Construction of novel pJRD215-derived plasmids using chloramphenicol acetyltransferase (cat) gene as a selection marker for Acidithiobacillus caldus

Background Acidithiobacillus caldus , a Gram-negative, chemolithotrophic sulfur-oxidizing bacterium, is widely applied in bioleaching. The absence of an ideal selection marker has become a major obstacle to achieve high efficiency of the gene transfer system for A . caldus . Plasmid pJRD215, widely used in Acidithiobacillus spp., has severe drawbacks in molecular manipulations and potential biosafety issues due to its mobility. Therefore, finding a new selection marker and constructing new plasmids have become an urgent and fundamental work for A . caldus . Results Effective inhibitory effect of chloramphenicol on the growth of A . caldus was elucidated for the first time. The P2- cat gene cassette, including a chloramphenicol acetyltransferase gene ( cat ) from plasmid pACBSR and a promoter (P2) upstream of the tetracycline resistance gene on pBR322, was designed, chloramphenicol acetyltransferase was expressed in A . caldus , and the enzyme activity was assessed. A new vector pSDU1 carrying the replication and mobilization regions derived from pJRD215, the P2- cat gene cassette and a multiple cloning site from pUC19 was successfully constructed. Compared with pJRD215, pSDU1 had a 27-fold increase in electrotransformation efficiency (30.43±0.88×10 4 CFU/μg DNA for pSDU1 and 1.09±0.11×10 4 CFU/μg DNA for pJRD215), better carrying capacity and could offer more convenience for the restriction enzyme digestion. In addition, the generated plasmid pSDU1Δmob, a novel non-mobilizable derivative of pSDU1 lacking some DNA sequences involved in the mobilization process, had increased copy number in A . caldus and lost its mobility for biosafety considerations. Both pSDU1 and pSDU1Δmob exhibited stable maintenance in A . caldus within 50 passages. However, further deletion of orfEF region involved in regulating repAC operon resulted in a negative effect on transformation efficiency, copy number and stability of plasmid pSDU1ΔmobΔorfEF in A . caldus . Conclusion Chloramphenicol was proved to be an ideal selection marker for A . caldus . Novel plasmids carrying cat gene were constructed. The utilization of these vectors will undoubtedly facilitate efficient genetic manipulations and accelerate the research progress in A . caldus .