Construction of vector of Brevibacterium lactofermentum and study of its stability in continuous culture

A 5.7-kb vector plasmid pBK2 was constructed by ligating the kanamycin resistance gene from Escherichia coli plasmid pACYC177 to an endogenous cryptic 4.4-kb plasmid of Brevibacterium lactofermentum ATCC 21086. The vector replicates efficiently and is stably maintained in the host and other coryneforms. However, the copy number varied from 50 to 10 per chromosome-equivalent under different culture conditions. Continuous culture studies showed instability when low dilution rates were used. Co-culture experiments were performed at various dilution rates to measure the growth rate ratio (alpha) of the plasmid-free cells to the plasmid-containing cells. It was observed that at low dilution rates the value of alpha was higher than that at high dilution rates. Thus, the instability of the plasmid can be attributed to the increase in alpha at low dilution rates. Modelling of instability using a random partitioning model of plasmid segregation and experimentally obtained values of alpha showed agreement with experimental data. This demonstrated that active partitioning is not the operative mechanism for plasmid segregation in this case.