Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani (original) (raw)
Related papers
PLoS ONE, 2012
Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.
Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani
Toxicon, 2013
Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In C. tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, three two-component systems have been found to positively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation. 1. INTRODUCTION Among the large number of Clostridium species, which are bacteria from the environment, some of them produce potent neurotoxins and are responsible for neurological disorders in human and animals. Botulinum neurotoxins (BoNTs) inhibit the release of acetylcholine at peripheral cholinergic nerve terminals causing the flaccid paralysis of botulism, whereas tetanus neurotoxin (TeNT) blocks neurotransmitter release at synapses at the central nervous system causing the spastic paralysis of tetanus. In the last decades, numerous works have been performed to elucidate the structure and mechanism of action of the clostridial neurotoxins as well as their genetic organization and regulation. 2. CLOSTRIDIA PRODUCING NEUROTOXINS Clostridia capable of producing BoNT display heterogeneous bacteriological characters and are divided into several species and groups. The taxonomic position of the C. botulinum species was originally based on only one phenotype, the production of a BoNT, and non-toxic variant strains, although genetically related to C. botulinum, were assigned to different species such as Clostridium sporogenes and Clostridium subterminale. It appeared
The gene for component-II of botulinum C2 toxin
Veterinary Microbiology, 1998
The gene encoding component-II of the Clostridium botulinum C2 toxin was cloned from the chromosomal DNA of C. botulinum type C strain (C)-203U28, and the nucleotide sequence was determined. The gene (bc2h) encodes a protein with 721 amino acid residues and is located at 247 bp downstream of the gene for component-I. The N-terminal 16 amino acids were identical to those obtained by analysis of the purified component-II toxin. The ORF for bc2h had only 39% homology at the amino acid level with the C. perfringens iota-Ib protein and an ATP/GTP binding site which is present in the iota-Ib protein is missing from the protein encoded by bc2h. Both genes had a homologous region that predicts a transmembrane segment. #
Sequence Diversity of Genes Encoding Botulinum Neurotoxin Type F
Applied and Environmental Microbiology, 2010
Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was comp...
PLOS One, 2007
Background. Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A-G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression. Methodology/ Principal Findings. Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid. Conclusions/Significance. Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum. Citation: Smith TJ, Hill KK, Foley BT, Detter JC, Munk AC, et al (2007) Analysis of the Neurotoxin Complex Genes in Clostridium botulinum A1-A4 and B1 Strains: BoNT/A3, /Ba4 and /B1 Clusters Are Located within Plasmids. PLoS ONE 2(12): e1271.
Current Microbiology, 2016
A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the ''toxin complex.'' Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the ''toxin complex'' produced by C-N71. The ''toxin complex'' was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the ''toxin complex.'' Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic ''toxin complex'' of C-N71 may be valuable for the development of an oral drug delivery system.
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 2014
The whole genomes for six botulinum neurotoxin-producing clostridial strains were sequenced to provide references for under-represented toxin types, bivalent strains or unusual toxin complexes associated with a bont gene. The strains include three Clostridium botulinum Group I strains (CDC 297, CDC 1436, and Prevot 594), a Group II C. botulinum strain (Eklund 202F), a Group IV Clostridium argentinense strain (CDC 2741), and a Group V Clostridium baratii strain (Sullivan). Comparisons of the Group I genomic sequences revealed close relationships and conservation of toxin gene locations with previously published Group I C. botulinum genomes. The bont/F6 gene of strain Eklund 202F was determined to be a chimeric toxin gene composed of bont/F1 and bont/F2. The serotype G strain CDC 2741 remained unfinished in 20 contigs with the bont/G located within a 1.15Mb contig, indicating a possible chromosomal location for this toxin gene. Within the genome of C. baratii Sullivan strain, direct r...
Universal and specific quantitative detection of botulinum neurotoxin genes
BMC Microbiology, 2010
Background: Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin.