Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release

• Published in: Applied and environmental microbiology Vol. 83; no. 9; p. E00126
• Main Authors: Park, Sangjin, Kim, Changhwan, Lee, Daesang, Song, Dong Hyun, Cheon, Ki Cheol, Lee, Hong Suk, Kim, Seong Joo, Kim, Jee Cheon, Lee, Sang Yup
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.05.2017
• Subjects: Animals; Antibiotic resistance; Antibiotics; Bacillus anthracis; Bacillus thuringiensis; Bacillus thuringiensis - genetics; Bacillus thuringiensis - growth & development; Bacteria; Chromosomes; Circuit design; Colonies; Disease Models, Animal; DNA Transposable Elements; Environmental Microbiology; Environmental release; Environmental studies; Gene Deletion; Gene expression; Genes; Genes, Reporter; Gram-Positive Bacterial Infections - immunology; Gram-Positive Bacterial Infections - microbiology; In vivo methods and tests; Lungs; Mice, Inbred BALB C; Microbial Viability - radiation effects; Microbiology; Mutagenesis; Mutagenesis, Insertional; Mutation; Pigments, Biological - genetics; Pigments, Biological - metabolism; Proteins; Recombination, Genetic; Safety; Solar simulators; Spo0A gene; Spores; Spores, Bacterial - genetics; Spores, Bacterial - growth & development; Sporulation; Synteny; Transposon mutagenesis; Ultraviolet Rays; Viability; Virulence; environmentally friendly strain; simulant; sporulation-dependent spo0A knockout; Bacillus thuringiensis; environmental release; small acid-soluble spore protein; biodefense
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.00126-17

For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, , a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two sites were inserted, one on each side of the gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted during sporulation, producing spores that lacked the gene. In addition, two major α/β-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment. has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem, especially when genetically engineered strains are used. To solve these problems, we report here the development of simulant strains that are capable of forming yellow colonies for easy detection, incapable of forming spores more than once due to a genetic circuit, and lacking in two major SASP genes. The genetic circuit to produce a spore without sporulation capability, together with the deletion of SASP genes, ensures the environmental and human safety of the simulant strains developed in this study. All of these features will allow wider use of as a simulant for in environmental release studies.