Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan1
Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan2
Department of Applied Biological Chemistry, The University of Tokyo, Bunkyoku, Tokyo 113-8657, Japan3
National Institute of Agro-Environmental Science, Tsukuba 305-8604 , Japan4
Author for correspondence: Takeshi Watanabe. Tel: +81 25 262 6647. Fax: +81 25 262 6854. e-mail: wata{at}agr.niigata-u.ac.jp
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ABSTRACT |
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Keywords: chitinase, Streptomyces, allosamidin
The GenBank accession numbers for the sequences determined in this work are AB031745AB031757 inclusive.
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INTRODUCTION |
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In addition to the difference in 3D structure, chitinases of the two families show several important differences in their biochemical properties. For example, family 18 chitinases hydrolyse the glycosidic bond with retention of the anomeric configuration (Armand et al. , 1994 ; Iseli et al., 1996
), whereas family 19 chitinases hydrolyse with inversion (Ohno et al. , 1996
; Fukamizo et al., 1995
). Family 18 chitinases are sensitive to allosamidin, but a family 19 chitinase from higher plants has been shown to be insensitive (Koga et al., 1987
). Family 18 chitinases hydrolyse GlcNAc- GlcNAc and GlcNAc-GlcN linkages, whereas family 19 chitinases hydrolyse GlcNAc-GlcNAc and GlcN-GlcNAc (Ohno et al., 1996
; Mitsutomi et al., 1996
, 1997
). These differences are probably common between all members of the two families and arise from the differences in their catalytic mechanisms. Substrate- assisted catalysis is the most widely accepted model for the catalytic mechanism of family 18 chitinases (Tews et al., 1997
; Brameld et al., 1998
), whereas a general acid-and-base mechanism has been suggested to be the catalytic mechanism for family 19 chitinases (Hart et al., 1995
; Garcia-Casado et al., 1998
).
Chitinase C from S. griseus HUT6037 is the first family 19 chitinase found in an organism other than higher plants (Ohno et al. , 1996 ). It has a catalytic domain homologous to those of plant class I, II and IV chitinases, and an N-terminal chitin- binding domain. Plant chitinases in classes I and IV have a cysteine- rich domain (also referred to as the wheatgerm agglutinin domain) at their N-termini which is involved in chitin binding (Iseli et al. , 1993
; Raikhel et al., 1993
; Shinshi et al., 1990
). Class IV chitinases are smaller than class I chitinases due to deletions in both the cysteine-rich domain and the catalytic domain (Collinge et al., 1993
). Class II chitinases are homologous to those of class I and IV but lack the cysteine-rich chitin-binding domain. The chitin-binding domains of plant chitinases and S. griseus chitinase C do not share significant sequence similarity. The chitin-binding domain of S. griseus chitinase C has obvious sequence similarity to the chitin- binding domains and probable chitin-binding domains of some bacterial family 18 chitinases, including chitinases A1 and D1 of Bacillus circulans WL-12 (Ohno et al., 1996
).
In the present study, we constructed an expression system for chitinase C in Escherichia coli for further characterization, and found some important characteristics of this chitinase. In addition, we demonstrated the general occurrence of family 19 chitinases in Streptomyces.
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METHODS |
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Expression of chiC and purification of its product.
E. coli BL21(DE3) cells harbouring pGC02 were grown at 30 °C on LB medium containing 100 µg ampicillin ml -1. When cells reached an OD600 of 0·7, IPTG (final concentration 0·4 mM) was added and the cells were cultivated for another 5 h at 30 °C. After collecting the cells by centrifugation, chitinase C secreted into the periplasmic fraction was extracted by the cold osmotic shock procedure as described by Manoil & Beckwith (1986) . The extracted periplasmic proteins containing chitinase C were collected by ammonium sulfate precipitation (60% saturation), dissolved in 20 mM phosphate buffer (pH 6·0), dialysed against 2 mM sodium phosphate buffer (pH 6·0) and lyophilized. Lyophilized proteins were dissolved in a small volume of 20 mM phosphate buffer (pH 6·0) and applied onto a hydroxylapatite column (3·5x13 cm) previously equilibrated with the same buffer. Proteins were eluted with the same buffer and the peak fractions containing chitinase C were collected, dialysed against 2 mM sodium phosphate buffer (pH 6·0), and lyophilized.
