Researchers developed spheroplast fusion as a method for genetic recombination in Mycobacterium aurum, addressing the limited success of conventional genetic transfer methods in mycobacteria. Two carotenoid pigment mutants (NgR9 and A13) were created through chemical mutagenesis and converted to spheroplasts. When mixed spheroplasts were treated with 40% polyethylene glycol (PEG-6000) for 5 minutes at 37°C, genetic recombination occurred at frequencies reaching 2.2-3.5 × 10⁻³. Recombinants were identified by altered carotenoid pigment profiles and mycolic acid patterns on Lowenstein-Jensen medium. Electron microscopy confirmed fusion events and showed various stages of spheroplast membrane fusion. The study established optimal fusion conditions: 40% PEG-6000, pH 6, 5-minute treatment at 37°C without added calcium. While some fusion products formed transient diploids that segregated rapidly, stable haploid recombinants were isolated, though none achieved the complete wild-type phenotype, likely due to the mutants being multiple-gene mutants. This work demonstrated that protoplast fusion could effectively generate genetic recombination in mycobacteria.

Key findings

• Spheroplast fusion induced by polyethylene glycol (PEG-6000) achieved genetic recombination in Mycobacterium aurum at frequencies of 2.2-3.5 × 10⁻³, significantly higher than previous mycobacterial genetic transfer methods.
• Optimal fusion conditions were identified: 40% PEG-6000, pH 6, 5-minute treatment at 37°C, with spheroplasts in 1:1 ratio and without added calcium.
• Electron microscopy confirmed complete fusion of spheroplasts and showed multiple fusion events that likely resulted in polyploid organisms that segregated rapidly upon subculture.
• Recombinants were confirmed through dual genetic markers (carotenoid pigments and mycolic acids), demonstrating genuine genetic exchange rather than spontaneous reversion.
• Although stable recombinants were isolated, none possessed complete wild-type phenotype, indicating the parental mutants carried multiple genetic mutations affecting carotenoid pigment synthesis.