RESUMEN
Expression of the chiB gene from Bacillus thuringiensis Bti75 was defined as inducible by the use of transcriptional fusions with the bgaB reporter gene. The transcription start site of the chiB gene was identified as the C base located 132 base pairs upstream of the start codon. Analysis of 5' and 3' deletions of the chiB promoter region revealed that the sequence from position -192 to +36 with respect to the transcription start site was necessary for wild-type levels of inducible expression of the chiB gene. The minimal promoter region for the expression of chiB gene was identified as the sequence from position -100 to +12. Furthermore, a 16-bp sequence (designated dre) downstream of the minimal promoter region of chiB was shown to be required for chitin induction. To confirm the function of this 16-bp sequence, 25 base substitutions were introduced into the dre site. Most of the mutations resulted in constitutive expression, or the efficiency of induction decreased. All mutations identified the dre sequence as a critical site for the inducible expression of chiB. In addition, the dre site was shown to interact with a sequence-specific DNA binding factor of strain Bti75 cultured in the absence of the inducer.
Asunto(s)
Bacillus thuringiensis/genética , Quitinasas/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Bacillus thuringiensis/enzimología , Secuencia de Bases , Quitinasas/biosíntesis , Inducción Enzimática , Datos de Secuencia Molecular , Eliminación de Secuencia , Sitio de Iniciación de la TranscripciónRESUMEN
To explore the influence of the direct repeat sequence (DRS) in Bacillus chitinase genes on heterogonous expression in Escherichia coli, we cloned and sequenced the entire open reading frame (ORF) and upstream sequences of the chitinase B (chiB) and chitinase MY75 (chiMY75) from Bacillus thuringiensis and Bacillus licheniformis. A pair of 8-bp DRS was found upstream of each chi gene. Chi ORFs with a series of truncated DRS were cloned and transformed into E. coli XL-Blue. The activity of the transformants without the DRS were significantly higher in chitinase assays than transformants containing the DRS. SDS-PAGE showed that part and full deletion of the DRS increased chi gene expression by approximately 1.7 and 3.8-fold, respectively. Northern blotting revealed deletion of the DRS regions increased chiB and chiMY75 mRNA expression. Specific binding of DNA-binding factors in the E. coli cell lyaste was observed to both the chiB and chiMY75 promoter regions and DRS elements. This is the first investigation to demonstrate that heterologous expression of Bacillus chi genes in E. coli is negatively regulated by their upstream DRS regions, which act as cis-acting elements.
Asunto(s)
Bacillus/genética , Quitinasas/genética , Elementos de Facilitación Genéticos/genética , Escherichia coli/enzimología , Regulación Enzimológica de la Expresión Génica , Secuencias Repetitivas de Ácidos Nucleicos/genética , Bacillus/clasificación , Bacillus/enzimología , Bacillus thuringiensis/enzimología , Bacillus thuringiensis/genética , Secuencia de Bases , Quitinasas/química , Quitinasas/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADNRESUMEN
The expression and application of Bacillus thuringiensis (Bt) chitinase genes have been extensively investigated. However, little information is available regarding the regulation of chitinase gene expression in Bt. In this study, a shuttle promoter-probe vector was constructed incorporating the thermostable ß-galactosidase gene bgaB of B. stearothermophilus as the reporter for the study of Bt promoters. Using this plasmid, the activity of the chiA gene promoter in Bt was investigated. Deletion analysis of the putative chiA promoter region revealed that the sequence located ~75 bp DNA from positions -116 to -42, with respect to the translation start site, is the core promoter of chiA gene. Furthermore, a site for chitin induction was identified near position -36. This site for negative regulation was indicated downstream of the RNA polymerase binding sites of the promoter of chiA. The expression of chiA started in cell grown for about 6 h and reached the maximum after 60 h of incubation. Induction of chiA expression by chitin was demonstrated by an increase in ß-galactosidase activity of ~2.5-fold.
Asunto(s)
Bacillus thuringiensis/enzimología , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Quitinasas/genética , Regiones Promotoras Genéticas , Bacillus thuringiensis/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Quitinasas/química , Quitinasas/metabolismo , Clonación Molecular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Chitinases, which can hydrolyze chitin, occur in a wide range of microorganisms including viruses, bacteria, and fungi. The derivatives of chitin are potentially useful in several areas such as food processing, medicines, and biological control in agriculture. Some bacteria can uptake and utilize chitin as carbon source by secreting chitinase. The chitin is degraded into chito-oligosaccharides [(GlcNAc)n] or N-acetylglucosamine (GlcNAc) by chitinases, and then the chitin derivatives are transferred into cells by specific transport systems of bacteria. The intracellular chitin derivatives activate or suppress the transcription of a series of chi genes and affect the amount of chitinase. The expression of chitinase genes are strictly regulated by various regulatory factors and responsive cis-acting elements. The present review will focus on the transport system and the regulation of chitinase genes expression in bacteria.
Asunto(s)
Bacterias/genética , Quitinasas/genética , Regulación Bacteriana de la Expresión Génica , Bacterias/enzimología , Quitina/metabolismo , HidrólisisRESUMEN
Strain MY75 is a gram-positive, aerobic, endospore-forming bacterium that can secrete high levels of extracellular chitinase (4.645 U/ml) when chitin powder exists as an inducer. This strain was identified as Bacillus licheniformis using the Biolog MicroLog microbial identification system and sequence analysis of 16S rDNA, gyrA and rpoB genes. Strain MY75 has the ability to inhibit the growth of Gibberella saubinetii and Aspergillus niger, two major pathogenic fungi in agriculture, and to restrain their spore germination completely. The chitinase was proved to play an important role in the strain's antifungal activity.