Purification of other chitinases.
Serratia marcescens chitinases A and C1 (Suzuki et al. , 1998 , 1999
), and B. circulans chitinases A1 and D1 (Armand et al., 1994
), were purified as described previously.
B. circulans chitinase C1 was produced by E. coli JM109 harbouring the recombinant plasmid pALC11 (Alam et al., 1995 ) and extracted from the periplasmic space of the cells by the cold osmotic shock procedure. Proteins in the periplasmic fraction were collected by ammonium sulfate precipitation (60% saturation) and applied to a hydroxyapatite column (1·5x15 cm) washed with 1 mM phosphate buffer (pH 6·0). Elution was done with a gradient of 1400 mM sodium phosphate buffer (pH 6·0) with a flow rate of 25 ml min-1. The fractions containing chitinase C1 were pooled, concentrated and further purified by HPLC with a Shim-pack PA-DEAE column in a liquid chromatograph (LC- 6A system, Shimadzu). Elution was performed with 20 mM Tris/HCl buffer (pH 7·5) with a 00·5 M NaCl gradient.
Serratia marcescens chitinase B was produced by E. coli DH5 cells harbouring plasmid pMCB7 carrying the cloned chiB gene (Watanabe et al., 1997
). The cells were grown for 24 h in LB medium supplemented with 100 µg ampicillin ml-1 and 0·4 mM IPTG, collected by centrifugation and disrupted by sonication. After removing unbroken cells and debris, proteins were collected by ammonium sulfate precipitation (2040% saturation). The precipitate was dissolved in a small volume of 1 mM sodium phosphate buffer (pH 6·0) and applied to a hydroxyapatite column (3x7 cm) previously equilibrated with the same buffer, and eluted with the same buffer. The unadsorbed protein fractions containing chitinase B were collected and lyophilized. Lyophilized chitinase B was dissolved in a small volume of 1 mM phosphate buffer (pH 6·0) and further purified by chitin affinity column chromatography as described previously (Suzuki et al., 1998
).
Antifungal activity.
A suspension of conidia of Trichoderma reesei was adjusted to 2·55x103 ml-1 and a paper disk placed on the centre of a potato dextrose agar plate was soaked with 40 µl of the suspension. After 24 h incubation at 30 °C, blank paper disks were placed around the T. reesei colony and solutions of various chitinases were added to these disks. The plates were incubated for approximately 12 h and inhibition of hyphal extension was evaluated by visual inspection.
Genomic DNA extraction and Southern hybridization.
Chromosomal DNAs of various Streptomyces species were extracted from the mycelia by the method described by Hopwood et al . (1985) with minor modifications. For Southern hybridization, restriction-enzyme-digested DNA was fractionated in a 0·7% agarose gel, transferred onto a nylon membrane (Hybond-N, Amersham) by the capillary method, and hybridized with a 32P- labelled chiC probe which included the entire catalytic domain of chitinase C.
PCR and nucleotide sequence determination of family 19 chitinases of Streptomyces species.
A portion of the genes encoding family 19 chitinases of various Streptomyces species was amplified by PCR. Primers for PCR were designed based on the regions of conserved amino acid sequences in the catalytic domains of chitinase C from S. griseus HUT6037 and plant family 19 chitinases. Forward and reverse primers corresponded to the amino acid sequences from Lys-134 to Ala-142 and from Ile-256 to Cys-262 of chitinase C, respectively. Amplified fragments were ligated with T-vector pT7Blue (Novagen) and maintained in E. coli JM109. Nucleotide sequences of amplified fragments in the T-vector were determined with an automated laser fluorescence sequencer (model 4000L; LI-COR). Sequencing reactions were done by using a Thermosequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham) according to the suppliers instructions with double- stranded templates. Nucleotide sequence data were analysed using the GENETYX system (Software Kaihatsu Co.).
SDS-PAGE.
