Correspondence: Akihiro Saito, Department of Materials and Life Science, Faculty of Science and Technology, Shizuoka Institute of Science and Technology, 2200-2 Toyosawa, Fukuroi, Shizuoka 437-8555, Japan. Tel.: +81538 45 0188; fax: +81538 45 0110; e-mail: email@example.com
The dasD gene is located just downstream of the dasABC gene cluster, encoding components of an ABC transporter for uptake of a chitin-degradation product N,N′-diacetylchitobiose [(GlcNAc)2] in Streptomyces coelicolor A3(2). To clarify the roles of the DasD protein in the degradation and assimilation of chitin, we obtained and characterized a recombinant DasD protein and a dasD-null mutant of S. coelicolor A3(2). The recombinant DasD protein produced in Escherichia coli showed N-acetyl-β-d-glucosaminidase (GlcNAcase) activity and its optimum temperature and pH were 40 °C and 7, respectively. dasD transcription was strongly induced in the presence of chitin, weakly by chitosan, but not by cellulose or xylan in S. coelicolor A3(2). Immuno-blot analysis demonstrated that DasD is a cytoplasmic protein. The dasD-null mutant exhibited cellular GlcNAcase activity which was comparable with that of the parent strain M145. DasD, thus, did not seem to be a major GlcNAcase. Induced extracellular chitinase activity in the dasD-null mutant was, interestingly, higher than M145, in the presence of colloidal chitin or (GlcNAc)2. In contrast to M145, (GlcNAc)2 temporally accumulated in the culture supernatant of the dasD-null mutant in the presence of colloidal chitin.
Chitin, a polymer of N-acetyl-β-d-glucosamine (GlcNAc), is the second most abundant biopolymer in nature after cellulose. Chitin is degraded into shorter oligo-, di-, and mono-saccharides by extracellular glycoside hydrolases which are represented by chitinase (EC 220.127.116.11), N-acetyl-β-d-glucosaminidase (EC 18.104.22.168), and N-acetyl-β-d-hexosaminidase (EC 22.214.171.124). A number of microbial genera are characterized as chitin-degraders, among which Streptomyces species are well-known decomporsers of chitin in soil.
Chitin-degradation systems in some Streptomyces species (S. lividans, S. coelicolor A3(2), S. olivaceoviridis, S. griseus, S. thermoviolaceus, and S. plicatus) have been intensively studied, and these species produce multiple extracellular chitinases which cleave chitin mainly into N,N′-diacetylchitobiose [(GlcNAc)2] (Blaak et al., 1993; Fujii & Miyashita, 1993; Kawase et al., 2001, 2006; Li et al., 2000; Miyashita & Fujii, 1993; Miyashita et al., 1991, 1997; Ohno et al., 1996; Robbins et al., 1988; Saito et al., 1999, 2000, 2003; Tsujibo et al., 1993, 2000a; Tsujibo et al., 2000b). Extracellular N-acetyl-β-d-hexosaminidase and N-acetyl-β-d-glucosaminidase (GlcNAcase), which release GlcNAc from (GlcNAc)2 and higher oligomers, have also been characterized in S. plicatus and S. thermoviolaceus, respectively (Mark et al., 1998; Tsujibo et al., 1998). In S. thermoviolaceus, an intracellular GlcNAcase has been reported as well (Kubota et al., 2004).
Chitin-degradation products are taken up via ABC (ATP-binding cassette) transporters [NgcEFG for GlcNAc and (GlcNAc)2 in S. olivaceoviridis (Xiao et al., 2002; Saito & Schrempf, 2004) and DasABC-MsiK for (GlcNAc)2 in S. coelicolor A3(2) (Saito et al., 2007, 2008)] and a PTS (phosphoenolpyruvate phosphotransferase system) for GlcNAc uptake (Wang et al., 2002; Nothaft et al., 2010). The dasD gene, which encodes a putative sugar hydrolase belonging to the family 3 of glycoside hydrolase classification, is located just downstream of the dasABC gene cluster of S. coelicolor A3(2). The amino acid sequence of the DasD protein is 34.4% identical with N-acetyl-β-d-glucosaminidase A of Altermonas sp. O-7 (Tsujibo et al., 1994) and is supposed to exhibit N-acetyl-β-d-glucosaminidase activity. To elucidate the biological roles of the DasD protein, we produced and characterized a recombinant DasD protein. We additionally obtained a dasD-null mutant and analyzed its chitin-degradation activity, to genetically characterize the biological role.
