Curing the Plasmid pMC1 from the Poly (γ-glutamic Acid) Producing Bacillus amyloliquefaciens LL3 Strain Using Plasmid Incompatibility

Abstract

Bacillus amyloliquefaciens LL3 is a glutamate-independent poly-γ-glutamic acid (γ-PGA) producing strain which consists of a circular chromosome (3,995,227bp) and an endogenous plasmid pMC1 (6,758bp). The study of the function of native plasmid and the genome-size reduction of the B. amyloliquefaciens LL3 strain requires elimination of the endogenous plasmid. Traditional plasmid-curing procedures using sodium dodecyl sulfate (SDS) or acridine orange combined with heat treatment have been shown to be ineffective in this strain. Plasmid incompatibility is an effective method for curing which has been studied before. In our research, the hypothetical Rep protein gene and the origin of replication of the endogenous plasmid were cloned into the temperature-sensitive vector yielding the incompatible plasmid pKSV7-rep-ori. This plasmid was transformed into LL3 by electroporation. The analysis of the strain bearing incompatible plasmids after incubation at 30°C for 30 generations showed the production of plasmid cured strains. High frequency of elimination was achieved with more than 93% of detected strains showing to be plasmid-cured. This is the first report describing plasmid cured in a γ-PGA producing strain using this method. The plasmid-cured strains showed an increase of γ-PGA production by 6% and led to a yield of 4.159g/l, compared to 3.918g/l in control and cell growth increased during the early stages of the exponential phase. Gel permeation chromatography (GPC) characterization revealed that the γ-PGA produced by plasmid-cured strains and the wild strains were identical in terms of molecular weight. What is more, the further study of plasmid function showed that curing of the endogenous plasmid did not affect its sporulation efficiency.