Molecular characterization of a novel chitinase ... - Wiley Online Library

19 downloads 1769 Views 359KB Size Report
cleavage site is presumably between A-34 and D-35. (Simomen and Palva 1993; Nielson et al. 1997). The deduced amino acid sequence FDGVDLDWE starting ...
Journal of Applied Microbiology 2005, 99, 945–953

doi:10.1111/j.1365-2672.2005.02639.x

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki F. Driss1, M. Kallassy-Awad2, N. Zouari1 and S. Jaoua1 1

Laboratory of Biopesticides, Centre of Biotechnology of Sfax, Sfax, Tunisia, and 2Faculty of Sciences Saint-Joseph University. Riad El Solh, Beirut, Lebanon

2004/1067: received 14 September 2004, revised 2 March 2005 and accepted 5 March 2005

ABSTRACT F . D R I S S , M . K A L L A S S Y - A W A D , N . Z O U A R I A N D S . J A O U A . 2005.

Aims: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. Methods and Results: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. Conclusions: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. Significance and Impact of the Study: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase structure-function relation. Keywords: Bacillus thuringiensis, chitibiosidase, cloning, expression, sequencing.

INTRODUCTION The chitinases, enzymes which catalyse the conversion of chitin (a linear homopolysaccharide of b-1, 4-N-acetylglucosamine) to its monomeric components, are widely distributed in nature (Gooday 1990). They are produced by a large variety of chitin-degrading organisms including bacteria, fungi, insects, plants and animals (Gooday 1990). They play an important role in normal life cycle functions such as morphogenesis and cell division, or in defence against pathogens. The Gram-positive bacterium Bacillus thuringiensis is used worldwide as a biological pest control agent acting on susceptible insects by producing during sporulation parasporal crystals containing delta-endotoxins (Ho¨fte and Whiteley 1989). Moreover, it is able to produce, under Correspondence to: Samir Jaoua, Laboratoire des Biopesticides, Centre de Biotechnologie de Sfax, B. P. K, 3038, Sfax, Tunisia (e-mail: [email protected]).

ª 2005 The Society for Applied Microbiology

adequate conditions, several biologically active molecules such as bacteriocins, insecticidal proteins and hydrolytic enzymes among which are chitinases. Indeed, B. thuringiensis is known to be chitin-degrading micro-organism producing chitinases to convert chitin into compounds that can serve as carbon and nitrogen sources. As chitin is the major structural component of fungal cell walls and exoskeletons and peritrophic membranes of insects (de Barjac 1990), pathogens have to cross the chitin-rich barrier to exert their virulence. Although the precise mechanism through which chitinases exert their effect on insects is still hypothetical, evidence of its synergistic effect with B. thuringiensis endotoxins was reported (Sneh et al. 1983; Regev et al. 1996; Sampson and Gooday 1998; Wiwat et al. 2000; Liu et al. 2002). As part of studies aimed to test and understand the synergistic effect of B. thuringiensis chitinases and Cry proteins, few B. thuringiensis chi genes have been cloned and sequenced (Thamthiankul et al. 2001; Arora et al. 2003;

946 F . D R I S S ET AL.

Barboza-Corona et al. 2003). The overexpression of B. thuringiensis endogenous chi genes and use of its product should contribute to the improvement of the insecticidal activity of commercial formulations. In the present work, we describe the selection of B. thuringiensis high chitinaseproducing strain, and the characterization and the heterologous expression of a novel chitinase encoding gene.

