PFGE experiments showing the presence of an extrachromosomal mega-sized DNA band of > 600 kb in the undigested genomic DNA preparations from 10 neurotoxigenic <i>C. butyricum</i> type E strains: ISS-145/1 (lane 2); ISS-20 (lane 3); ISS-21 (lane 4); ISS-109 (lane 5); ISS-86 (lane 6); ISS-190 (lane 7); KZ-1886 (lane 8); KZ-1890 (lane 9); LCL-063 (lane 10); LCL-155 (lane 11).

<p>An additional extrachromosomal smaller band is evident in the DNA preparations from neurotoxigenic type E strains ISS-145/1, ISS-20, ISS-21, ISS-109, ISS-86, ISS-190 (lanes 2–7), and KZ-1890 (lane 9). Lane 1, undigested genomic DNA from non-neurotoxigenic <i>C. butyricum</i> strain ATCC 19398. S.e., <i>XbaI</i>-digested genomic DNA fragments of <i>Salmonella enterica</i> serotype Braenderup strain H9812 (ref. 35). S.c., <i>Saccharomyces cerevisiae</i> chromosomal DNA size marker (Biorad). PFGE conditions: 5–60 s pulse at 6 V/cm for 18 h (<b>1a</b>), and 50–90 s pulse at 6 V/cm for 20 h (<b>1b</b>). At the different PFGE conditions applied, the apparent sizes of the extrachromosomal mega-sized DNA bands (lanes 2–11) did not change in relation to the linear bands of the molecular size standards, consistent with the behaviour expected for linear DNA molecules. The mega-band sizes were estimated as follows (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021706#pone-0021706-g001" target="_blank">Figure 1b</a>): >610 kb for strains ISS-20, ISS-21, ISS-109, ISS-145/1 (lanes 2–5); ∼825kb for strains ISS-86, ISS-190, LCL-063 and LCL-155 (lanes 6, 7, 10 and 11); and between 750 and 785 kb for strains KZ-1886 and KZ-1890 (lanes 8, 9). Migration of the extrachromosomal smaller DNA bands relative to the linear DNA markers was inconsistent (lanes 2–7 and lane 9 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021706#pone-0021706-g001" target="_blank">Figures 1a and 1b</a>), consistent with the behavior expected for circular super-coiled DNA molecules: as a consequence, the actual size of the extrachromosomal smaller bands of strains ISS-145/1, ISS-20, ISS-21, ISS-109, ISS-86, ISS-190 and KZ-1890 could not be determined.