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In recent years, the prospect of using Mycobacterium bovis BCG as a live vaccine carrier for foreign antigens has prompted a rapid evolution of the molecular biology of Mycobacteria. This progress was considerably facilitated by the development of the electroporation technique allowing high levels of transformation rates of this organism (1). Nowadays, Escherichia coliMycobacteria shuttle plasmids are commonly used to take full advantage of the molecular biological tools developed in E.coli, before transferring the final constructions into the mycobacterial species. However, plasmid analysis of mycobacterial transformants still presents major difficulties. The slow growth rate of cell cultures impedes rapid analysis of recombinant bacteria (see Table 1). Moreover, yields of extracted plasmid DNA are usually poor, essentially due to the high resistance of Mycobacteria to standard cell lysis in addition to generally low plasmid copy numbers. We report here a simple technique allowing a significantly faster analysis of plasmid DNA from mycobacterial transformants. The method is based on two basic features, i.e. the inherent ability of shuttle plasmids to replicate in at least two distant bacterial species, and the fact that DNA molecules may be introduced or released from a bacterial cell submitted to electroporation. Indeed, both inter(2, 3) and intraspecies (4) DNA transfers have already been demonstrated during electroporation. Interspecies transfer usually requires two pulses. We demonstrate direct plasmid transfer between Mycobacterium smegmatis and M.bovis BCG, and E.coli, two very distant bacterial genera, following a single electrical pulse. M. smegmatis m c 2 ^ and M.bovis BCG 1173P2 were transformed (1) with the shuttle plasmid pRR3 (5) and plated on Middlebrook 7H10 agar supplemented with Middlebrook ADC Enrichment (Difco) + 15 /tg/ml kanamycin. Visible colonies were obtained after incubation for 4 days for M. smegmatis or 14 days for M.bovis BCG at 37 °C. Twelve growing colonies were picked and mixed with 20 fi\ of ice-cold 10% glycerol. The tubes were briefly vortexed before placing on ice for 10 min. These mixtures were added to E.coli electro-competent cells (at a concentration of 10'° cells per ml) prepared as described previously (6). After a single electric pulse using parameters optimal for E.coli electrotransformation (135 W, 2500 V, 45 /iF on the Cellject apparatus, Eurogentech), cells were incubated for 1 hour at 37°C before plating on LB medium supplemented with 25 /ig/ml of kanamycin. Resistant E.coli cells appeared after 12 — 16 hours at 37 °C and plasmid DNA was extracted and analysed the next day using standard procedures. Direct DNA transfer between BCG and E.coli resulted in 10-100 transformants per M.bovis colony. This technique was even more efficient between M.smegmatis and E.coli; a mean of 10 transformants obtained per M.smegmatis colony. Moreover, contamination risks inherent to long term mycobacterial cultures are avoided, as are the laborious and low yield DNA extractions from this organism. This protocol should be applicable to general use, especially for slow-growing or fastidious bacteria or for those that are resistant to standard cell lysis procedures.

Rapid mycobacterial plasmid analysis by electroduction between Mycobacterium spp. and Escherichia coli