MATERIAL AND METHODS Bacterial strains, plasmids and media All the 256 B. thuringiensis strains consecutively named from BUPM1 to BUPM256 and used for the screening of chitinase activity, were isolated by S. Jaoua, N. Zouari and S. Tounsi (unpublished data) in the Laboratory of Biopesticides of the Centre of Biotechnology of Sfax (Tunisia). BUPM255 is a B. thuringiensis subsp. kurstaki strain, which was isolated in Byblos (Lebanon). pMOSBlue Blunt Ended Cloning kit (Amersham, Paris, France) was used for the construction of the plasmids pMOSchi255 and pMOSORFchi255, carrying, respectively, the entire chi255 with its regulatory regions and the corresponding open reading frame (ORF). Plasmid pBADchi255 was constructed in this work by cloning the ORF of chi255 in pBAD33-GFPuv (Clontech, Palo Alto, CA, USA; GenBank accession no. U62637). LB broth (Sambrook et al. 1989) was used for growth of B. thuringiensis and Escherichia coli. NYSMC is NYSM medium (Li and Yousten 1975) supplemented with 1% colloidal chitin. It is a chitinaseinducing medium used for the investigation of chitinase production by B. thuringiensis. Screening of chitinase-producing strains of B. thuringiensis and selection of an over-producing one Screening of B. thuringiensis chitinase-producing strains was made according to the method of Barboza-Corona et al. (1999) with slight modifications. A single colony from an overnight-incubated plate containing solid LB medium, was used to inoculate by sticking Castan˜eda’s solid medium supplemented with 2 g l)1 casaminoacids and 0Æ6% colloidal chitin (w/v). Plates were incubated at 30C for 2 days, and then transferred to 37C for a week, a suitable period for the appearance of clearing zones. Translucent haloes because of chitin degradation appear around the chitinase-producing colonies. High chitinase-producing strains were tested for their antifungal activity. Strains were cultured in NYSM medium for 72 h and the supernatants recovered by centrifugation for 10 min at 17 500 ·g were poured in wells obtained by punching solid LB medium, supplemented with streptomycin (100 lg ml)1) used to avoid bacterial

growth and containing Aspergillus niger spores. The plates were incubated at 30C. The antifungal activity was estimated by evaluation of the inhibition zones surrounding the colonies. A 0Æ6% colloidal chitin-containing medium was used to assess the chitinase activity. Culture conditions The strain BUPM255 was cultured in the chitinaseinducing medium (NYSMC), with an initial OD 600 of 0Æ15. The pH was adjusted to 7 before sterilization at 121C for 20 min. The 1000 ml flasks, containing 100 ml of culture medium, were incubated for 146 h at 30C in a rotary shaker set at 200 rev min)1. Total biomass, spores and chitinolytic activities were followed at various incubation periods indicated in the Results section. The direct plate count technique consisting in determining colony forming units was used for the estimation of viable cell and spore numbers. Appropriate culture dilutions were plated on a solid LB medium and incubated at 30C overnight. For spore counts, samples were heated at 80C for 10 min before plating. Assays for chitinase activity and specificity determination Supernatants from B. thuringiensis cultures were harvested by centrifugation at 17 500 ·g for 10 min at various periods indicated with results. Samples were stored at )20C until used for chitinase assay. The reaction mixture consisted of equal volumes (400 ll) of supernatant samples and 0Æ5% colloidal chitin in 50 mmol l)1 acetate buffer, pH 7Æ0. The reaction was performed at 37C for 1 h. The remaining chitin was removed by centrifugation at 17 500 ·g for 10 min. Chitinase activity was evaluated by monitoring the reduction of 3,5-dinitrosalicylic acids by action of N-acetylglucosamine (NAG) issued from hydrolysis of chitin (Miller 1959). The absorbance in the reaction medium was recorded at 550 nm. Readings were compared with a standard curve prepared with a series of dilutions of NAG (from 0 to 2Æ5 mmol l)1). One unit was defined as the amount of enzyme that catalyses the release of 1 lmol of NAG per minute of incubation at 37C. Specificity of Chi255 activity to hydrolyse chitin was determined by the HPLC analysis of the end products of Chi255 expressed in E. coli hydrolysis reaction. Twofold dilutions of 200 ll of recombinant extract proteins were reacted at 37C with 400 ll of either 2 g l)1 chitobiose or 0Æ5% colloidal chitin in 50 mmol l)1 acetate buffer, pH 7Æ0. The reaction performed with colloidal chitin was carried out for different periods (0, 5, 10, 25 min and 2 h) and that performed with chitobiose was carried out for 2 h only. Reaction was stopped by adding 1/6 volume TCA 10% (w/v). The remaining chitin was removed by centrifugation at 17 500 ·g for 10 min. The supernatant containing

