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Title: Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

Abstract

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. In this paper, we sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7more » toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ 70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. Finally, this TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.« less

Authors:
 [1];  [1];  [1];  [2];  [2];  [1]
  1. California Dept. of Public Health, Richmond, CA (United States). Infant Botulism Treatment and Prevention Program
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Bioscience Division
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States); California Dept. of Public Health, Richmond, CA (United States)
Sponsoring Org.:
California Dept. of Public Health (United States)
OSTI Identifier:
1321719
Report Number(s):
LA-UR-12-23980
Journal ID: ISSN 1932-6203
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 9; Journal Issue: 5; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; biological science; toxins; clostridium botulinum; botulinum toxin; gene regulation; sequence alignment; sequence motif analysis; clostridium; RNA polymerase

Citation Formats

Dover, Nir, Barash, Jason R., Burke, Julianne N., Hill, Karen K., Detter, John C., and Arnon, Stephen S. Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters. United States: N. p., 2014. Web. doi:10.1371/journal.pone.0097983.
Dover, Nir, Barash, Jason R., Burke, Julianne N., Hill, Karen K., Detter, John C., & Arnon, Stephen S. Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters. United States. doi:10.1371/journal.pone.0097983.
Dover, Nir, Barash, Jason R., Burke, Julianne N., Hill, Karen K., Detter, John C., and Arnon, Stephen S. Thu . "Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters". United States. doi:10.1371/journal.pone.0097983. https://www.osti.gov/servlets/purl/1321719.
@article{osti_1321719,
title = {Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters},
author = {Dover, Nir and Barash, Jason R. and Burke, Julianne N. and Hill, Karen K. and Detter, John C. and Arnon, Stephen S.},
abstractNote = {Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. In this paper, we sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. Finally, this TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.},
doi = {10.1371/journal.pone.0097983},
journal = {PLoS ONE},
number = 5,
volume = 9,
place = {United States},
year = {Thu May 22 00:00:00 EDT 2014},
month = {Thu May 22 00:00:00 EDT 2014}
}

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  • Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2x10/sup +6/ viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6C, but only the terrestrial strains grew at 12.8C. None of the strains grew at 10C during prolonged incubation. The sodiummore » chloride tolerance and the pH sensitivity of the marine and the terestrial strains were determined at 30C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2x10/sup +6/ unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin.« less
  • A model system (chicken skins with chicken exudate) was used to determine if Clostridium botulinum type E (Beluga) spores, stressed by low dose irradiation, would develop and produce toxin at abuse temperatures of 10 and 30/sup 0/C in the absence of characteristic spoilage. Unstressed spores germinated, multiplied, and produced toxin on vacuum-packed chicken skins, stored at either 30 or 10/sup 0/C. Cell numbers increased faster and toxin was evident sooner at 30/sup 0/C than at 10/sup 0/C. At 30/sup 0/C, growth occurred and toxin was produced more slowly when samples were incubated aerobically than anaerobically. When samples were incubated aerobicallymore » at 10/sup 0/C, no toxin was detected within a test period of 14 days. An irradiation dose of 0.3 Mrad at 5/sup 0/C reduced a spore population on vacuum-sealed chicken skins by about 90%. The surviving population produced toxin at 30/sup 0/C under either aerobic or anaerobic conditions, at 10/sup 0/C no toxin was detected even on skins incubated anaerobically. Under the worst conditions (30/sup 0/C, vacuum packed) toxin was not detected prior to characteristic spoilage caused by the natural flora surviving 0.3 Mrad.« less