The 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, theta-replicating, non-mobilizable shuttle plasmid (pAEX-5E) was determined in fully virulent (pX01(+)/pX02(+) ), partially cured (pX01(+)/pX02(-) and pX01(-)/pX02(+)) and fully cured (p X01(-)/pX02(-)) derivatives of Bacillus anthracis var. Nem Hampshire. Under the growth conditions used (L-broth, 37 degrees C, aerobic, batch culture) , pAEX-SE remained segregationally stable in the pW01(-)/pX02(+) and pX01-/ pX02- derivatives for in excess of 100 culture generations, but was expelle d from the pX01(+)/pX02(+) and pX01(+)/pX02(-) derivatives (100% loss occur red after 101 +/- 3.8 and 51+/-6.0 culture generations, respectively). In t he presence of antibiotic selection pressure to maintain pAEX-SE (5 mu g er ythromycin ml(-1)) no comparable loss of pX01 or pX02 was observed over 100 generations of growth:th in any of the derivatives of B. anthracis. Under these conditions the pX01(+)/pX02(-) - derivative had an extended culture d oubling time (t(d), +/- S.E. of the mean) of 75.3 +/- 1.4 min compared with 47.3 +/- 1.1, +/- 6.2 +/- 0.86 and 43.2 +/- 1.2 min for the pX01(-)/pX02() , pX01(-)/pX02(+) and pX01-/pX02- derivatives, respectively. That antibio tic resistance was pAEX-5E-mediated was confirmed using a second antibiotic marker (kanamycin). After 100 generations of growth in the presence of ery thromycin, colonies mere shown to have retained kanamycin resistance. South ern blot analysis, in conjunction with plasmid rescue to Escherichia coli c onfirmed that, after 100 culture generations in the presence of antibiotic selection pressure, pAEX-5E had remained structurally stable and had not in tegrated into the B. anthracis genome.