| Chitinases, which can hydrolyze chitin, are potentially useful in several areas such asindustry, medicines, and biological control in agriculture. Bacillus thuringiensis (Bt),which is used worldwide, can produce chitinase into the culture media. Theexpression and application of Bacillus thuringiensis (Bt) chitinase genes have beenextensively investigated. However, little information is available regarding theregulation of chitinase gene expression in Bt. In this study, the characteristics ofchitinase synthesis in Bt were investigated and the genetic elements in the regulatoryregion of chitinase genes were identified.To reveal the characteristics of chtinase synthesis in Bt,77Bt strains were grownin the medium with or without chitin and the chitinase activities of the cultures werequantitatively measured. The results showed that all tested B. thuringiensis strainscould essentially produce chitinase without chitin. After induced, the chitinolyticactivity of41%tested strains had no obvious response to the inducer, whereas56%strains increased in different degree. The efficiency of chitin induction was differentfrom each other while the amount of chitinase in most of the strains did not increasemarkedly after added inducer. To find out the effect of glucose on synthesis ofchitinase, four Bt strains were investigated to find out the relationship between chitininduction and glucose suppression on chitinase synthesis. The investigationdemonstrated that the glucose inhibited the efficiency of chitin induction. But theglucose could not inhibit the basal expression of chitinase. The regulation of synthesisof chitinase in Bacillus thuringiensis was polymorphism. Among the77Bt strains,the strain Bt75showed the most efficiency of chitin induction with the chitinaseactivity increased approximately9.7-fold.To investigate the regulation of chitinase gene in Bacillus thuringiensis, a shuttlepromoter-probe vector was constructed. While the Bt75show no β-galactosidaseactivity, the thermostable β-galactosidase gene bgaB of B. stearothermophilus was selected as the reporter. The construction of promoter-probe vector was started fromthe shuttle vector pHT315, which was used widely in Bt. The fragment containing thepromoter-less bgaB gene preceded by the translational stops and the modifiedribosome binding site was ligated to the vector pHT315to construct the plasmidpHT-bgaB, in which the bgaB and lacZ genes were transcribed in the oppositedirections. Initial expression studies of pHT-bgaB showed low but significantexpression of β-galactosidase in E. coli DH5α and Bt75, even in the absence of anupstream promoter. To prevent possible read-through into the promoterless cassette,the rrnBT1T2transcriptional terminators of E. coli were inserted upstream of thebgaB gene to generate plasmid pCB. Transformants of E. coli and Bt containing thevector pCB did not exhibit β-galactosidase activity. This suggested that the vectorpCB can be used to study the functions of promoters in Bacillus.Using the plasmid pCB, the chiA promoter was subjected to deletion analysis todetermine the minimal region required for stimulating chiA gene expression and toidentify other cis-acting elements in this region. The results showed that5′deletion to-116and3′deletions to-42had no effect on expression of chiA. More DNAsequences deleted resulted in the complete loss of expression from chiA. And theDNA sequence from-116to-42, with respect to the translation start site, wassufficient for expression from the promoter. These reveal that the sequence locatedapproximately75bp DNA from positions-116to-42, with respect to the translationstart site, is the core promoter of chiA gene. The region upstream of position-116andthe region downstream the position-21is not necessary for the expression andregulation of chiA. Furthermore, a site for chitin induction was identified nearposition-36and upstream the position-20. This site for negative regulation wasindicated downstream of the RNA polymerase binding sites of the promoter of chiA.Transcription of chiA is not affected by the inducer when the operator is disruptedwhile the core promoter is not. Using5′RACE (Rapid amplification of cDNA ends),the base A-46upstream of the chiA translation start site was identified as thetranscription start site of the chiA gene. The DNA sequence TTACAA (-35) andTATCAT (-10), with a spacing of17bp, is supposed to bind the RNA polymerase.The chiB promoter was also subjected to deletion analysis to determine the minimal region required for stimulating chiB gene expression and to identify othercis-acting elements in the regulatory region. The results showed that5′deletion to-324and3′deletions to-41had no obviously effect on expression or regulation ofchiB.5′deletions to-232or3′deletions to-121showed decreased expression.5′deletions to202or3′deletions to-135resulted in the complete loss of expressionfrom chiB. While the DNA sequence from-116to-42, with respect to the translationstart site, was sufficient for expression from the chiB promoter, even though theactivity of promoter was low. These results of this analysis indicate that the sequencelocated approximately112bp DNA from positions-232to-121, with respect to thetranslation start site, is the core promoter of chiB gene. The sequences upstream anddownstream the core promoter seems to be necessary for the sufficient activity ofchiB promoter, while the region upstream of position-324and the region downstreamthe position-41is not necessary for the expression and regulation. Also, a site forchitin induction, which is downstream of the core promoter of chiB, was identifiedupstream the position-97. This site termed dre for regulation was indicatedcompletely downstream of the RNA polymerase binding sites of the chiB promoter.Transcription of chiB is not affected by the inducer when the dre site is disruptedwhile the core promoter is not. The dre site may be a palindromic sequence(AGACTTCGTGATGTCT) located between position-112and-97. Also thetranscription start site of the chiB gene was identified to be base C-132upstream of thechiB translation start site by using5′RACE. The-10boxes and-35boxes of chiBpromoter are indicated to be the sequence TTTACA and TACGAT, with a spacing of17bp. In vitro, a DNA binding factor interacts with the chiB promoter region wasfound in the cell of Bt75cultured in the medium without inducer. The binding of therepressor to the operator is displacement when the Bt75strain is cultured in themedium with inducer.To identify the role of the dre site in the regulation of chiB, seven bases at-98,-99,-103,-106,-109,-110and-111were changed by site-directed substitution using PCR.The effects of the changed bases on chiB expression were evaluated byβ-galactosidase assays when the transformants were cultured in the medium with orwithout chitin. The results improved that the dre site played a critical role in the regulation of chiB expression. Among the twenty mutations, fourteen substitutions inthe dre site resulted in constitutive expression from chiB. These suggest that somebases in the dre site are necessary for the inducible expression of chiB gene. Thenorthern blot also revealed that the amount of the mRNA increased due to thetransversion from T-99to G. It suggests that the dre site regulates the transcription ofthe chitinase gene chiB.Additionally, transcription from the chiA and chiB promoter did not occursimultaneously with bacterial growth either in the presence or absence of inducer.Expression of chiA and chiB started in cell grown for about6h and almost reachedthe maximum after14h of incubation. Subsequent period the enzyme didn’t increasedramatically. The capacity of GlcNAc to induce transcription from the chiA and chiBpromoter was investigated. The results suggest that GlcNAc isn’t the efficientinducers of the chitinase genes in Bt. |
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