Transposon Tn916 mutagenesis in Clostridium botulinum

Lin, Wei‐Jen; Johnson, Eric A.

doi:10.1128/aem.57.10.2946-2950.1991

Cited by 26 publications

(8 citation statements)

References 23 publications

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“…Mutagenesis of C. botulinum 62A was performed with the tetracycline-resistant (Tc r ) transposon Tn916. Tn916 was transferred from Enterococcus faecalis CG110 to C. botulinum by a filter-mating procedure as previously described (26). Fourteen Tc r transconjugants were selected from independent mating experiments, purified by single colony isolation, and examined by the following PFGE analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Fourteen Tc r transconjugants were selected from independent mating experiments, purified by single colony isolation, and examined by the following PFGE analysis. The copy number of Tn916 in each Tc r transconjugant was determined by Southern analysis as previously described (26). Briefly, Tn916 is known to contain one internal HindIII site; therefore, each copy of Tn916 in the chromosome will generate two hybridized bands when chromosomal DNA is probed with HindIII-digested Tn916.…”

Section: Methodsmentioning

confidence: 99%

“…After electrophoresis, DNA fragments in the gels were depurinated in 0.2 N HCl for 30 min and vacuum transferred onto nylon membranes (Gelman, Ann Arbor, Mich.) by the alkaline transfer procedure (31). Plasmid pAM120 (26), which contains Tn916, was labeled with 32 P by using a nick translation kit (Bethesda Research Laboratories). A plasmid control, pAM120LT, demonstrated that the plasmid not containing Tn916 did not hybridize to C. botulinum strains, as was demonstrated in a previous study (26).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid pAM120 (26), which contains Tn916, was labeled with 32 P by using a nick translation kit (Bethesda Research Laboratories). A plasmid control, pAM120LT, demonstrated that the plasmid not containing Tn916 did not hybridize to C. botulinum strains, as was demonstrated in a previous study (26). A bacteriophage was cured from strain 62A by adding 0.2% mitomycin in early log growth.…”

Section: Methodsmentioning

confidence: 99%

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Genome analysis of Clostridium botulinum type A by pulsed-field gel electrophoresis

Lin

Johnson

1995

Appl Environ Microbiol

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Genomic DNA from type A Clostridium botulinum was digested with restriction endonucleases that cut at rare sites, and the large fragments were separated by pulsed-field gel electrophoresis. Of 15 restriction enzymes tested, MluI, RsrII, SmaI, NruI, KspI, NaeI, and XhoI generated satisfactory digestion patterns of genomic DNA of various C. botulinum strains, enabling the use of the method for genomic fingerprinting. The genomes of four group I (type A) C. botulinum strains examined had similar restriction patterns. However, each strain had unique digestion patterns, reflecting genotypic differences. The genome size of C. botulinum strain 62A was estimated to be 4,039 ؎ 40 kbp from the summation of restriction fragments from MluI, RsrII, and SmaI digestions. Genes encoding proteins involved in the toxinogenicity of C. botulinum, including neurotoxin, hemagglutinin A, and genes for a temperate phage, as well as various transposon Tn916 insertion sites in C. botulinum 62A, were mapped by pulsed-field gel electrophoresis. The genes encoding neurotoxin and hemagglutinin A-1, were located on the same fragment in several cases, indicating their probable physical linkage. The macrorestriction analysis established here should be useful for genetic and epidemiological studies of C. botulinum. Clostridium botulinum is currently classified into four physiological groups (I to IV) (6, 16, 39) that differ widely in phenotypic properties and nucleic acid relatedness (16, 22). Although strains in the four groups are quite different physiologically and genetically, all have the common property of producing a characteristic botulinum neurotoxin (BoNT), of which seven serotypes of toxin (A through G) are recognized. When grown in media, foods, or the human intestine, C. botulinum produces toxin complexes where the BoNT is noncovalently linked with nontoxic proteins, including hemagglutinins (Hns) and a nontoxic nonhemagglutinating protein (NTNH) (10, 30, 32, 38). Strains in groups I and II, forming complexes of A, B, E, or F toxins, have been responsible for nearly all cases of human botulism (16). Although the nucleotide sequences of the genes encoding all seven serotypes of the BoNTs as well as tetanus toxin and some Hns and NTNHs have been determined (3, 4, 9, 13, 15, 17, 19, 21, 22a, 40, 41, 46), little is known regarding the regulation of expression of the various genes and the organization of the genes for the toxin complexes on host DNA. The genes for BoNT, Hn, and NTNH are closely linked on their host DNAs in C. botulinum types A (9), B (9), C (15, 18, 41), D (11), E (15), F (9), and G (50). In type C, the neurotoxin gene may form an operon with a gene for a nontoxic component (17), which operon may also exist in type A (48). The locations of the toxin-associated genes have been studied for certain serotypes. In types C and D, genes for BoNT, Hn, and NTNH are located on bacteriophages (tox ϩ bacteriophages) (11, 12, 41). The genes encoding the toxin complex are

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Genome analysis of Clostridium botulinum type A by pulsed-field gel electrophoresis

Lin

Johnson

1995

Appl Environ Microbiol

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“…Total genomic DNA was isolated from the C. botulinum strains by lysozyme–proteinase K treatment as described previously [15]. Fragments encoding BoNT cluster genes were amplified from C. botulinum strains Kyoto‐F and NCTC 2916 using PCR as outlined in Table 1 and Fig.…”

Section: Methodsmentioning

confidence: 99%

Nucleotide sequence and transcriptional analysis of the type A2 neurotoxin gene cluster inClostridium botulinum

Dineen

Bradshaw

Karasek

et al. 2004

FEMS Microbiology Letters

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The nucleotide sequences of the upstream regions of the botulinum neurotoxin type A1 (BoNT/A1) cluster of Clostridium botulinum strain NCTC 2916 and the BoNT/A2 cluster of strain Kyoto-F were determined. A novel gene, designated orfx3, was identified following the orfx2 gene in both clusters. ORF-X2 and ORF-X3 exhibit similarity to the BoNT cluster associated P-47 protein. The BoNT/A1 and BoNT/A2 clusters share a similar gene arrangement, but exhibit differences in the spacing between certain genes. Sequences with similarity to transposases were identified in these intergenic regions, suggesting that these differences arose from an ancestral insertion event. Transcriptional analysis of the BoNT/A2 cluster revealed that the genes of the cluster are primarily synthesized as three polycistronic transcripts. Two divergent polycistronic transcripts, one encoding the orfx1, orfx2, and orfx3 genes, the second encoding the p47, ntnh, and bont/a2 genes, are transcribed from conserved BoNT cluster promoters. The third polycistronic transcript, expressed at low levels, encodes the positive regulatory botR gene and the orfx genes. This is the first complete analysis of a botulinum toxin A2 cluster.

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