SDS-PAGE in slabs was conducted as described by Ames (1974) , using the buffer system of Laemmli (1970)
. When necessary, renaturation of enzymes in polyacrylamide gels and detection of chitinase activity was performed as described previously (Watanabe et al., 1990
).
Enzyme and protein assay.
Chitinase activity was measured by a modification of Schales procedure (Imoto & Yagishita, 1971 ) with either colloidal chitin or soluble chitin as the assay substrate. One unit of chitinase activity was defined as the amount of enzyme that produced 1 µmol reducing sugar min-1. Protein concentration was measured by the Lowry method, using bovine serum albumin as the standard.
Chemicals.
Glycol chitin and colloidal chitin were prepared from powdered crab shell chitin purchased from Funakoshi Chemical Co. by the methods described by Yamada & Imoto (1981) and Jeuniaux (1966)
, respectively. Chitin EX (powdered prawn shell chitin) was purchased from Funakoshi Chemical Co. Regenerated chitin was prepared from chitosan 8B (80% deacetylated chitin) purchased from Funakoshi Chemical Co. as described by Molano et al. (1977)
. Soluble chitin was obtained from Yaizu Suisan Chemical Co. The degree of deacetylation and approximate molecular mass of the soluble chitin were 38·8% and 2030 kDa, respectively. Allosamidin was purified from Streptomyces sp. 1713 by extraction with methanol and then column chromatography with active carbon, Dowex-50 and SP-Sephadex C-25 by a method described previously (Sakuda et al. , 1986
).
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RESULTS |
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Chitinase C was extracted from the periplasmic space of BL21(DE3) cells and precipitated with ammonium sulfate (60% saturation). Chitinase C in the precipitated proteins was purified by hydroxylapatite column chromatography, as shown in Fig. 1 . The proteins were eluted from the column with 20 mM sodium phosphate buffer, pH 6·0, and the peak fractions containing chitinase C were collected and combined. The collected fraction exhibited a single protein band of chitinase C in SDS-PAGE analysis, with a size identical to that of chitinase C from S. griseus HUT6037, as shown in Fig. 1(b)
. The N-terminal amino acid sequence of the purified chitinase C was analysed and shown to be identical to that of chitinase C detected in the culture supernatant of S. griseus HUT6037. Recovery of the purified chitinase C from the periplasmic protein fraction was approximately 30%. Approximately 10 mg purified chitinase C was obtained from a 1 litre culture of E. coli BL21(DE3) carrying pGC02.
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The effects of pH and temperature on the activity of chitinase C were measured using colloidal chitin as the assay substrate. Chitinase C maintained almost the same level of activity from pH 4 to 8·5; it showed practically no activity at pH values below 2·5 or over 10·5. When measured at pH 6·0, maximum activity was observed at around 55 °C. The enzyme was stable at up to 55 °C during a 15 min incubation at pH 6·0.
The hydrolysing activity of chitinase C against various chitinous substrates was studied and compared with those of bacterial family 18 chitinases. As shown in Table 2, chitinase C of S. griseus HUT6037 exhibited the highest activity against glycol chitin among the tested chitinous substrates. It hydrolysed all other substrates much less efficiently: hydrolysing activity against colloidal chitin, for example, was one-tenth of that activity against glycol chitin. However, when compared with other bacterial chitinases of family 18, the specific hydrolysing activity of chitinase C was remarkably high against both soluble and insoluble chitin, including colloidal chitin, glycol chitin and powdered chitin (chitin EX).
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Detection of allosamidin-resistant chitinases in the culture supernatant of various Streptomyces species
S. griseus chitinase C produced in E. coli was not inhibited by allosamidin, whereas the bacterial family 18 chitinases were severely inhibited. This meant that family 19 chitinases similar to chitinase C could be detected specifically as chitinases which were active in the presence of allosamidin. Therefore, in order to study whether family 19 chitinase is unique to S. griseus HUT6037 or common to Streptomyces species, we first attempted to detect allosamidin-insensitive chitinases in the culture supernatants of various Streptomyces species.
