-exchange chromatography (DX-500, Dionex) with pulsed amperometric detection (HPAEC-PAD; Dionex) and a CarboPac PA1 column (Dionex). GlcNAc and (GlcNAc)2 were separated under isocratic conditions (18 mM sodium hydroxide) at a flow rate of 1.0 ml min–1 and identified by their respective retention times.
Production of His-tagged MsiK protein in the msiK mutant.
msiK and its flanking region (including the putative promoter sequence) were amplified by using the primers msiKCf and msiKCr (Table 2) to produce C-terminally His-tagged MsiK protein under the control of the putative native promoter. The amplified DNA fragment (1295 bp) was cloned into pGEM-T Easy (Table 1), and its sequence was confirmed to be identical to that registered in the genome database (http://www.sanger.ac.uk/Projects/S_coelicolor/). The EcoRI–HindIII fragment was integrated into pWHM3 (Table 1) to obtain the resulting plasmid pCMK02. The msiK null mutant ASC3 was transformed with pWHM3 or pCMK02, which was prepared from E. coli ET12567 by the method of Kieser et al. (2000).
RT-PCR.
DNA-free total RNA was prepared from mycelia by our method (Saito et al., 2007) and by using an SV Total RNA Isolation System (Promega). To characterize transcripts, RT-PCR analysis was done by using AccuPower RT-PCR Premix (Bioneer), as reported previously (Saito et al., 2007). Sets of primers specific for each transcript were designed to give PCR products ranging from 375 to 545 bp (Table 2). For PCR, the number of cycles was set to 20 to avoid saturation of PCR product formation. RT-PCR experiments without prior reverse transcription were done to ensure that no residual DNA was present in the RNA samples.
Chitinase assay.
Chitinase activity was measured by using the fluorescent substrate 4-methylumbelliferyl-N,N'-diacetylchitobioside (Sigma) according to a previously described method (Miyashita et al., 1991). One unit of chitinase activity was defined as the amount of enzyme that liberated 1 µmol of 4-methylumbelliferone from the substrate in 1 min at 37 °C.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), on chitin utilization in S. coelicolor A3(2) M145, msiK (SCO4240) was disrupted by homologous recombination in the bacterium. The profile of the obtained strain ASC3, which was analysed by Southern blot hybridization using the msiK and hyg probes, clearly demonstrated the msiK disruption and the hyg insertion on the genome (Fig. 1b). The growth of the obtained msiK null mutant ASC3 was tested on minimal agar medium containing starch, cellulose, chitin and chitosan, and related mono- and disaccharides, and we judged the ability of the mutant to utilize those sugars by examining growth on each supplemented medium (Table 3). ASC3 did not seem to utilize any of the polysaccharides or maltose, whereas its growth on glucose-containing medium was comparable with that of the parental organism, M145. ASC3 seemed to utilize cellobiose but apparently less effectively than did M145. These data indicate that the protein product of msiK is essential for the ability to utilize polysaccharides and maltose, and that msiK is also involved in cellobiose utilization. The poor growth of the msiK mutant on a medium containing maltose or cellobiose suggests that msiK is involved in the uptake of those disaccharides in S. coelicolor A3(2), as demonstrated in S. lividans (Schlösser et al., 1997). The defect of chitosan utilization in the msiK mutant may imply that msiK is involved in the uptake of chitosan-degradation products, such as chitobiose.
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), suggesting that msiK is not involved in GlcNAc uptake. The data supported the finding of Nothaft et al. (2003) that S. coelicolor A3(2) exclusively uses the PTS for GlcNAc. In contrast, mutation of msiK dramatically altered the pattern of utilization of (GlcNAc)2 in the culture supernatant (Fig. 2b). In M145, (GlcNAc)2 was depleted within 4 h, as reported previously (Saito et al., 2007). However, in the msiK mutant ASC3, the (GlcNAc)2 concentration remained higher than 200 µM even 8 h after addition of the disaccharide. These data clearly demonstrate that the msiK gene product is required for (GlcNAc)2 uptake in S. coelicolor A3(2). The slight decrease in the (GlcNAc)2 concentration in the ASC3 culture supernatant may indicate the presence of a low level of N-acetylglucosaminidase (chitobiase) activity. The poor GlcNAc uptake activity of the msiK mutant might reflect inhibition of the PTS for GlcNAc uptake (Nothaft et al., 2003) due to the presence of (GlcNAc)2 (Fig. 2b).
