Botulinum neurotoxigenic <i>C. butyricum</i> type E strains used in this study.

1<p>References 2, 6, 7, 8, 9.</p>2<p>Reference 6.</p

<i>Nru</i>I restriction map analysis of the <i>C. butyricum</i> type E strain BL5262 contig 1 sequence (http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1) (757,653 bp) as a linear DNA molecule (2a) and as a circular DNA molecule (2b), and relative positions of the <i>S1</i>, <i>latt</i>, <i>bl</i>, <i>mb</i>, and <i>dp</i> gene probes.

<p>The shaded area (∼ 168 kb) corresponds to the genetic region missing from the smaller megaplasmids of strains ISS-20, ISS-21, ISS-109, ISS-145/1.</p

Organization of the CRISPR-<i>cas</i> locus in contig 1 (http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1) of <i>C. butyricum</i> type E strain BL5262.

<p>The whole CRISPR-<i>cas</i> locus is 11,514 bp (nucleotides 175,848 through 187,362 of contig 1). ♦ CRISPR conserved repeat sequences. CRISPR hypervariable spacer sequences numbered according to their acquisition order, with the more recently added spacers having the highest numbers. The additional distinct spacers of strains ISS-190 and ISS-86 are coloured in yellow and green, respectively. ¹The 21st CRISPR spacer sequence matches a conserved sequence from putative phage terminase large subunit genes within the genomes of <i>C. botulinum</i> type E strain Alaska E43 and <i>C. botulinum</i> type B strain Eklund 17B (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071324#pone-0071324-t003" target="_blank">Table 3</a>).</p

PFGE patterns of <i>C. butyricum</i> type E strains digested with <i>Nru</i>I restriction enzyme (1a) and Southern hybridization analysis using gene probes <i>dp</i> (1b) and <i>S1</i> (1c).

<p>Strains: ISS-20 (lane 1); ISS-21 (lane 2); ISS-109 (lane 3); ISS-145/1 (lane 4); ISS-86 (lane 5); ISS-190 (lane 6). m.s. (lane 7): molecular standard, <i>Xba</i>I-digested DNA from <i>Salmonella</i> Braenderup strain H9812 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071324#pone.0071324-Hunter1" target="_blank">[13]</a>.</p

Gene specific probes and primer sets within the <i>C. butyricum</i> type E strain BL5262 contig 1 sequence (http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1) (757,653 bp).

<p>Gene specific probes and primer sets within the <i>C. butyricum</i> type E strain BL5262 contig 1 sequence (<a href="http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1" target="_blank">http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1</a>) (757,653 bp).</p

Analysis of the CRISPR spacers in <i>C. butyricum</i> type E strains BL5262 (http://www.ncbi.nlm.nih.gov/nuccore/NZ_ACOM01000001.1), ISS-86 (GenBank: KF150773) and ISS-190 (GenBank: KF150772).

1<p>Spacers are numbered according to their acquisition order, i.e. the more recently added spacers have the highest numbers.</p>2<p>Spacers 40, 43 and 44 are those of the CRISPR array of strain ISS-86.</p>3<p>Bold underlined nucleotides match the target sequences.</p>4<p>Putative prophage sequences within bacterial genomes were identified through the program Prophinder (<a href="http://aclame.ulb.ac.be/prophinder" target="_blank">http://aclame.ulb.ac.be/prophinder</a>).</p

Plasmid-borne <i>bont</i>/B PCR-RFLP subtype genes among <i>C. botulinum</i> strains.

<p>Plasmid-borne <i>bont</i>/B PCR-RFLP subtype genes among <i>C. botulinum</i> strains.</p

Southern blot membrane before (a) and after (b) S1 nuclease treatment.

<p>Strains: CDC-MDb2 (lanes 1, 11); CDC-1758 (lanes 2, 12); CDC-5078 (lanes 3, 13); CDC-1852 (lanes 4, 14); CDC-1872 (lanes 5, 15); CDC-628 (lanes 6, 16); CDC-1588 (lanes 7, 17); CDC-2978 (lanes 8, 18); CDC-706 (lanes 9, 19); CDC-4848 (lanes 10, 20). M.S. (Molecular standard: DNA isolated from <i>Salmonella enterica</i> serotype Braenderup strain H9812 and restricted with <i>Xba</i>I) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004829#pone.0004829-Hunter1" target="_blank">[23]</a>.</p

<i>C. botulinum</i> strains analyzed in this study, and genomic location of their <i>bont</i>/B and /A PCR-RFLP subtype genes.

1<p>CDC (Centers for Disease Control and Prevention, USA); ISS (Istituto Superiore di Sanità, Italy).</p>2<p>IB (infant botulism); FB (foodborne botulism); AB (animal botulism); uB (unknown botulism).</p>3<p>Bnp (non-proteolytic B); Bbv (bivalent B).</p>4<p>The size of the closest molecular standard bands are indicated.</p>5<p>Non-proteolytic <i>C. botulinum</i>.</p>6<p>n.d. = Not determined, because of DNA degradation.</p

Molecular size of the plasmids carrying the different <i>bont</i>/B subtype genes.

a<p>The plasmid sizes were deduced by comparison with a molecular standard <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004829#pone.0004829-Hunter1" target="_blank">[23]</a>.</p><p>Slight differences between the sizes of plasmids included in the same group were observed.</p