Materials and methods
Bacterial strains, plasmids, and media
Streptomyces coelicolor A3(2) strain M145 (Kieser et al., 2000) and its dasA mutant ASC2 (Saito et al., 2007) were used. Escherichia coli JM109 (Yanisch-Perron et al., 1985) was used as the host for gene manipulation. Escherichia 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. Escherichia coli BL21(DE3)(Novagen) was used to overproduce DasD protein. Plasmid vectors used are listed in Table 1. Luria–Bertani (LB) medium (Sambrook & Russell, 2001) was used to culture S. coelicolor A3(2); E. coli transformants were grown in LB medium supplemented with 50 μg mL−1 ampicillin or 10 μg mL−1 gentamicin. 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 (Kieser et al., 2000) was used to prepare spores of S. coelicolor A3(2) strains.
Table 1. Plasmids and primers used in this study
Cloning vector for PCR products in Escherichia coli
Promega (Madison, WI)
Expression vector to overproduce recombinant proteins in Escherichia coli
Plasmid preparation and restriction enzyme digestion were carried out as described by Sambrook & Russell (2001). DNA fragments were ligated using a DNA ligation kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions.
Production and purification of the recombinant DasD protein
The whole dasD gene was amplified by PCR using a primer set (dasDBamHIf and dasDEcoRIr. See Table 1) and cloned into the plasmid pGEM-T Easy (Table 1). After asserting the nucleotide sequence of the cloned gene, the BamHI-EcoRI fragment was integrated into the corresponding sites of the expression plasmid vector pGEX-4T-1 (Table 1) to obtain the plasmid pGEX-4T-1dasD. Escherichia coli BL21(DE3) carrying the constructed plasmid was cultivated in LB containing 100 μg mL−1 ampicillin. DasD production was induced by adding 1 mM IPTG to the culture. Escherichia coli cells were harvested by centrifugation and disrupted by sonication on ice. The cytoplasmically soluble fraction, which was obtained by centrifugation, was subjected to Glutathione Sepharose 4B to purify the GST-fused DasD protein, by following the manufacturer's instructions. To remove the GST tag, the recombinant DasD protein was treated with thrombin and subjected to an anion-exchange column (Resource Q, GE Healthcare). DasD protein without the GST tag was eluted by NaCl gradient (0–1 M) in 20 mM Tris-HCl buffer (pH 8.5). Purified DasD protein was subjected to anti-serum preparation as well as to enzymatic and biochemical characterization.
Proteins were separated on 12.5% polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) (Laemmli, 1970) and were visualized by staining SDS-polyacrylamide gels (SDS-PAGs) with Coomassie Brilliant Blue (CBB) R-250. For immuno-blot analysis, proteins separated on an SDS-PAG were blotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). Anti-DasD antiserum was prepared by injecting the purified recombinant DasD protein into a rabbit. Anti-DasA and anti-MsiK antisera were prepared as described previously (Saito et al., 2007, 2008).
To investigate the enzymatic activity of DasD, the purified DasD protein was incubated with 30 μM 4-methylumbelliferyl-N-acetyl-β-d-glucosamine (4MU-GlcNAc) or 4-methylumbelliferyl-N-acetyl-β-d-galactosamine (4MU-GalNAc) (Sigma, St. Louis, MO), and the activity was evaluated by measuring the concentration of liberated 4-methylumbelliferone (4-MU) based on its fluorescence. 50 mM Tris-HCl (pH 6.8) was routinely used, unless otherwise mentioned. To investigate the cleavage activity of DasD against (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, the purified DasD protein was incubated with 1.25 mg mL−1 of the di-, tri-, and tetra-saccharides and applied to a silica gel-60 plate (Merck), which was then developed four times with a 2 : 1 (by volume) mixture of 1-propanol/ammonia water. The spots were visualized by heating the plate with sulfuric acid. Chitinase activity was measured using 4-methylumbelliferyl-N,N′-diacetylchitobioside (Sigma, St. Louis, MO) 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-MU from the substrate in 1 min at 37 °C.
Disruption of the dasD gene
Regions (approximately 1 kb) upstream and downstream of the dasD (SCO5235) gene were amplified by PCR using specific primers that we designed (Table 1). The products were cloned into pGEM-T Easy (Table 1), 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 dasD downstream region was isolated with HindIII and EcoRI and cloned into the corresponding sites of pBlueScript SK+ (Table 1) to obtain the plasmid pDDD03. The HindIII-XhoI fragment of the dasD upstream region was then inserted into pDDD03 to obtain pDDD04. The HindIII fragment of the aacC4 gene cassette (Blondelet-Rouault et al., 1997) was integrated into the corresponding site on pDDD04. A plasmid clone in which the aacC4 gene was oriented opposite to the residual dasD gene was selected and named pDDD06. pDDD06 was digested with XhoI and PstI, and the fragment containing the upstream and downstream fragments of dasD and the aacC4 gene cassette was inserted into the SalI–PstI-digested pIJ2925 (Table 1) to obtain pDDD07. The BglII fragment of pDDD07, which includes the entire XhoI–PstI fragment, was inserted into the BamHI site of the temperature-sensitive plasmid vector pAS100 (Table 1) to obtain the dasD-disruption plasmid pDDD08. Streptomyces coelicolor A3(2) M145 was transformed with pDDD08, 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 5 μg mL−1 gentamicin. After streaking the obtained colonies on SFM agar medium containing gentamicin and culturing at 30 °C, we obtained strains that were resistant to gentamicin but sensitive to thiostrepton. Disruption of dasD was verified by Southern blot analysis, using the labeled dasD and aacC4 genes as probes.
