Confirmation of tagging <i>C. botulinum</i> BoNT-encoding plasmids pBotCDC-A3-Erm (strain CDC-A3), pCLJ-Erm (strain 657Ba) and pCLL-Erm (strain Eklund 17B) by PFGE and Southern hybridization analysis.

<p>(<b>B</b>) Ethidium bromide stained PFGE of nondigested DNA samples from <i>C. botulinum</i> strains: wild type CDC-A3 (Lane 1), CDC-A3580s1 (Lane 2), wild type 657Ba (Lane 3), 657Ba-CT4 (Lane 4), wild type Eklund 17B (Lane 5) and Eklund17B-CT11 (Lane 6); Lambda PFG Marker (Lane M), (New England Biolabs). The position of BoNT-encoding plasmids is indicated with arrows. (<b>A</b>) Southern hybridization with the <i>bont/A3</i> probe (Lanes 1 and 2); the <i>bont/bvB</i> probe (Lanes 3 and 4) and the <i>bont/npB</i> probe (Lanes 5 and 6); (<b>C</b>) Southern hybridization with the <i>ermB</i> probe. PFGE conditions: 6V/cm, 12°C, 1–20 s pulse time, 24 h.</p

Comparison of predicted ORFs of pCLL with plasmids of <i>Clostridium perfringens</i>.

<p>Comparison of predicted ORFs of pCLL with plasmids of <i>Clostridium perfringens</i>.</p

Confirmation of plasmid pBotCDC-A3-Erm transfer from <i>C. botulinum</i> strain CDC-A3580s1 to strain LNT01 by PFGE and Southern hybridization analysis.

<p>(<b>A</b>) Ethidium bromide stained PFGE of <i>C. botulinum</i> DNA samples: <i>Sma</i>I digested DNA of <i>C. botulinum</i> strain LNT01 (Lanes 1 and 7), CDC-A3 wild type (Lanes 2 and 8), CDC-A3580s1 (Lanes 3 and 9), and LNT01 transconjugants (pBotCDC-A3-Erm) (Lanes 4–6 and 10–12); Lanes 1–6, nondigested DNA samples; Lanes 7–12, <i>Sma</i>I digested DNA samples. Lambda PFG Marker (Lane M), New England Biolabs. The position of the pBotCDC-A3 plasmid is indicated with an arrow. Southern hybridization with: (<b>B</b>) the <i>ermB</i> probe, and (<b>C</b>) the <i>bont/A3</i> probe. PFGE conditions: 6V/cm, 12°C, 1–26 s pulse time, 24 h.</p

Confirmation of plasmid pCLL-Erm transfer from <i>C. botulinum</i> strain Eklund 17BCT11 to strain LNT01.

<p>(<b>A</b>) Ethidium bromide stained PFGE of <i>C. botulinum</i> strains: LNT01 (Lanes 1 and 7), wild type strain Eklund 17B (Lanes 2 and 8); Eklund 17BCT11 (Lanes 3 and 9) and LNT01 transconjugants (pCLL-Erm) (Lanes 4–6 and Lanes10–12). Lanes 1–6, nondigested DNA samples; Lanes 7–12, <i>Nar</i>I digested DNA samples. Lambda PFG Marker (Lane M), New England Biolabs. The position of the pCLL plasmid is indicated with an arrow. Southern hybridization with: (<b>B</b>) the <i>ermB</i> probe, and (<b>C</b>) the <i>bont/npB</i> probe. PFGE conditions: 6V/cm, 12°C, 1–20 s pulse time, 24 h.</p

Confirmation of tagging <i>C. botulinum</i> BoNT-encoded plasmids pBotCDC-A3-Erm (strain CDC-A3), pCLJ-Erm (strain 657Ba) and pCLL-Erm (strain Eklund 17B) by PCR analysis.

<p>PCR products of wild-type <i>C. botulinum</i> strains CDC-A3 (Lane 1), 657Ba (Lane 4) and Eklund 17B (Lane 7) and two erythromycin resistant, thiamphenicol sensitive clones of each of CDC-A3 (Lanes 2 and 3), 657Ba (Lanes 5 and 6) and Eklund 17B (Lanes 8 and 9) strains; 1 kb Plus ladder (Invitrogen) (Lane M).</p

Confirmation of plasmid pCLK-Erm and pCLJ-Erm transfer to <i>C. botulinum</i> Hall A-<i>hyper</i>-Tn<i>916</i> mutant strain.

<p>(<b>A</b>) Ethidium bromide stained PFGE of <i>C. botulinum</i> strains: wild type Hall A-<i>hyper</i> (Lane 1), Hall A-<i>hyper</i>/Tn<i>916</i> mutant (Lane 2); Hall A-<i>hyper</i>/Tn<i>916</i>/pBotCDCA3-Erm (Lane 3); CDC-A3 plasmid-cured (Lane 4); wild type CDC-A3 (Lane 5). (<b>D</b>) Ethidium bromide stained PFGE of <i>C. botulinum</i> strains: wild type Hall A-<i>hyper</i> (Lane 6), Hall A-<i>hyper</i>/Tn<i>916</i> mutant (Lane 7); Hall A-<i>hyper</i>/Tn<i>916</i>/pCLJ-Erm (Lanes 8 and 9); 657Ba-CT4 (Lane 10); wild type Hall A-<i>hyper</i> (Lane 11). Nondigested DNA and <i>Sma</i>I digests were loaded on the gels as indicated below the lanes. Lambda PFG Marker (Lane M), New England Biolabs. The position of the pBotCDC-A3 and pCLJ plasmids is indicated with arrows. Southern hybridization with: (<b>B</b> and <b>E</b>) the <i>ermB</i> probe; and (<b>C</b> and <b>F</b>) the <i>tet</i> probe. PFGE conditions: 6V/cm, 12°C, 1–26 s pulse time, 24 h.</p

Oligonucleotide primers used in this study.

<p>Oligonucleotide primers used in this study.</p

Confirmation of plasmid pCLJ-Erm transfer from <i>C. botulinum</i> strain 657BaCT4 to strain LNT01.

<p>(<b>A</b>) Ethidium bromide stained PFGE of <i>C. botulinum</i> strains: LNT01 (Lanes 1 and 7), wild type strain 657Ba (Lanes 2 and 8); 657BaCT4 (Lanes 3 and 9) and LNT01 transconjugants (pCLJ-Erm) (Lanes 4–6 and 10–12); Lanes 1–6, nondigested DNA samples; Lanes 7–12, <i>Xho</i>I digested DNAsamples. Lambda PFG Marker (Lane M), New England Biolabs. The position of the pCLJ plasmid is indicated with an arrow. Southern hybridization with: (<b>B</b>) the <i>ermB</i> probe and (<b>C</b>) the <i>bont/bvB</i> probe. PFGE conditions: 6V/cm, 12°C, 1–26 s pulse time, 24 h.</p