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x

NEW CHITINASE OF B. THURINGIENSIS

the hydrolysis products was filtered and passed through a polypore H column (250 · 4Æ6 mm) that had been equilibrated with H2SO4 (0Æ01 mol l)1) at a flow rate of 0Æ3 ml min)1. Identification of chitin oligomers was performed by UV absorbance at 205 nm, giving separated peaks. Purified chitobiose and NAG (both from Sigma) were used as controls. Cloning and sequencing of chi255 from BUPM255 The putative chitinase gene designated chi255 was amplified by PCR using chromosomal DNA isolated from strain BUPM255 as template, Taq DNA polymerase (Amersham) and primers CHI0 (5¢AAGCTTTTTCCTCCCATACCAA3¢) and CHI4 (5¢GATGTTTTAATTTTAGACGAACGGAC3¢) complementary to the regions located, respectively, at 255 bp upstream and 627 bp downstream from the ORF. Primers CHI1 (5¢ATGGCTATGAGGTCTCAAAAATTCA3¢) and CHI3 (5¢CTAACAGGTGACTATCTTCTTATAT3¢) located, respectively, at the initiation codon (ATG) and 288 bp downstream from the stop codon (TAG) were used for the PCR amplification and the cloning of the ORF of chi255. These primers were designed on the basis of published sequence data of chitinase chiA71 of B. thuringiensis (GenBank accession number: BTU89796). The plasmid DNA of positive clones, respectively, designated pMOSchi255 and pMOSORFchi255, were purified and subjected to sequencing with both Thermosequenase Cycle Sequencing kit (Amersham) and ABI model 3100 automated sequencer. The sequence of chi255 is registered under the accession number AJ635226 at EMBL nucleotide sequence database. Sequence comparisons with other chitinases obtained from GenBank were performed using BLAST (Altschul et al. 1997) and CLUSTALW, version 1Æ82 (Thompson et al. 1994). PHYLIP program (http://evolution.genetics.washington.edu/phylip.html) was used to build the phylogenetic tree.

947

to a final concentration of 1% (w/v). After a 2-h incubation, the culture was centrifuged at 6000 ·g for 10 min and the pellet was washed twice with sonication buffer (phosphate buffer 50 mmol l)1, pH 7Æ0, NaCl 300 mmol l)1). Cells were disrupted with sonication buffer supplemented with 5 g l)1 lysozyme by three rounds of sonication using model Vibro-cellTM 72405 sonicator (Bioblock Scientific, Illkirch, France). Unbroken cells and debris were removed by centrifugation at 6000 ·g for 10 min. For the chitinase activity detection, 100 ll of the supernatant were poured in the wells of chitin-containing plates; and the latter were incubated at 37C.

RESULTS Chitinase production Among 256 B. thuringiensis strains, isolated in the laboratory and screened for chitinolytic activity, 87% exhibited different sizes of zones of degradation of colloidal chitin on solid chitinase-inducing medium evidencing differences in their chitinolytic activities (data not shown). The strain BUPM255 was selected among those giving diameters of clearing zones higher than 2 cm, on such a medium. It showed the highest antifungal activity performed against A. niger (Fig. 1), and therefore was (a)

1

2

1 cm (b)

Heterologous expression of chi255 in E. coli The overexpression of chi255 was achieved by cloning its ORF downstream from the very strong promoter (inducible by arabinose) of the E. coli vector pBAD33-GFPuv. Targeted digestion products of the latter with SacI and NheI, and pMOSORFchi255 with SacI and XbaI were purified using purification kit (Qiagen, Hilden, Germany), ligated and introduced in E. coli by chemical transformation. An overnight culture of a transformant in LB broth supplemented with 60 lg ml)1 ampicillin was used for the inoculation of a 50 ml fresh medium with an initial OD 450 of 0Æ05. The culture was incubated at 37C until the OD 450 reached 0Æ6, when induction of chi255 expression, from the pBAD promoter, was performed by addition of L-arabinose

1 2

1 cm

Fig. 1 Chitinase activity of BUPM255. Wells were filled up with 100 ll of 72-h culture supernatant of Bacillus thuringiensis strain BUPM255. (a) Clearing zones of colloidal chitin: 1, non-producing strain used as a negative control; 2, BUPM255. (b) Antifungal activity against Aspergillus niger: 1, non-producing strain used as a negative control; 2, BUPM255

