Genetic transfer of IncP, IncQ and IncW plasmids to four Thiobacillus ferrooxidans strains by conjugation

The physiological function of some proteins involved in Thiobacillus ferrooxidans energetic metabolism has been difficult to assess. Our long-term goal is to construct and to analyse mutants in which these proteins are no longer synthesized in order to determine in which respiratory chains they are involved. However, up to now, there is no report on the construction of mutants in this microorganism because of the lack of genetic tools. Recently, the transfer by conjugation from Escherichia coli of IncP plasmids and by mobilization of IncQ plasmids into private T. ferrooxidans strains has been reported, but the transfer frequency was very low. We have extended this research by establishing a genetic transfer system by conjugation of IncQ (pJRD215), IncP (pJB3Km1) and IncW (pUFR034) group plasmids from E. coli not only to two private T. ferrooxidans strains (BRGM1 and Tf-49), but more interestingly, to the two collection strains ATCC33020 and ATCC19859, from which most of the T. ferrooxidans genes characterized to date, originate. IncQ plasmid pJRD215 had the higher transfer frequency and was stable in these four T. ferrooxidans strains. The conjugation conditions were optimized for the ATCC33020 strain by analysing the influence on the transfer frequency of: (i) the donor strain; (ii) the plasmid to be transferred; (iii) the ratio of donor/recipient cells; (iv) the adaptation of the donor and the recipient cells to the mating medium; (v) the composition of the mating medium. Under the best conditions, the transfer frequency could be increased from 10_7 to 5×10_5.