Detection and validation of a small broad-host-range plasmid pBBR1MCS-2 for use in genetic manipulation of the extremely acidophilic Acidithiobacillus sp.

An efficient genetic system for introducing genes into biomining microorganisms is essential not only to experimentally determine the functions of genes predicted based on bioinformatic analysis, but also for their genetic breeding. In this study, a small broad-host-range vector named pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups, was studied for the feasibility of its use in conjugative gene transfer into extremely acidophilic strains of Acidithiobacillus. To do this, a recombinant plasmid pBBR-tac-Sm, a derivative of pBBR1MCS-2, was constructed and the streptomycin resistant gene (Smr) was used as the reporter gene. Using conjugation, pBBR-tac-Sm was successfully transferred into three tested strains of Acidithiobacillus. Then we measured its transfer frequency, its stability in Acidithiobacillus cells, and the level of resistance to streptomycin of the transconjugants and compared this with the IncQ plasmid pJRD215 control. Our results indicate that pBBR1MCS-2 provides a new and useful tool in the genetic manipulation of Acidithiobacillus strains.