Spheroplast Fusion as a Mode of Genetic Recombination in Mycobacteria

Service de la Tuberculose et des Mycobactérias, Institut Pasteur, 25 Rue du Dr Roux, 75015 Paris, France Centre de Recherche de Biochimie et de Génétique Cellulaire du CNRS, 118 Route de Narbonne, 31062 Toulouse Cedex, France ABSTRACT SUMMARY: Spheroplasts were prepared from two carotenoid pigment mutants of Mycobacterium aurum named NgR 9 and A 11, which were obtained by the chemical mutagenesis of the wild type strain A + with N -methyl- N’ -nitro- N -nitrosoguanidine. The carotenoid pigments and the - and β-mycolic acids were taken as genetic markers and the recombinants were selected on the basis of their colour on Löwenstein-Jensen medium. Spheroplasts of the two mutants were mixed in a 1:1 ratio and were treated with 40% (w/v) polyethylene glycol 6000 for 5 min at 37 °C. The frequency of NgR 9 x A 11, recombination in optimal conditions was about 2.5 x 10 -3 . The recombinants selected on the basis of their carotenoid pigment profile were also tested for their - and β-mycolic acids as a second genetic marker. The results were further confirmed by electron microscopy. The optimal conditions, for spheroplast fusion as a mode of genetic recombination in M. aurum are described.