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x

948 F . D R I S S ET AL.

40 120 30 90 20

60

10

30 0

0

24

48 72 96 Culturing time (h)

120

chitinase activity (mU ml–1)

Total cell and spore count (107 CFU ml–1)

150

0 144

Fig. 2 Kinetic of chitinase production by Bacillus thuringiensis strain BUPM255 in a chitinase-inducing medium. s, total cell count; n, spore count; d, chitinase activity

chosen for further studies. First, secretion of chitinase was followed by culturing the strain BUPM255 in the NYSMC medium. The results in Fig. 2 showed that the chitinase production increased steadily during the vegetative growth to reach a maximum of 28Æ31 mU ml)1 after 70 h of incubation. Then, a sharp decrease of the chitinase activity in the medium was observed, which might be attributed to proteolytic activities of released proteases (Zouari and Jaoua 1999).

Fig. 3 Comparison of the amino acid sequence encoded by chi255 with other chitinases. Chitinases were listed by their accession numbers corresponding to the amino acid sequence: Chitinase Chi255 encoded by chi255 from Bacillus thuringiensis subsp. kurstaki BUPM255 (CAG25670), chitinase CW from Bacillus cereus (AAM48520), chitinase from B. cereus (NP_830268), chitinase from B. thuringiensis subsp. kenyae (AAL17867), chitinase from B. thuringiensis subsp. israelensis (AAM88400), chitinase from B. thuringiensis subsp. kurstaki (AAO34713) and chitinase from B. thuringiensis subsp. pakistani (AAB58579). Only the regions containing differences brought about by Chi255 are presented. The vertical downward arrows indicate amino acid positions. The black boxes represent the residues showing variations

Cloning and nucleotide sequence analysis of chi255 Due to the interest provided by the chitinolytic activity exhibited by the B. thuringiensis strain BUPM255, one chitinase gene named chi255 was subjected to characterization and comparison with the few published genes. The PCR fragment containing the ORF of chi255 flanked by its expression regulatory upstream and the downstream regions was cloned in the vector pMOSBlue. The sequencing of the corresponding fragment was performed. The ORF of chi255 consists of 2031 nucleotides encoding a protein of 676 amino acids with a calculated molecular mass of 74 kDa. A potential ribosome-binding (Shine-Dalgarno) sequence (5¢GAAAGG3¢) precedes 5 bp upstream from the initiation codon. Putative promoter consensus sequences are identical to those reported for chiA71 of B. thuringiensis subsp. pakistani (Thamthiankul et al. 2001) and for chiA74 of B. thuringiensis subsp. kenyae (Barboza-Corona et al. 2003). The )10 sequence (5¢TTAATA3¢) is 164 bp upstream from the initiation codon and is 14 bp distant from the )35 sequence (5¢TTGAGA3¢). Downstream from the TAG terminator codon, a possible transcription terminator was found.

Chi255 amino acid sequence analysis Alignment made by CLUSTALW (Fig. 3) revealed that the sequence of Chi255 is different at several sites from those of five other chitinases which share the highest similarity shown by alignment using BLAST program and that of ChiA71 which was reported to be an exochitinase (Thamthiankul et al. 2001). The sequence similarity of Chi255 (CAG25670) was 98% with chitinase from B. thuringiensis subsp. kurstaki (AAO34713), 97% with chitinase from B. thuringiensis subsp. kenyae (AAL17867), chitinase CW from Bacillus cereus (AAM48520) and chitinase from B. cereus (NP_830268), 96% with chitinase from B. thuringiensis subsp. israelensis (AAM88400), and 71% with chitinase from B. thuringiensis subsp. pakistani (AAB58579). Although chitinase Chi255 has the same number of amino acid residues as the endochitinase ChiA74 of B. thuringiensis subsp. kenyae (Barboza-Corona et al. 2003), it shares 97% identity with it. Indeed, residues I-257, V-503, I-550, T-607 and N-670 in the sequence of ChiA74 were substituted, respectively, by the residues V, E, V, N and K, in that of Chi255 at the same positions. Moreover, the deduced amino acid sequence DHGLMLPNR of Chi255 starting at D-121