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). The dasA null mutant ASC2 retains the ability to take up (GlcNAc)2, although the level of activity is markedly reduced (25 % of that of the parental strain M145), thereby implying the presence of other (GlcNAc)2-uptake systems (Saito et al., 2007). In contrast, the disruption of msiK leads to a virtually complete absence of (GlcNAc)2 uptake (Fig. 2b). The data thus suggest that the (GlcNAc)2-uptake systems other than DasABC must also be ABC transporters dependent on MsiK. (GlcNAc)2 uptake therefore appears to be governed by ABC transporters in S. coelicolor A3(2), in contrast to E. coli (Keyhani et al., 2000) and the Gram-negative chitin degrader S. marcescens (Uchiyama et al., 2003), in which the uptake of (GlcNAc)2 is mediated by a PTS. The gene cluster (SCO6005, SCO6006 and SCO6007) might be a good candidate for a (GlcNAc)2 ABC uptake system (Bertram et al., 2004) other than DasABC, although the putative products of the three ORFs share relatively low amino acid identity (31–48 %) with NgcE, F and G, which are components of the ABC transporter Ngc specific for GlcNAc and (GlcNAc)2 in S. olivaceoviridis (Saito & Schrempf, 2004; Xiao et al., 2002).
msiK is required for the induction of chitinase production
In S. coelicolor A3(2), production of chitinase is induced by the chitin-degradation product (GlcNAc)2 (Saito et al., 2000). The defect of chitin utilization in the msiK null mutant (Table 3, Fig. 3a) implied that msiK might be required for the induction of chitinase production as well as for (GlcNAc)2 uptake. To investigate the effect of msiK mutation on this induction, M145 and the msiK null mutant ASC3 were grown in LB medium and then incubated in minimal medium supplemented with 250 µM GlcNAc or (GlcNAc)2. Chitinase production in M145 was induced by (GlcNAc)2 but not by GlcNAc (Fig. 3b). In contrast, chitinase activity was not detected in the culture supernatant of ASC3 in the presence of either GlcNAc or (GlcNAc)2 (Fig. 3b). Chitinase activity was detectable in the culture supernatant of M145 6 days after the addition of 0.1 % (w/v) powdered chitin, whereas chitinase activity was not induced in the msiK null mutant ASC3 even after 13 days (Fig. 3c). The defect in chitinase production was rescued by introducing the multicopy plasmid pCMK02, which carries the gene for His-tagged MsiK protein with its putative native promoter (Figs 1 and 3d). These data demonstrate that the product of msiK is essential for the induction of chitinase production by (GlcNAc)2 or chitin in S. coelicolor A3(2). Intracellular (GlcNAc)2 would act as an inducer.
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). Our preliminary experiments suggested that msiK also is required for the induction of amylase production by maltose in S. coelicolor A3(2) (A. Saito and others, unpublished data). Therefore, we assume that uptake of cellobiose and xylobiose as well as maltose and (GlcNAc)2 is necessary for the induction of the corresponding polysaccharide-hydrolysing enzymes. Just how disaccharide molecules that are taken into cells trigger the production of the corresponding glycosylhydrolases remains an open question. MalR, CebR and BxlR, which respectively regulate the transcription of the malEFG, cebEFG and bxlEFG operons (Schlösser et al., 1999; Tsujibo et al., 2004; van Wezel et al., 1997b), and/or a pleiotropic regulator Reg1 (Nguyen et al., 1997), which shares 95 % amino acid identity with MalR of S. coelicolor A3(2), would also indirectly control amylase, cellulose and xylanase production.
msiK is constitutively transcribed
To elucidate the conditions for msiK expression in S. coelicolor A3(2), the msiK transcript was investigated by RT-PCR after growth in the presence of 250 µM glucose, maltose, cellobiose, xylobiose, GlcNAc or (GlcNAc)2. For comparison, we also analysed the transcription of genes for the following oligosaccharide-binding proteins of ABC transporters of OSP (oligosaccharide and polyols) family members: dasA [SCO5232, (GlcNAc)2-binding protein gene; Saito et al., 2007]; malE (SCO2231, putative maltose/maltotriose-binding protein gene; van Wezel et al., 1997a); cebE1 (SCO2795, similar to the cellobiose/cellotriose-binding protein gene cebE in S. reticuli; Bertram et al., 2004; Schlösser et al., 1999); and bxlE1 (SCO7028, similar to the xylobiose-binding protein gene bxlE of S. thermoviolaceus; Bertram et al., 2004; Tsujibo et al., 2004). The expression of msiK transcripts seemed to be constitutive under the various test conditions. In contrast, the transcription of dasA, malE, cebE1 and bxlE1 was induced in the presence of the corresponding (putative) ligand, although there did appear to be some background expression in each case (Fig. 4). The data extend the finding by Schlösser et al. (1999) that in S. reticuli, the regulation of MsiK production differs from that of the cellobiose/cellotriose-binding protein CebE. It is assumed that the MsiK protein interacts with the other components (such as DasBC and MalFG) of the ABC transporters, whose genes form clusters with the genes of the corresponding SBPs (i.e. dasA and malE, respectively) (Bertram et al., 2004). This uptake machinery, which is unusual among bacteria, may be energetically efficient for streptomycetes; these organisms utilize a variety of oligosaccharides after degrading their cognate polysaccharides, which are present in soil.
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ACKNOWLEDGEMENTS |
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Edited by: W. H. Schwarz
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 14 April 2008;
revised 11 August 2008;
accepted 18 August 2008.
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X174/HinfI) are also shown. As for msiK, the result with 15 cycles of PCR and that without RT reaction are indicated. The rRNA bands within RNA (0.15 µg) on the ethidium bromide-stained gel are shown as a control. BP, binding protein; RT, reverse transcription.