Researches On Plasmid Genetic Transformation In Bacillus Subtilis

Bacillus subtilis is a spore forming Gram-positive bacterium,which is considered as a safe microorganism,because its cell wall does not contain endotoxin(lipopolysaccharide,LPS).It has been widely used in agriculture as a plant growth promoter and biological control for plant diseases.It has also been used in industry as a probiotic and as a host for expression of industrial enzymes and heterologous proteins.The introduction of exogenous plasmids into B.subtilis to construct suitable genetic engineering systems further increases its applications.Due to its broad applications,development of highly efficient genetic engineering tools has become one of the research focuses of B.subtilis.Several methods for plasmid transformation in B.subtilis have been developed so far,including protoplast transformation,traditional two-step transformation and electroporation.Although these approaches have been widely used and optimized,the procedures are complicated and the transformation efficiency is relatively low.Moreover,these approaches often fail when used in wide strains of B.subtilis.Therefore,improving the efficiency and simplifying the experimental procedures of plasmid transformation are keys to increase the applications of B.subtilis.Previous studies have reported that the transformation efficiency of unmethylated DNA or DNA concatemer was much higher than that of methylated plasmid DNA.In this study,we quantified the transformation efficiency of unmethylated DNA concatemer and tested whether this modification would allow plasmid transformation in wide strains of B.subtilis.We amplified plasmids into continuous,unmethylated,long polymers by using the phi29 DNA polymerase amplification kit(Evomic Science),which amplifies DNA through a rolling circle amplification mechanism(RCA).We found that the transformation efficiency of the RCA products of a given plasmid was about 10~2 times higher than that of the original plasmids,confirming that demethylation and concatenation could greatly improve the transformation efficiency.However,we failed to obtain transformant in wild strains of B.subtilis using the RCA product by the two-step method or electroporation,suggesting that this approach cannot solve the transformation problem of wide strains of B.subtilis.Of the plasmids tested,we found that the transformation efficiency of the chloramphenicol resistant plasmid p GK12 and its RCA product were the highest.Increasing the length of p GK12(4.5 kb-8.5 kb)by inserting exogenous fragments did not significantly affect the transformation efficiency of their RCA products.Taken together,these results suggest that demethylation and concatenation of plasmids can significantly improve the transformation efficiency in laboratory strains but not in wild strains of B.subtilis.In this study,we also developed a novel method for plasmid transfer that is based on the donor-cell dependent cell-to-cell natural transformation(CTCNT)phenomenon we previously discovered in B.subtilis.In that study,we found that transformation of chromosomal DNA could occur between two different B.subtilis strains when they were in close proximity and under stress.Here we found that plasmids could be transferred by CTCNT as well.We chose the multiple auxotrophic strain TLPHMC?amy E as the donor strain for the plasmid to achieve unidirectional transfer of the target plasmid from the donor to a recipient strain.In this transformation method,the donor and the recipient strains were cultured separately in appropriate mediums and then were mixed and co-cultured on a microporous filter membrane on top of solid medium with antibiotics to stress the cells and allow the transfer of plasmids.Transformants started to appear after co-cultured for 3 hours and the number of transformants increased as the co-culturing time increased.We tested how environmental conditions affect the transformation efficiency of this cell-to-cell plasmid transformation.We found that different stresses caused by antibiotics have significant impact on the transformation efficiency.When the recipient was strain 168 and the plasmid was chloramphenicol resistant,the optimal concentration of chloramphenicol to generate the stress during the co-culture period was C50(50?g/ml).When the plasmid was erythromycin resistant,the optimal concentration of erythromycin to generate the stress was E2(2?g/ml),whereas when the plasmid was kanamycin resistant,the optimal concentration of kanamycin to generate the stress was K30(30?g/ml).When the frame of the target plasmids was the same but the antibiotic resistance gene was different,the number of transformants obtained with kanamycin was 100 times and 10 times higher than that of chloramphenicol and erythromycin,respectively.Moreover,the transformation efficiency appeared to decrease as the length of the plasmid increased(reduced by 3times when the length of plasmid doubled).Additionally,we found that the ratio of the donor/recipient cells during the co-culture period strongly affected the transformation efficiency:the transformation efficiency was optimal when the ratio of donor/recipient was 1/1.Using this cell-to-cell plasmid transformation approach,we not only transferred plasmids into the laboratory strain 168 efficiently,but also successfully transferred plasmids into wild strains of B.subtilis and Bacillus amylogenus.These results suggest that this approach can be employed for genetic engineering in wild strains of Bacillus.B.subtilis is a promising host for many industrial applications,but it is difficult to introduce plasmid into wild strains of B.subtilis.This study not only greatly improves the plasmid transformation efficiencies of the two-step method and electroporation by using unmethylated and concatenated DNA,but also develops a novel cell-to-cell plasmid transformation system that is applicable to wild strains of B.subtilis.This approach is easy to operate and convenient for genetic screening.We envision that it will help establish new expression systems of Bacillus spp.