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-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.

Keywords: MinION; biocontainment; insertion sequence; synthetic biology.

FIG 1

Schematic of the functioning of a biocontained toluene-degrading bacterium. Acinetobacter sp. Tol 5 is a Gram-negative bacterium that metabolizes various carbon sources, including toluene. BamA, a widely conserved outer membrane protein, is essential for cell growth in most Gram-negative bacteria. A bamA-deficient mutant of Tol 5 was complemented with a plasmid harboring a bamA gene controlled by a XylR-Pu promoter system. XylR is a transcriptional regulatory protein-binding toluene. Toluene-bound XylR activates the Pu promoter. In the presence of toluene, the biocontained Tol 5 can grow due to the transcription and translation of the bamA gene from the plasmid. As XylR switches to an inactive form after the complete degradation of toluene, the transcription and translation of the bamA gene stop. As a result, the cell growth of biocontained Tol 5 ceases due to the depletion of newly synthesized BamA.

FIG 2

Construction of Acinetobacter sp. Tol 5 mutants expressing the bamA gene in a specific condition and analysis of mutants escaping from the biological containment system. (A) Schematic diagrams representing the genomic organization around the bamA gene in the chromosome of Tol 5 and the plasmid for complementation. Half-arrowheads indicate the locations where primers anneal. (B) PCR confirmation of bamA disruption. PCR was performed using the primers BamA_p1_F and BamA_p2_R. The target sizes were 5,701 bp (wild type [WT]) and 3,392 bp (bamA-deficient mutants). (C) Schematic of the procedure to enrich cultures for mutants escaping from the biocontainment system. The PuBamA strain was serially passaged in LB medium not supplemented with toluene. (D) Schematic of insertion sequence (IS) insertion into the Pu promoter on the pPuBamA plasmid. Half-arrowheads indicate the positions where the primers IS-P-ChecK_F and IS-P-Check_R anneal. PCR using these primers amplifies a 663-bp DNA fragment in the absence of an IS element. (E) PCR amplification of the DNA region around the Pu promoter.

FIG 3

Cell growth of Acinetobacter sp. Tol 5 strains with conditional expression of bamA on agar plates. (A) Growth of a bamA-deficient mutant carrying the bamA gene under the control of an arabinose-inducible promoter. (B) Growth of a bamA-deficient mutant carrying the bamA gene under the control of a toluene-inducible promoter. Cell suspensions of the wild-type (WT) and the bamA conditional expression mutant strains were serially diluted 1:10. Each serial dilution was spotted onto LB agar plates.

FIG 4

Biocontainment of a toluene-degrading bacterium. (A) Toluene degradation by the wild type (WT) and by a ΔbamA mutant carrying the bamA gene under the control of a toluene-inducible system (PuBamA). Data are presented as mean ± standard deviation (n = 3). (B) Bacterial viability after toluene degradation. After a 3-day incubation of the toluene degradation reaction shown in panel A, cell suspensions of the WT and PuBamA strains were spotted on LB agar plates supplemented or not supplemented with toluene vapor for a CFU test. Data are presented as mean ± standard error (n = 3).

FIG 5

Types of inserted ISs and their insertion sites in the PU promoter. (A) Nucleotide sequence of the region surrounding the Pu promoter in the pPuBamA plasmid. Gray backgrounds, boxes, the dot, underlines, and arrowheads indicate the upstream regulatory sequences, the promoter sequence (−24 and −12), the transcription start site (+1), the direct repeats created by IS elements, and the positions where IS elements were inserted (+12, +17, +120, and +146), respectively. The upstream regulatory sequences, the promoter sequence, and transcription start site correspond to those found by Inouye et al. (44). (B) Distribution of the IS insertion sites. Deep sequencing of PCR amplicons (lane of the third passage in Fig. 2E) revealed that three types of ISs were inserted at different sites in the Pu promoter.