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

Abstract

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

Genome analysis of thirteen Colombian clostridial strains by pulsed field gel electrophoresis

“…This fact was probably related to the formation of larger pores increasing the diffusion of enzymes, reagents and detergents which led to obtaining high purity DNA. It was also observed in this study that three proteinase K treatments led to agarose inserts becoming more translucent (suggesting efficient protein degradation by enzymes and thereby leading to obtaining DNA which had no remaining proteins), whilst one or two treatments were not enough to obtain suitable DNA (Wilkinson and Young, 1993;Lin and Johnson, 1995;Hielm et al 1998).…”

Genetic Characterization of Clostridium botulinum Type A Containing Silent Type B Neurotoxin Gene Sequences

“…The DNA preparations were digested with restriction endonucleases, electrophoresed through agarose gels, blotted onto nylon membranes, and hybridized with specific probes as described previously (8). PFGE was performed as described previously (8,9) at 180 V for 18 h, with a ramped pulse time of 0.5-10 s for BamHI, PvuII, and NdeI digests, and at 200 V for 22 h, and a ramped pulse time of 1-40 s for SmaI or XhoI digests.…”

Molecular Typing of Pasteurella pneumotropica Isolated from Rodents by Amplified 16S Ribosomal DNA Restriction Analysis and Pulsed‐Field Gel Electrophoresis

“…Although we tried a method which prevents DNA degradation by adding a free radical scavenger such as thiourea to the PFGE buffer (18), the DNA banding patterns were not obtained in this study. We further examined the formaldehyde fixation method (12), and heat treatment (22,24). However, the DNA banding patterns were not obtained.…”

“…For the further discrimination of the isolates that showed an indefinite banding pattern in the PFGE gels, we used 3 different methods including the formaldehyde fixation method (12), addition of 50 or 100 µM thiourea into the PFGE buffer (18) and heat treatment for 10 min at 50 C to inactivate DNase activity before embedding in a low-melting agarose (22,24).…”