Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection
ABSTRACT Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-depen...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2021-09-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/Spectrum.00259-21 |
| _version_ | 1798035762540707840 |
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| author | Masahito Ishikawa Takaaki Kojima Katsutoshi Hori |
| author_facet | Masahito Ishikawa Takaaki Kojima Katsutoshi Hori |
| author_sort | Masahito Ishikawa |
| collection | DOAJ |
| description | ABSTRACT Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10−7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection. |
| first_indexed | 2024-04-11T21:02:44Z |
| format | Article |
| id | doaj.art-2e2acd53dd6641768afe76fe46422664 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-11T21:02:44Z |
| publishDate | 2021-09-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-2e2acd53dd6641768afe76fe464226642022-12-22T04:03:27ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972021-09-019110.1128/Spectrum.00259-21Development of a Biocontained Toluene-Degrading Bacterium for Environmental ProtectionMasahito Ishikawa0Takaaki Kojima1Katsutoshi Hori2Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, JapanLaboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, JapanDepartment of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, JapanABSTRACT Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10−7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.https://journals.asm.org/doi/10.1128/Spectrum.00259-21biocontainmentinsertion sequencesynthetic biologyMinION |
| spellingShingle | Masahito Ishikawa Takaaki Kojima Katsutoshi Hori Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection Microbiology Spectrum biocontainment insertion sequence synthetic biology MinION |
| title | Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection |
| title_full | Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection |
| title_fullStr | Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection |
| title_full_unstemmed | Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection |
| title_short | Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection |
| title_sort | development of a biocontained toluene degrading bacterium for environmental protection |
| topic | biocontainment insertion sequence synthetic biology MinION |
| url | https://journals.asm.org/doi/10.1128/Spectrum.00259-21 |
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