Cloning of the Bacillus thuringiensis serovar sotto chitinase ( Schi ) gene and characterization of its protein

Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi) gene from Bacillus thuringiensis serovar sotto (Bt sotto) chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF) of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions) of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed.


Introduction
Chitin, a (1,4)-b-linked homopolysaccharide made up of N-acetylglucosamine residues, is absent from vertebrates and plants but is commonly found in the exoskeletons of insects and crustaceans as well as in fungi and some algae.Chitinase (EC 3.2.1.14)is a glycosyl hydrolase that catalyzes the hydrolytic degradation of chitin, and is found in a wide variety of organisms including bacteria, fungi, invertebrates, plants and animals (Goody, 1990), but the roles of chitinases in different organisms are diverse.Bacteria produce chitinases to digest chitin for use as a carbon and energy sources, while fungi produce this enzyme to modify the important cell wall component chitin and invertebrates require chitinases for the partial degradation of old exoskeletons.In plants, however, chitinases are part of the plants defense mechanisms against fungal pathogens.Some chitinolytic bacteria have been shown to be potential agents for the biological control of both phytopathogenic fungi and insect pests (Chernin et al., 1997;Sampson and Gooday, 1998).One of the most impor-tant insecticidal microbes used in biological control is Bacillus thuringiensis (Bt), a gram-positive, rod-shaped, spore-forming bacterium that forms insecticidal protein(s) during the stationary phase of its growth cycle.Insecticidal proteins, mainly Cry (crystal) proteins, play the leading role in controlling of insect pests.It has been reported that chitinases are widely distributed in Bt strains and that some of the chitinase-producing strains can enhance the insecticidal activity of Bt (Liu et al., 2002), although, as far as we know, only a few chitinase genes have yet been cloned (Thamthiankul et al., 2001;Barboza-Corona et al., 2003;Zhong et al., 2003;Lin and Guan, 2004) and furthermore, the synergistic effect of purified Bt chitinase and Cry proteins has still not been quantitatively demonstrated (Barboza-Corona et al., 2003).
It is known that B. thuringiensis serovar sotto (Bt sotto) has strong larvicidal activity against lepidopteran pests because of Cry proteins (Zhong et al., 2004).We cloned the cry1Aa13 gene from Bt sotto plasmid and expressed it in Escherichia coli, but expression of the cry1Aa13 gene product was low (Zhong et al., 2004).In order to further determine the synergistic action between the Bt sotto Cry1Aa13 protein and Bt sotto Schi chitinase, it is necessary to clone and sequence the Schi chitinase.This re-port deals with the cloning, sequencing and partial characterization of Bt sotto Schi gene.

DNA manipulation and cloning of the Bt sotto Schi gene
Touchdown PCR avoids the need for complicated optimization of the annealing temperature of the primers by using a 0.5-1 °C sequential decrease from a high annealing temperature in the first PCR cycle to a "touchdown" annealing temperature which is then used for several more cycles (Don et al., 1991).Touchdown PCR was carried out in a TGradient Thermocycler (Biometra) using the following program: 94 °C for 3 min, 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1.5 min, followed by 24 cycles at decreasing annealing temperatures in decrements of 0.5 °C per cycle, then 10 cycles of 1 min at 94 °C, 1 min at 52 °C, 1.5 min at 72 °C, and final extension at 72 °C for 10 min.The reaction mixture contained 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl 2 , 200 mM dNTPs, 0.2 mM of each primer, 1U Taq plus DNA polymerase (TaKaRa) and 30 ng of Bt sotto total genomic DNA, extracted according to Zhong et al. (2003), as template.Small-scale plasmid isolation, transformation, restriction enzyme digestion and agarose gel electrophoresis were performed by standard methods, i.e. 1% (w/v) agarose gel electrophoreses and purification using the Wizard SV Gel and PCR Clean-Up System (Promega), the resulting PCR product being ligated to the T-easy vector which was used to transform Escherichia coli DH5a competent cells with the Schi gene.The positive recombinant clone was screened and used for nucleotide sequencing.

Nucleotide sequence analysis
The Bt sotto Schi gene nucleotide sequence was determined at least once in each strand by the dideoxy chain termination method using an ABI 377 sequencer.DNA and deduced amino acid sequences were compared with those of other chitinase genes from GenBank, using both the BLAST (http://ncbi.nlm.nih.gov/BLAST/) and CLUSTAL W multiple sequence alignment tool.

