ABSTRACT
A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518 bp PCR product, and analysis of the sequence revealed the presence of 1482 bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398 bp. Theoretical molecular weight (51.7 kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-ß-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.
Subject(s)
Bacillus cereus , Insecticides/chemistry , Membrane Glycoproteins , Bacillus cereus/chemistry , Bacillus cereus/genetics , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Glycoproteins/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
The biochemical characterization of novel antimicrobial peptides (AMPs) and the determination of ligand molecules that induce AMP production are essential for understanding the host innate immune response in insects. Here, we purified a new 14-kDa AMP, named tenecin 4, from the larval hemolymph of the beetle Tenebrio molitor. Tenecin 4 contains 14% glycine residues and has moderate similarities both to the C-terminal region of Drosophila attacin and to silk-moth gloverin proteins. Purified tenecin 4 showed bactericidal activity against Gram-negative Escherichia coli but not against Gram-positive Bacillus subtilis or the fungus Candida albicans. Tenecin 4 production was induced by Toll cascade-activating ligands, such as ß-1,3-glucan, lysine-type peptidoglycan and active Spätzle, and by the probable Imd pathway-activating ligand monomeric meso-diaminopimelic acid-type peptidoglycan. Taken together, these data show that tenecin 4 is a defense protein against Gram-negative pathogens and is induced by multiple ligands in Tenebrio larvae.