Growth conditions for S. coelicolor A3(2) M145 and its derivatives
To investigate cell response to various sugars, we cultivated S. coelicolor A3(2) strains according to a method described previously (Saito et al., 2000), with some modifications. Spores 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 18–20 h at 30 °C on a rotary shaker at 150 rpm. Mycelia were harvested by centrifugation (1000 g, 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 (GlcNAc)2 and 0.05%(w/v) colloidal chitin, colloidal chitosan, cellulose nanofiber, or xylan powder. After sugar supplementation, cultures were again grown at 30 °C on a rotary shaker at 150 rpm. 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 protein analysis, GlcNAcase assay, or total RNA preparation.
Reverse transcription (RT)-PCR
DNA-free total RNA was prepared from S. coelicolor A3(2) mycelia following the method of Saito et al. (2007), using an SV Total RNA Isolation System (Promega). To characterize transcripts, RT-PCR analysis was carried out using a PrimeScript RT-PCR Kit (Takara Bio Inc., Otsu, Japan). A primer set specific for the dasD gene transcript was designed (Table 1). Transcripts of the chitinase gene chiC were also investigated for comparison. For PCR, the number of cycles was set to 25 to avoid saturation.
Determination of (GlcNAc)2 concentration
To measure the concentrations of (GlcNAc)2, culture supernatant obtained by centrifugation was subjected to an HPLC with a refractive index detector RID-6A (Shimadzu, Kyoto, Japan) using a normal phase column Inertsil-NH2 (4.6 × 250 mm, GL Science Inc., Tokyo, Japan). (GlcNAc)2 was separated under isocratic conditions 85% (v/v) acetonitrile in water at a flow rate of 1.0 mL min−1 and identified by the retention time.
DasD fused with GST at the C-terminus was successfully produced in the cytoplasmic fraction of E. coli in a soluble form. The purified recombinant DasD protein without the GST tag appeared to be homogeneous in an SDS-PAGE analysis (Fig. 1a). The purified DasD protein showed hydrolytic activity against 4MU-β-d-GlcNAc, whereas it did not against 4MU-β-d-GalNAc (Fig. 1b). The result suggests that DasD acts as N-acetyl-β-d-glucosaminidase β-d-GlcNAcase) (EC 126.96.36.199) or chitobiase (EC 188.8.131.52), but not as N-acetyl-β-d-hexosaminidase (EC 184.108.40.206). DasD liberated GlcNAc residue not only from (GlcNAc)2 but from (GlcNAc)3 and (GlcNAc)4 (Fig. 1c). It was thus demonstrated that DasD is a β-d-GlcNAcase. The purified DasD protein showed the highest hydrolyzing activity against 4MU-β-d-GlcNAc at pH 7 and at 40 °C (Fig. 1d ane e).
DasD is a cytoplasmic protein whose gene expression is induced by chitin
To investigate the localization of DasD, the mycelia of S. coelicolor A3(2), which were grown a liquid minimal medium supplemented with colloidal chitin, were disrupted by sonication and separated into cytoplasmic and envelope fractions using ultra centrifugation (100 000 g, 1 h, 4 °C). The MsiK protein, which is an ATP-binding component of ABC sugar transporters (Saito et al., 2008), was expectedly detected in the envelope fraction. In contrast, the DasD protein was detected in the cytoplasmic fraction. DasD composed of 496 amino acids (aa) shares 46% identical aa sequence with the GlcNAcase NagA (632 aa) of S. thermoviolaceus (Tsujibo et al., 1998), in 262 aa overlapping. NagA is the unique GH3 GlcNAcase from streptomycetes, which has been experimentally characterized so far, and is likely secreted via its N-terminal signal peptide (Tsujibo et al., 1998). In contrast, DasD does not possess an N-terminal hydrophobic amino acid sequence which would act as a Sec- or Tat-dependent signal peptide. Additionally, GlcNAcase activity was not detected in the culture supernatant of S. coelicolor A3(2), using 4MU-β-d-GlcNAc as a substrate (Saito et al., 2007). We, thus, concluded that DasD is a cytoplasmic protein.
