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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. thuringiensis 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. TwoloxP sites were inserted, one on each side of thespo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deletedspo0A during sporulation, producing spores that lacked thespo0A 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 enhancedin vivo 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.IMPORTANCE B. thuringiensis 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 ofB. thuringiensis 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 ofB. thuringiensis 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 ofB. thuringiensis as a simulant forBacillus anthracis in environmental release studies.

Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release