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x

NEW CHITINASE OF B. THURINGIENSIS

AAM48520 NP_830268 CAG25670 AAB58579 AAL17867 AAO34713

949

chitinases, as reported for E-315 of Serratia marcescens ChiA (Perrakis et al. 1994). Three conserved aromatic amino acids (one W and two Y) characteristic of a fibronectin-like domain (FLD) were found twice at the middle of the Chi255 amino acid sequence, which lead to suppose the presence of two FLD type III: FLD1 from K-350 to Y-435 and FLD2 from I-479 to T-574 by analogy with those reported for ChiA74 of B. thuringiensis subsp. kenyae at the same positions (Barboza-Corona et al. 2003). At the C-terminal region corresponding to the fourth domain of Chi255, the residues W-591, Y-595 and W-626 were also present in chitin binding domains (CBD) of chitinases and C-termini of bacterial cellulases (Thamthiankul et al. 2001).

AAM88400

Fig. 4 Phylogenetic tree of chitinase Chi255 from Bacillus thuringiensis subsp. kurstaki (CAG25670) in relation to chitinase CW from Bacillus cereus (AAM48520), endochitinase from B. cereus (NP_830268), endochitinase from B. thuringiensis subsp. kenyae (AAL17867), chitinase from B. thuringiensis subsp. israelensis (AAM88400), chitinase from B. thuringiensis subsp. kurstaki (AAO34713) and chitinase from B. thuringiensis subsp. pakistani (AAB58579). The tree was constructed by using PHYLIP program

was only 11% similar to the corresponding sequence in ChiA74. It is worth noticing that this sequence is 88% identical to the corresponding one in the exochitinase ChiA71 of B. thuringiensis subsp. pakistani (Thamthiankul et al. 2001). Furthermore, Chi255 and ChiA71 have both a residue V at the position 550 whereas the corresponding residue in the sequence of ChiA74 is I. In addition, the phylogenetic tree showed high homology between Chi255 and ChiA71 as they have the same root (Fig. 4). However, the analysis of amino acid sequence of Chi255 revealed different domains. At the N-terminus of the deduced amino acid sequence of the premature Chi255, a typical signal peptide composed of 34 amino acids was evidenced (Fig. 5). As B. thuringiensis is a Gram-positive bacterium, the cleavage site is presumably between A-34 and D-35 (Simomen and Palva 1993; Nielson et al. 1997). The deduced amino acid sequence FDGVDLDWE starting at the F-203 from the start codon is similar to the active site motif of enzymes in family 18 of glycosyl hydrolases (Henrissat and Bairoch 1993). The amino acid residues D-207, D-209 and E-211 of Chi255 which correspond perfectly to those reported for ChiA74 of B. thuringiensis subsp. kenyae at the same positions (Barboza-Corona et al. 2003) and ChiA71 of B. thuringiensis subsp. pakistani at the positions 204, 207 and 209, respectively (Thamthiankul et al. 2001), might play the essential role for the chitinase activity as it was shown by Watanabe et al. (1993) working on chitinase ChiA1 of B. circulans. Moreover, the amino acid E-211 seems to be involved in the acid-base catalysis by

Investigation of chi255 functionality and specificity of the encoded chitinase In order to prove the functionality of chi255 and to determine the specificity of the encoded chitinase, chi255 was cloned and expressed downstream from a very strong promoter of the E. coli vector (pBAD33-GFPuv). Expression of chi255 in E. coli was proved using colloidal chitincontaining media. Colloidal chitin degradation zone was observed with the crude preparation of Chi255 expressed in E. coli (pBADchi255) under inducing conditions (Fig. 6) demonstrating that the new chitinase gene of BUPM255 is functional. No activity was observed under non-inducing conditions. Investigation of the Chi255 specificity was performed by the HPLC analysis. When colloidal chitin was used as substrate for Chi255, chromatograms of HPLC analysis of soluble products showed that peak corresponding to dimeric units (chitobiose ¼ (NAG)2) appeared after 5 min of the enzymatic reaction. The surface of the peak continued to increase with no apparition of others (Fig. 7a). This could evidence that the reaction products did not include detectable amounts of monomers (NAG) and oligomers readily separable on HPLC columns (Brunner et al. 1998). As NAG was not a principal product of chitin hydrolysis, Chi255 was assayed on (NAG)2 which was not cleaved into NAG (Fig. 7c). These results confirm that Chi255 should be a chitinase that releases chitobiose as a reaction end product of chitin hydrolysis. DISCUSSION Regardless of the number of previous studies dealing with the enhancement of the biological insecticidal activity of B. thuringiensis by addition of exogenous chitinases (Sneh et al. 1983; Regev et al. 1996; Tantimavanich et al. 1997; Sampson and Gooday 1998; Wiwat et al. 2000) and the role of B. thuringiensis endogenous ones in such insecticidal activity (Sampson and Gooday 1998; Thamthiankul et al. 2001; Liu et al. 2002), few reports dealt with the cloning and

