title: Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release.
abstract: For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently,Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains ofB. thuringiensiswith 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. TwoloxPsites were inserted, one on each side of thespo0Agene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deletedspo0Aduring sporulation, producing spores that lacked thespo0Agene. 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 enhancedin vivosafety. 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.IMPORTANCEB. thuringiensishas recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features ofB. thuringiensisas 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 ofB. thuringiensissimulant 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 ofB. thuringiensisas a simulant forBacillus anthracisin environmental release studies.
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.|The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Sangjin
authors.initials: S
authors.lastname: Park
authors.affiliation: The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Changhwan
authors.initials: C
authors.lastname: Kim
authors.affiliation: The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Daesang
authors.initials: D
authors.lastname: Lee
authors.affiliation: The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Song
authors.affiliation: The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Ki Cheol
authors.initials: KC
authors.lastname: Cheon
authors.affiliation: The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Hong Suk
authors.initials: HS
authors.lastname: Lee
authors.affiliation: The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Seong Joo
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: The 5th R&D Institute, Agency for Defense Development (ADD), Daejeon, Republic of Korea.
authors.forename: Jee Cheon
authors.initials: JC
authors.lastname: Kim
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea leesy@kaist.ac.kr.|BioProcess Engineering Research Center, KAIST, Daejeon, Republic of Korea.|BioInformatics Research Center, KAIST, Daejeon, Republic of Korea.
authors.forename: Sang Yup
authors.identifier: http://orcid.org/0000-0003-0599-3091
authors.initials: SY
authors.lastname: Lee
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000254:growth & development*; D004251:DNA Transposable Elements; D004195:Disease Models, Animal; D004783:Environmental Microbiology; D017353:Gene Deletion; D017930:Genes, Reporter; D016908:Gram-Positive Bacterial Infections / Q000276:immunology / Q000382:microbiology; D008807:Mice, Inbred BALB C; D050296:Microbial Viability / Q000528:radiation effects; D016254:Mutagenesis, Insertional; D010860:Pigments, Biological / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013171:Spores, Bacterial / Q000235:genetics* / Q000254:growth & development*; D014466:Ultraviolet Rays; D014774:Virulence
chemical_list: D004251:DNA Transposable Elements; D010860:Pigments, Biological
keywords: Bacillus thuringiensis; biodefense; environmental release; environmentally friendly strain; simulant; small acid-soluble spore protein; sporulation-dependent spo0A knockout