Streptomyces species were grown for 72 h in the presence of 0·2% (w/v) colloidal chitin and proteins in the culture supernatants were collected and subjected to SDS-PAGE analysis. After renaturation of proteins in the polyacrylamide gel, chitinase activity was detected by zymogram analysis in the presence and absence of allosamidin. The presence of allosamidin during the course of zymogram analysis was expected to reduce the intensity of activity bands or erase the activity bands of family 18 chitinases on the agar replica of the SDS-PAGE gel. As shown in Fig. 3(a) , in the absence of allosamidin, several chitinase bands with a variety of sizes were detected in the culture supernatants of all Streptomyces species. On the other hand, as shown in Fig. 3(b)
, larger chitinase bands disappeared or were weakened drastically in the presence of allosamidin, and only one or two chitinase band(s) with sizes similar to that of chitinase C of S. griseus remained unaffected. The chitinase band in lane 1 of Fig. 3(b)
corresponds to chitinase C1 (27 kDa), a proteolytic derivative of chitinase C (32 kDa) lacking its N-terminal chitin-binding domain. These results strongly suggest that all Streptomyces species tested in this study produce family 19 chitinases with sizes similar to that of S. griseus chitinase C in addition to family 18 chitinases.
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DISCUSSION |
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To visualize the relationship among Streptomyces family 19 chitinases and plant family 19 chitinases in terms of amino acid sequence similarity, a phylogenetic tree was constructed (Fig. 7). To avoid the effects of deletions, the amino acid segments of family 19 chitinases corresponding to positions 53102 as indicated in Fig. 6
were used to make the sequence alignment and construct the tree. Class I/II chitinases and Streptomyces chitinases each formed a discrete cluster, whereas class IV chitinases were rather dispersed throughout the tree. The divergency of Streptomyces chitinases was somewhat lower than that among class I/II chitinases and markedly lower than that among class IV chitinases. Two possible evolutionary relationships of Streptomyces family 19 chitinases with plant chitinases can be imagined. One is that a common ancestral chitinase was already present prior to divergence of plant and bacteria, and family 19 chitinases then evolved independently in plants and bacteria. In this case, class IV type is the original form of family 19 chitinases. This idea does not seem to be likely because amino acid sequence similarities among Streptomyces family 19 chitinases and certain plant chitinases are extremely high. For example, the amino acid identity between the catalytic domain of S. griseus HUT6037 chitinase C (213 amino acids) and that of maize chitinase is 44%. The other possibility, which is more likely to be the case, is that family 19 Streptomyces chitinases were acquired from plants by horizontal gene transfer. Streptomyces species are important soil micro-organisms, and higher plants and Streptomyces share a common habitat in many places. In addition, some strains of Streptomyces are plant pathogens; S. scabies and S. ipomoeae included in the present study are examples of such Streptomyces. Such a close relationship, including direct interactions between Streptomyces and higher plants, would increase the chances for horizontal gene transfer from plants to Streptomyces. The high sequence similarity among Streptomyces family 19 chitinases and the limited distribution of family 19 chitinases in prokaryotic organisms appear to support the idea that the acquisition of family 19 chitinases by Streptomyces occurred relatively recently. Hamel et al . (1997)
suggested that classes I and IV were derived from a common ancestral sequence that predates the divergence of dicots and monocots. It is not clear whether the gene transfer event occurred before or after divergence of class IV and class I/II chitinases. More detailed evolutionary analysis will be required to clarify this point.
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The general occurrence of family 19 chitinases in Streptomyces was demonstrated in this study. Our latest results suggest the presence of family 19 chitinases in a few bacterial species in addition to Streptomyces (data not shown). This means that family 19 chitinases are relatively rare in prokaryotic organisms, but not restricted only to Streptomyces species. These chitinases are expected to have different properties in different prokaryotic organisms, including the strength and specificity of the antifungal activity. Therefore, prokaryotic organisms may be a good source of highly active chitinases and chitin-binding domains with respect to antifungal activity and efficiency of chitin degradation.
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ACKNOWLEDGEMENTS |
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Received 6 April 1999;
revised 9 September 1999;
accepted 16 September 1999.