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x

950 F . D R I S S ET AL.

Fig. 5 Nucleotide sequence (EMBL accession number: AJ635226) and deduced aminoacid sequence of chi255. The underlined ATG and TAG correspond respectively to the start and stop codons. The secretion signal peptide has been italicized. , CD; j, FLD1; (, FLD2; , CBD; , codon and its corresponding residue specific to chi255

the sequencing of B. thuringiensis chitinase encoding genes (Thamthiankul et al. 2001; Arora et al. 2003; BarbozaCorona et al. 2003). In this report, chi255 was cloned by PCR and sequenced from a B. thuringiensis subsp. kurstaki strain named BUPM255, and both the nucleotide and the deduced amino acid sequences were analysed. The putative corresponding promoter showed homology with the deltaendotoxin encoding gene cry3A of B. thuringiensis (Agaisse and Lereclus 1995) that is active during the vegetative growth phase. This corroborates well with the maximal production of chitinase activity during the vegetative growth of BUPM255. The analysis of the deduced amino acid sequence of Chi255 showed that the mature enzyme, resulting after the cleavage of the signal peptide, was

composed of four domains. The first domain at the N-terminal region had sequence similarity with the catalytic domain (CD) of chitinases belonging to family 18 of the glycosyl hydrolases by involving an active site motif typical of this family (Henrissat and Bairoch 1993). In the highly conserved region of this domain, D-207, D-209 and E-211 might include the proton-donor site, essential to hydrolyse the biopolymer (chitin), by homology with those reported for ChiA74 (Barboza-Corona et al. 2003). At the middle region of the mature enzyme, two FLD domains were found. They contained the conserved aromatic residues, typical of fibronectin (type III). Such domains, found in enzymes that degrade different types of insoluble polymers (Candussio et al. 1990; Meink et al. 1991; Thamthiankul

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x

NEW CHITINASE OF B. THURINGIENSIS

1

1

12

t = 0 min

t = 5 min

12

12

951

12

2

12 3

t = 10 min

t = 25 min

12

t=2 h 12

Fig. 6 Chitinase activity of Escherichia coli (pBADchi255) recombinant strain, evidenced on colloidal chitin plate. Wells were filled up with crude protein extracts from cells cultured, 1: without induction, 2: with induction by L-arabinose

et al. 2001) such as bacterial chitinases, cellulases and amylases, were considered to be related to the substrate attachment (Barboza-Corona et al. 2003). The last domain located at the C-terminus was defined as CBD. Its sequence was similar to the CBDs of other chitinases. The aromatic amino acids residues W-591, Y-595 and W-626 were well conserved. Their presence in the CBDs, cellulase-binding domains and the corresponding domains in xylanases indicates that they are essential for the hydrophobic binding of the enzyme to its substrate (Morimoto et al. 1997). Furthermore, on the basis of published chi gene sequences and the corresponding deduced amino acid ones, novelty of chi255 was confirmed. In fact, the sequence of chi255 is different at several sites from those of five other chi genes that share the highest similarity by alignment using BLAST program. Regarding the localization of some variant residues, like E-503, V-550 in FLD2 and N-607 in CBD, differences in binding to chitin are expected and subsequently in the hydrolysis activity of Chi255 compared with those of other chitinases. Deep investigation of such effects should be performed. The results obtained by the HPLC analysis of the end-products of Chi255 reaction suggest that a Chi255 exo-type action was splitting (NAG)2 units from the non-reducing end. This chitinolytic activity was named chitiobiosidase by Tronsmo and Harman (1993), which is more precise than exochitinase, used to describe the activity of enzymes that release monomeric units from chitin (Robbins et al. 1988). Actually, Chi255 was expected

