Development of a homologous expression system for and systematic site-directed mutagenesis analysis of thurincin H, a bacteriocin produced by Bacillus thuringiensis SF361

• Published in: Applied and Environmental Microbiology 2014-06, Vol.80 (12), p.3576-3584
• Main Authors: Wang, Gaoyan, Manns, David C, Churey, John J, Worobo, Randy W
• Format: Article
• Language: English
• Subjects: Amino acids; Bacillus thuringiensis; Bacillus thuringiensis - genetics; Bacillus thuringiensis - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriocins - biosynthesis; Biosynthetic Pathways; Chromosomes; E coli; Escherichia; Food Microbiology; Gene Expression; Mutagenesis, Site-Directed; Mutation; Peptides; Promoter Regions, Genetic; Protein Engineering - methods
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.00433-14

Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared.