Table 1.
Clostridia strains used in this study.
Figure 1.
PFGE experiments showing the presence of an extrachromosomal mega-sized DNA band of > 600 kb in the undigested genomic DNA preparations from 10 neurotoxigenic C. butyricum 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).
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 C. butyricum strain ATCC 19398. S.e., XbaI-digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 35). S.c., Saccharomyces cerevisiae chromosomal DNA size marker (Biorad). PFGE conditions: 5–60 s pulse at 6 V/cm for 18 h (1a), and 50–90 s pulse at 6 V/cm for 20 h (1b). 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 (Figure 1b): >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 Figures 1a and 1b), 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.
Figure 2.
PFGE analysis of genomic DNA of six neurotoxigenic C. butyricum type E strains representative of the different megaplasmids sizes: ISS-109 (lanes 1 and 2); ISS-190 (lanes 3 and 4); KZ-1886 (lanes 5 and 6); KZ-1890 (lanes 7 and 8); LCL-063 (lanes 9 and 10); LCL-155 (lanes 11 and 12).
S.e., XbaI-digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 35). PFGE conditions: 5–60 s pulse at 6 V/cm for 18 h. − and + indicate absence and presence of treatment with ATP-dependent exonuclease, respectively. The megaplasmid bands disappeared after treatment with the ATP-dependent nuclease, an enzyme that selectively digests linear DNA molecules, indicating that all megaplasmids had a linear structure (lanes 2, 4, 6, 8, 10, 12). The smaller plasmids of strains ISS-109 (lanes 1 and 2) and ISS-190 (lanes 3 and 4) were not susceptible to the ATP-dependent nuclease treatment, indicating that they had a circular structure. The ∼167 kb band of strain KZ-1890 disappeared after the ATP-dependent nuclease treatment (lanes 7 and 8), indicating that it was a linear DNA molecule; however, the same plasmid of strain KZ-1890 showed inconsistent PFGE migration typical of supercoiled DNA (lane 9 of Figures 1a and 1b). Hence, the smaller plasmid of strain KZ-1890 can exist in both linear and supercoiled forms, consistent with the behavior expected for circular DNA.
Figure 3.
PFGE of undigested genomic DNA of non-neurotoxigenic C. butyricum strains (lanes 1–4) and C. botulinum type E strains (lanes 5–7).
Strains: UC-9035 (lane 1); UC-9041 (lane 2); GP1 (lane 3); ATCC 19398 (lane 4); CDC-5234 (lane 5); CDC-4581 (lane 6); CDC-5380 (lane 7). S.e., XbaI-digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 35). PFGE conditions: 5–60 s pulse at 6 V/cm for 18 h. Extrachromosomal bands close to the 54.7 kb band of the molecular standard (S.e.) are evident in lane 3 (non-neurotoxigenic C. butyricum strain GP1) and lane 6 (C. botulinum type E strain CDC-4581).
Figure 4.
Clustering analysis of the PFGE profiles of the ten neurotoxigenic C. butyricum type E strains obtained by XhoI (4a) and SmaI (4b) endonucleases.
The similarity of PFGE profiles was evaluated using the Bionumerics software (version 4.0) (Applied Maths, Sint-Martens-Latem, Belgium); a similarity level of ≥ 90% was used to define PFGE groups. The 10 neurotoxigenic C. butyricum type E strains were divided in 6 PFGE groups with both endonucleases.
Figure 5.
PFGE of undigested genomic DNA of strains ISS-145/1 (lane 1); ISS-20 (lane 2); ISS-21 (lane 3); ISS-109 (lane 4); ISS-86 (lane 5); ISS-190 (lane 6); KZ-1886 (lane 7); KZ-1890 (lane 8); LCL-063 (lane 9); LCL-155 (lane 10).
S.e., XbaI-digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 34). PFGE conditions: 5–60 s pulse at 6 V/cm for 18 h (5a). Southern hybridization with a bont/E gene probe showing that the gene probe hybridized to the chromosome bands of strains (5b). Southern hybridization with a β-lactamase gene probe showing that the gene probe hybridized to the megaplasmids of strains ISS-145/1 (lane 1); ISS-20 (lane 2); ISS-21 (lane 3); ISS-109 (lane 4); ISS-86 (lane 5); ISS-190 (lane 6); LCL-063 (lane 9); LCL-155 (lane 10) (5c).
Figure 6.
PFGE of XhoI-digested DNA of strains ISS-145/1 (lane 1); ISS-20 (lane 2); ISS-21 (lane 3); ISS-109 (lane 4); ISS-86 (lane 5); ISS-190 (lane 6); KZ-1886 (lane 7); KZ-1890 (lane 8); LCL-063 (lane 9); LCL-155 (lane 10).
S.e., XbaI-digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 34). PFGE conditions: 4–40 s pulse at 6 V/cm for 18 h (6a). Southern hybridization with a bont/E gene probe showing that the gene probe hybridized to single restriction bands, as indicated by the black arrows (6b).
Figure 7.
PFGE of undigested genomic DNA of strains ATCC 19398 (lane 1); 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) (7a).
S.e., XbaI-digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 35). PFGE conditions: 5–60 s pulse at 6 V/cm for 18 h. Southern hybridization with a specific mobA gene probe showing that the gene probe hybridized to the smaller plasmids observed in lanes 2–7 (strains ISS-145/1, ISS-20, ISS-21, ISS-109, ISS-86, ISS-190) (7b).
Table 2.
Primers used to generate the specific gene probes for the Southern blot experiments.