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Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

ABSTRACT: Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

SUBMITTER: Wang X

PROVIDER: S-EPMC92410 | biostudies-literature | 2000 Nov

REPOSITORIES: biostudies-literature

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Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

Wang X X Maegawa T T Karasawa T T Kozaki S S Tsukamoto K K Gyobu Y Y Yamakawa K K Oguma K K Sakaguchi Y Y Nakamura S S

Applied and environmental microbiology 20001101 11

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E ge ...[more]

PMID: 11055954

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Project description:Clostridium butyricum, the type species of the genus Clostridium, is considered an obligate anaerobe, yet it has been shown to grow in the presence of oxygen. C. butyricum strains atypically producing the botulinum neurotoxin type E are the leading cause of type E human botulism in Italy. Here, we show that type E botulinum neurotoxin-producing C. butyricum strains growing exponentially were able to keep growing and producing toxin in vitro upon exposure to air, although less efficiently than under ideal oxygen-depleted conditions. Bacterial growth in air was maintained when the initial cell density was higher than 103 cells/ml. No spores were detected in the cultures aerated for 5 h. To understand the biological mechanisms allowing the adaptation of vegetative cells of C. butyricum type E to oxygen, we compared the proteome and metabolome profiles of the clostridial cultures grown for 5 h under either aerated or anaerobic conditions. The results indicated that bacterial cells responded to oxygen stress by slowing growth and modulating the expression of proteins involved in carbohydrate uptake and metabolism, redox homeostasis, DNA damage response, and bacterial motility. Moreover, the ratio of acetate to butyrate was significantly higher under aeration. This study demonstrates for the first time that a botulinum neurotoxin-producing Clostridium can withstand oxygen during vegetative growth. IMPORTANCE Botulinum neurotoxins, the causative agents of the potentially fatal disease of botulism, are produced by certain Clostridium strains during vegetative growth, usually in anaerobic environments. Our findings indicate that, contrary to current understanding, the growth of neurotoxigenic C. butyricum strains and botulinum neurotoxin type E production can continue upon transfer from anaerobic to aerated conditions and that adaptation of strains to oxygenated environments requires global changes in proteomic and metabolic profiles. We hypothesize that aerotolerance might constitute an unappreciated factor conferring physiological advantages on some botulinum toxin-producing clostridial strains, allowing them to adapt to otherwise restrictive environments.

Project description:BackgroundClostridium botulinum is a taxonomic designation for at least four diverse species that are defined by the expression of one (monovalent) or two (bivalent) of seven different C. botulinum neurotoxins (BoNTs, A-G). The four species have been classified as C. botulinum Groups I-IV. The presence of bont genes in strains representing the different Groups is probably the result of horizontal transfer of the toxin operons between the species.ResultsChromosome and plasmid sequences of several C. botulinum strains representing A, B, E and F serotypes and a C. butyricum type E strain were compared to examine their genomic organization, or synteny, and the location of the botulinum toxin complex genes. These comparisons identified synteny among proteolytic (Group I) strains or nonproteolytic (Group II) strains but not between the two Groups. The bont complex genes within the strains examined were not randomly located but found within three regions of the chromosome or in two specific sites within plasmids. A comparison of sequences from a Bf strain revealed homology to the plasmid pCLJ with similar locations for the bont/bv b genes but with the bont/a4 gene replaced by the bont/f gene. An analysis of the toxin cluster genes showed that many recombination events have occurred, including several events within the ntnh gene. One such recombination event resulted in the integration of the bont/a1 gene into the serotype toxin B ha cluster, resulting in a successful lineage commonly associated with food borne botulism outbreaks. In C. botulinum type E and C. butyricum type E strains the location of the bont/e gene cluster appears to be the result of insertion events that split a rarA, recombination-associated gene, independently at the same location in both species.ConclusionThe analysis of the genomic sequences representing different strains reveals the presence of insertion sequence (IS) elements and other transposon-associated proteins such as recombinases that could facilitate the horizontal transfer of the bonts; these events, in addition to recombination among the toxin complex genes, have led to the lineages observed today within the neurotoxin-producing clostridia.

Dataset Information

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

Publications

Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains.

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