Standards

t = 0 min

t=2 h

Fig. 7 Chromatograms of the HPLC analysis of end products hydrolysis reactions of Chi255 (expressed in Escherichia coli). (a) Colloidal chitin used as substrate; (b) standards: 1, peak coming from the reaction buffer; 2, chitobiose 0Æ5 mmol l)1; 3, NAG 0Æ1 mmol l)1; (c) chitobiose used as a substrate

to have an endo-type action as it shared the same number of amino acid residues with ChiA74 that was reported to be an endochitinase (Barboza-Corona et al. 2003). But our results are more consistent with an exo-type action. This difference in the chitinase activity with ChiA74 might be a consequence of the presence in Chi255 of a unique sequence, starting at D-121 close to the CD, in addition to other differences at positions 257, 503, 550, 607 and 670, elsewhere. This unique sequence was only 11% similar to the corresponding one in ChiA74, but 88% similar to the contiguous one in ChiA71 reported to be an exochitinase (Thamthiankul et al. 2001). Moreover, Chi255 and ChiA71 have the same residue (V) at position 550 of Chi255 that is different from the corresponding one (I) in most of the other chitinases reported to be endochitinases. The phylogenetic tree supports our results by proving the homology of Chi255 to the exochitinase ChiA71. All these results corroborate

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x

952 F . D R I S S ET AL.

with the exo-type action of Chi255, and would require a further investigation of the chitinase structure-function relation. ACKNOWLEDGEMENTS We thank Mrs Najeh Belguith-Ben Hassen and Mr Hedi Issaoui for their technical assistance. We extend our thanks to Pr. Jamil Jaoua, an English teacher at the Sfax Faculty of Science, for kindly accepting to proofread this manuscript. This work was supported by grants from Tunisian Ministe`re de la Recherche Scientifique et de la Technologie et du De´veloppement des Compe´tences, and from the Third World Academy of Sciences (TWAS). REFERENCES Agaisse, H. and Lereclus, D. (1995) How does Bacillus thuringiensis produce so much insecticidal crystal proteins?. J Bacteriol 177, 6027– 6032. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402. Arora, N., Ahmad, T., Rojagopal, R. and Bhatnagar, R.K. (2003) A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochem Biophys Res Commun 307, 620–625. Barboza-Corona, J.E., Contreras, J.C., Velazquez-Robledo, R., Bautista-Justo, M., Gomez-Ramirez, M., Cruz-Camarillo, R. and Ibarra, J.E. (1999) Selection of chitinolytic strains of Bacillus thuringiensis. Biotech Lett 21, 1125–1129. Barboza-Corona, J.E., Nieto-Mazzocco, E., Vela’zquez-Robledo, R., Salcedo-Harnandez, R., Bautista, M., Jime’nez, B. and Ibarra, J.E. (2003) Cloning, sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis. Appl Environ Microbiol 69, 1023– 1029. de Barjac, H. (1990) Characterization and prospective view of Bacillus thuringiensis israelensis. In Bacterial Control of Mosquitoes and Blackflies ed. de Barjac, H. & Sutherland, D.J. pp. 10–15. Rutgers: Rutgers University Press. Brunner, F., Stintzi, A., Fritig, B. and Legrand, M. (1998) Substrate specificities of tobacco chitinases. Plant J 14, 225–234. Candussio, A., Schmid, G. and Bock, A. (1990) Biochemical and genetic analysis of a maltopentose-producing amylase from an alkaliphilic Gram-positive bacterium. Eur J Biochem 191, 177–185. Gooday, G.W. (1990) The ecology of chitin decomposition. Adv Microb Ecol 11, 378–430. Henrissat, B. and Bairoch, A. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293, 781–788. Ho¨fte, H. and Whiteley, H.R. (1989) Insecticidal proteins of Bacillus thuringiensis. Microbiol Rev 53, 242–255. Li, E. and Yousten, A.A. (1975) Metalloprotease from Bacillus thuringiensis. Appl Microbiol 30, 354–361. Liu, M., Cai, Q.X. Liu, H.Z., Zhang, B.H., Yan, J.P. and Yuan, Z.M. (2002) Chitinolytic activities in Bacillus thuringiensis and their

