RESUMO
Thurincin H is a bacteriocin produced by Bacillus thuringiensis, it is encoded in a group of ten genes, most of which have been characterized experimentally or by homology. However, the activity of the thnI gene encoding a 95 amino acid ORF remains unknown. In this work, the thnI gene was cloned under the regulation of two promoters and transformed into a sensitive strain to determine if it acts as an immunity protein. In addition, a deletion mutant without the thnI gene was used to test whether thnI is required or not for the biosynthesis of thurincin H. It was concluded that thnI does not provide immunity and is not required to produce thurincin H.
Assuntos
Bacillus thuringiensis , Bacteriocinas , Bacillus thuringiensis/química , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Regiões Promotoras GenéticasRESUMO
ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect enzymatic activity of ChiA74 in crude preparations of bacterial secretomes. In the present study, we cloned the chiA74 open reading frame (ORF) lacking the 5' sequence coding for its secretion signal peptide (chiA74Δsp) into a cold shock expression vector (pColdI) for production of the enzyme in Escherichia coli BL21-Rosetta 2. As a result, the N-terminal end of ChiA74Δsp ORF was fused to an artificial sequence of 28 amino acid, including a 6× histidine tag for purification of recombinant 6×His tagged-ChiA74Δsp (rChiA74, â¼74kDa). Along with a protein of â¼74kDa, we co-purified its â¼55kDa processed form which was confirmed by Western blot analysis. Optimal endochitinase activity of purified rChiA74 occurred at pH 7 and 40°C. Most divalent cations (e.g. Ba(+2), Ca(+2), Mn(+2), Mg(+2), Zn(+2) and Cu(+2)) at concentration of 10mM reduced chitinase activity by â¼30%, and Hg(+2) (10mM) drastically inhibited ChiA74 activity by â¼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11±0.01nmol/min, 2.15µM±0.45 and 3.81s(-1), respectively, using 4-MU-GlcNAc3 as substrate. Using purified rChiA74 and colloidal chitin as substrate, chitin-derived oligosaccharides with degree of polymerization of 2 and 1 were detected.
Assuntos
Bacillus thuringiensis/enzimologia , Bioquímica/métodos , Quitinases/isolamento & purificação , Quitinases/metabolismo , Expressão Gênica , Western Blotting , Cátions Bivalentes/farmacologia , Quitina/metabolismo , Cromatografia em Camada Fina , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Proteínas Recombinantes de Fusão/isolamento & purificação , TemperaturaRESUMO
The hydrolysis of beta-D: -glucosidic bonds which is required for the liberation of many physiologically important compounds is catalyzed by the enzyme beta-glucosidase (BGLU, EC 3.2.1.21). BGLUs are implicated in several processes in plants, such as the timely response to biotic and abiotic stresses through activation of phytohormones and defense compounds. We identified 26 BGLU isozymes in the genome of the maize inbred B73 and propose a standardized nomenclature for all Zea mays BGLU paralogs (Zmbglu1-Zmbglu26). We characterized their intron-exon structure, protein features, phylogenetic relationships, and measured their expression and activity in various tissues under different environmental conditions. Sequence alignments revealed some characteristic motifs (conserved amino acids) and specific differences among different isozymes. Analysis of putative signal peptides suggested that some BGLUs are plastidic, whereas others are mitochondrial, cytosolic, vacuolar or secreted. Microarray and RT-PCR analysis showed that each member of the Zmbglu family had a characteristic expression pattern with regard to tissue specificity and response to different abiotic conditions. The source of variance for gene expression was highest for the type of organ analyzed (tissue variance) than for the growth conditions (environmental variance) or genotype (genetic variance). Analysis of promoter sequences revealed that each Zmbglu paralog possesses a distinct set of cis elements and transcription factor binding sites. Since there are no two Zmbglu paralogs that have identical molecular properties, we conclude that gene subfunctionalization in maize occurs much more rapidly than gene duplication.