Cloning and nucleotide sequencing of the Bt sotto Schi chitinase gene
We successfully cloned the Bt sotto Schi chitinase gene using touchdown PCR with primers P1 and P2.The Bt sotto Schi gene structural and flanking region sequence was deposited in the GenBank database under accession number AY129671.The manuscript has Schi gene region contains an open reading frame (ORF) with a base composition of 33.9% adenine and 28.4% thymine (A + T = 62.3%) and 19.7% guanine 18.0% cytosine (G + C = 37.7%) consisting of 2067 nucleotides encoding 688 amino acid residues with a deduced molecular weight of 75.75 Da and a theoretical isoelectric point of 5.74.The codon usage is shown in Table 1, from which it can be seen that because of the relatively high A + T content there is a preference for A and T at the third base position in the codons, the A:G ratio being 3.67:1 and the T:C ratio 3.08:1.
The putative promoter consensus region, spanning nucleotide positions 59 to 64 (TTGAGA, -35) and 79 to 84 (TTAATA, -10) of the Schi gene are identical to those of the chiA71 gene from B. thuringiensis serovar Pakistani (Thamthiankul et al., 2001), the chiA74 gene from B. thuringiensis serovar kenyae (Barboza-Corona et al., 2003), and the ichi gene from B. thuringiensis serovar israelensis (Zhong et al., 2003).The putative promoters also show obvious homology to the -35 (TTGACA) and -10 (TATAAT) consensus sequence for an E. coli promoter and Bacillus subtilis s A .Two stem-loop structures (one at 25 to 41 bp and the other at 69 to 94 bp from the TAG stop codon) of the putative transcriptional terminator were found at the 3' end, these structures which causing the polymerase to pause and subsequently cease transcription, similar results having been reported by us for the Bt ichi sequences (Zhong et al., 2003).

Amino acid sequence analysis of the Schi
The predicted 46 N-terminal amino acids of the Schi chitinase precursor exhibited a typical feature of the signal peptide characteristic of Gram-positive bacteria.There was a positively charged hydrophilic N-terminal segment ending in lysine with a net charge of +3, followed by a hydrophobic amino acid sequence.Cleavage of the signal sequence of the Schi chitinase precursor occurred at between A-46 and D-47.Interesting, the signal peptide may also be recognized by gram-negative bacterial and eukaryotic organisms according to the SignalP server (http://www.cbs.dtu.dk/services/SignalP/).We estimated the mature Schi chitinase molecular weight to be 70.50 kDa and the isoelectric point to be 5.55.Computer analysis of the deduced mature Schi chitinase amino acid sequence re-vealed that it consists of three discrete domains, an N-terminal catalytic domain, a fibronectin type III-like domain (FLD) and a chitin-binding domain (ChBD).

Discussion
According to our catalytic domain analysis, the Bt chitinase Schi gene we cloned and sequenced during this study seems to be the chiA gene encoding the chitinase precursor.The phenomenon of possible promoter showed homology with B. subtilis s A indicated that the expression of Schi take place during the vegetative (i.e.exponential) growth phase.The signal sequence of the Bt sotto Schi chitinase precursor contains an extremely long N-terminal hydrophilic segment, with the signal peptide cleavage in the Schi precursor occurring between amino acid residues A-46 and D-47.The signal peptide is the same length as that of the Bt ichi chitinase but longer than that of the chiA71 and chiA74 chitinases.
The high homology (97% identity) between the Bt sotto Schi chitinase and the B. cereus strain CH chiB chitinase (Mabuchi and Araki, 2001) supports the view that even though B. thuringiensis and B. cereus belong to different species they are closely related evolutionary.
The mature Bt sotto Schi chitinase seems to be composed of a glycosyl-hydrolase family 18 catalytic domain, a fibronectin type III-like domain and a chitin-binding domain.Chitinases are acid-base catalysts and from sequence comparison with B. circulans chitinase A1 (Watanabe et al., 1994), it seems that the catalytic residues acting as proton donors are the amino acid residues D-219, D-221 and E-223.Fibronectin is a multifunctional extracellular matrix and plasma protein that plays a significant role in cell adhesion.Fibronectin type III-like domains (FLD) have been found in chitinase, cellulases, a-amylase, and poly-3hydroxybutyrate (PHB) depolymerase.In the chitinbinding domain (ChBD) of many chitinase, the aromatic amino acids tryptophan (W) and tyrosine (Y) are highly conserved and may play a crucial role during binding to the pyranosyl rings of N-acetylglucosamine residues in chitin (Morimoto et al., 1997).In Bt sotto Schi chitinase we found that tryptophan and tyrosine residues (e.g.W-603, Y-607, W-624 and W-638) were well conserved.It is also known that Schi chitinases show homology with the cellulosebinding domain of some cellulases and some researchers consider that chitinases and these cellulases have common substrate-binding mechanisms (Morimoto et al., 1997).The work presented in this paper has partially elucidated the role of each domain but the structure of the Bt sotto Schi chitinase is still not fully understood because of the limitations inherent in attempting to characterization of enzyme from amino acid sequences.
*Amino acid or punctuation one-letter code in parenthesis.Nr: Number of residues.