synergistic effects on larvicidal activity. J Appl Microbiol 93, 374– 379. Meink, A., Braam, C., Gilkes, N.R., Kiburn, D.G., Miller, R.C. and Warren, E.A.J. (1991) Unusual sequence organization in CenB. An inverting endoglucanase from Cellulomonas fimi. J Bacteriol 173, 308–314. Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31, 426–428. Morimoto, K., Karita, S., Kimura, T., Sakka, K. and Ohmya, K. (1997) Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the function of novel cadherin-like domains and a chitin-binding domain. J Bacteriol 179, 7306–7314. Nielson, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1–6. Perrakis, A., Tewes, I., Dauter, Z., Oppenheim, A.B., Chet, I., Wilson, K.S. and Vorgias, C.E. (1994) Crystal structure of a bacterial chitinase at 2Æ3 A resolution. Structure 2, 1169–1180. Regev, A., Keller, M., Strizhov, N., Sheh, B., Prudovsky, E., Chet, I., Ginzberg, I., Koncz-Kalman, Z. et al. (1996) Synergistic activity of a Bacillus thuringiensis d-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae. Appl Environ Microbiol 62, 3581–3586. Robbins, P.W., Albright, C. and Benfield, B. (1988) Cloning and expression of a Streptomyces plicatus chitinase (chitinase- 63) in Escherichia coli. J Biol Chem 263, 443–447. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning. A Laboratory Manual, 2nd edn. NY: Cold Spring Harbor Laboratory, Cold Spring Harbor. Sampson, M.N. and Gooday, G.W. (1998) Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects. Microbiology 144, 2189–2194. Sneh, B., Schuster, S. and Gross, S. (1983) Improvement of the insecticidal activity of Bacillus thuringiensis var. entomocidus on larvae of Spodoptera littoralis (Lepidoptera–Noctuidae) by addition of chitinolytic bacteria, a phagostimulant and a UV-protectant. Z Angew Entomol 96, 77–83. Simomen, M. and Palva, I. (1993) Protein secretion in Bacillus species. Microbiol Rev 57, 109–137. Tantimavanich, S., Pantowatana, S., Bhumiratana, A. and Panbangred, W. (1997) Cloning of a chitinase gene into Bacillus thuringiensis subsp. aizawai for enhanced insecticidal activity. J Gen Appl Microbiol 43, 31–37. Thamthiankul, S., Suan-Nday, S., Tantimavanich, S. and Panbangred, W. (2001) Chitinase from Bacillus thuringiensis subsp. pakistani. Appl Microbiol Biotechnol 56, 395–401. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673– 4680. Tronsmo, A. and Harman, G.E. (1993) Detection and quantification of N-Acetyl-B-D-glucosaminidase, chitobiosidase, and endochitinase in solution and on gels. Anal Biochem 208, 74–79. Watanabe, T., Kobori, K., Miyachita, K., Fujii, T., Sakai, M., Uchida, M. and Tanaka, H. (1993) Identification of glutamic acid 204 and

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x

NEW CHITINASE OF B. THURINGIENSIS

aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J Biol Chem 268, 18567– 18572. Wiwat, C., Thaithanun, S., Pantuwatana, S. and Bhumiratana, A. (2000) Toxicity of chitinase-producing Bacillus thuringiensis sp. kurstaki HD-1 toward Plutella xylostella. J Invertebr Pathol 79, 270– 277.

953

Zouari, N. and Jaoua, S. (1999) Production and characterization of metalloproteases synthesized concomitantly with delta-endotoxin by Bacillus thuringiensis subsp. Kurstaki strain grown on gruel-based media. Enzyme Microb Technol 25, 364–371.

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 945–953, doi:10.1111/j.1365-2672.2005.02639.x