Transcripts of the dasD gene were detected in the presence of colloidal chitin, slightly in the presence of colloidal chitosan, but hardly detected with cellulose nanofiber or xylan powder (Fig. 2b). dasD was also induced by (GlcNAc)2 or (GlcNAc)3, but not by GlcNAc (Fig. 2c), as observed in some chitinase genes and the dasR and dasA genes (Saito et al., 2000, 2007). The polycistronic transcripts of dasCD were detected by RT-PCR analysis (Fig. 2d). The data suggested that the dasD gene was transcribed together with dasC, whose polycistronic transcripts with dasB were observed (Saito et al., 2007). It is thus assumed that dasB, dasC, and dasD were transcribed together as one polycistronic unit. We recently indicated that the dasR disruption upregulated the transcription of dasB, dasC, and dasD in the presence of chitin (Nazari et al., 2013), suggesting that DasR might act as a transcriptional repressor for the dasBCD transcription.
DasD is not likely the main GlcNAcase in S. coelicolor A3(2)
To investigate the biological role of DasD by a genetic approach, we disrupted the dasD gene and obtained the dasD-null mutant MRC8. A cell-free extract of the dasD mutant, which was grown in the presence of colloidal chitin, still exhibited cleavage activity against 4MU-β-d-GlcNAc, which was comparable with the parent strain M145 (data not shown). The data suggest that S. coelicolor A3(2) possesses other protein(s) exhibiting GlcNAcase activity. Three genes (SCO2758, SCO2786, SCO2943) have been predicted to encode proteins possessing GlcNAcase activity (Świątek et al., 2012). Among them, SCO2758 encodes a protein whose aa sequence exhibits striking similarity with that of the intracellular GH20 GlcNAcase NagC of S. thermoviolaseus (Kubota et al., 2004); 69% identity and 87% similarity in 563 aa overlapping. Main GlcNAcases must remain to be elucidated in S. coelicolor A3(2).
dasD mutation enhances the chitinase production in S. coelicolor A3(2)
(GlcNAc)2, which could be a substrate for the DasD protein, is a minimum unit that induces chitinase production in S. coelicolor A3(2) (Saito et al., 2000). We elucidated the chitin-degradation activity in the dasD-null mutant, because dasD disruption would affect the (GlcNAc)2 consumption rate. The dasD mutant formed a larger and clearer clearing zone, than its parent strain M145, around its colony on the minimal agar medium containing colloidal chitin as a carbon source (Fig. 3a), suggesting that the dasD mutation enhanced the induction level of chitinase production. The dasD mutant showed higher chitinase activity in the presence of colloidal chitin or (GlcNAc)2 than strain M145 (Fig. 3b and c). It was thus concluded that the dasD disruption increased the level of chitinase production in the presence of an inducer (GlcNAc)2 or colloidal chitin. In the presence of colloidal chitin, (GlcNAc)2 temporarily accumulated in the culture supernatant of the dasD mutant, but it was not detected in that of the parent strain M145 (Fig. 3d).
Chitinase activity in the dasA mutant was also higher than that in the parent strain M145, as reported previously (Saito et al., 2007; Colson et al., 2008) and was much higher than that in the dasD mutant (Fig. 3a–c). The temporal accumulation level of (GlcNAc)2 was higher in the dasA mutant than in the dasD mutant (Fig. 3d). It has been reported that the (GlcNAc)2-consumption rate of the dasA mutant was about 40% of the parent strain (Saito et al., 2007). Immuno-blot analysis revealed that DasA was produced in the dasD mutant, but the amount of DasA was apparently lower than in the parent strain M145 (Fig. 2e). The lowered production of DasA in the dasD mutant may decrease the (GlcNAc)2-consumption rate and enhance chitinase production. It remains to be elucidated why DasA production was lowered by the dasD mutation.
GlcNAc is an important signaling molecule for streptomycetes and a major decision point toward the onset of development and antibiotic production (Rigali et al., 2006, 2008). In the dasD-null mutant, pigmentation was apparently enhanced in the liquid minimal medium supplemented with colloidal chitin (A. Saito, T. Fujii & K. Miyashita, unpublished data). Although DasD was unlikely the main enzyme cleaving (GlcNAc)2, DasD might be involved in controlling the intracellular GlcNAc/(GlcNAc)2 concentration. Streptomyces coelicolor A3(2) and probably other streptomycetes should have an elaborated chitin-assimilation system which can adjust the intracellular GlcNAc concentration to regulate primary and secondary metabolism.
This work was partly supported by JSPS KAKENHI Grant Numbers 21380050 and 17780056, and by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science and Technology, Japan (MEXT), 2010-2014 (S1001032). The authors thank Shinsuke Ifuku at Tottori University and Ayano Masui at Yaizu Suisankagaku Industry for providing cellulose nanofiber and N,N′-diacetylchitobiose, respectively.