native virulence plasmid combination affects the segregational stability of a theta-replicating shuttle vector in Bacillus anthracis var. New Hampshire

The segregational stability of a small, θ‐replicating, non‐mobilizable shuttle plasmid (pAEX‐5E) was determined in fully virulent (pX01+/pX02+), partially cured (pX01+/pX02− and pX01−/pX02+) and fully cured (pX01−/pX02−) derivatives of Bacillus anthracis var. New Hampshire. Under the growth conditions used (L‐broth, 37 °C, aerobic, batch culture), pAEX‐5E remained segregationally stable in the pX01−/pX02+ and pX01−/pX02− derivatives for in excess of 100 culture generations, but was expelled from the pX01+/pX02+ and pX01+/pX02− derivatives (100% loss occurred after 101±3·8 and 54±6·0 culture generations, respectively). In the presence of antibiotic selection pressure to maintain pAEX‐5E (5 μg erythromycin ml−1) no comparable loss of pX01 or pX02 was observed over 100 generations of growth in any of the derivatives of B. anthracis. Under these conditions the pX01+/pX02− derivative had an extended culture doubling time (td±S.E. of the mean) of 75·3 ± 1·4 min compared with 47·3 ± 1·1, 46·2 ± 0·86 and 43·2 ± 1·2 min for the pX01+/pX02+, pX01−/pX02+ and pX01−/pX02− derivatives, respectively. That antibiotic resistance was pAEX‐5E‐mediated was confirmed using a second antibiotic marker (kanamycin). After100 generations of growth in the presence of erythromycin, colonies were shown to have retained kanamycin resistance. Southern blot analysis, in conjunction with plasmid rescue to Escherichia coli confirmed that, after 100 culture generations in the presence of antibiotic selection pressure, pAEX‐5E had remained structurally stable and had not integrated into the B. anthracis genome.