Investigation of plasmid instability in amylase-producing B. subtilis using continuous culture

Continuous culture was employed to study plasmid instability in an amylase-producing Bacillus subtilis 1A289 that was genetically manipulated. No true steady state could be obtained with 1A289(pEAA)-strain (plasmid)-due to its structural instability, which occurred both with glucose and Maltrin-100 as limiting carbon sources. The plasmid, pEAA (Cm(R), amy(+), i.e., chloramphenicol resistant, amylase positive) degenerated into a smaller plasmid, pEAA1 (CM(R), amy(-)) that was stable. There was a direct correlation between amylase-producing ability and this structural instability since f(amy) (fraction of cells with amylase-producing ability) reached zero at the same time that f (fraction of cells that are resistant to chloramphenicol) reached its maximum level. Since the deletion in pEAA was larger than the original amylase-gene insert, either all of part of the insert is absent from pEAA1. Though on discernible change in 1A289(pHV33), where pHV33 is the vector plasmid, was observed during continuous cultivation, its behavior was different from that of the stable 1A289(pEAA1).