Genomic insights into the evolution and ecology of botulinum neurotoxins.

Clostridial neurotoxins, which include botulinum neurotoxins (BoNTs) and tetanus neurotoxins, have evolved a remarkably sophisticated structure and molecular mechanism fine-tuned for the targeting and cleavage of vertebrate neuron substrates leading to muscular paralysis. How and why did this toxin evolve? From which ancestral proteins are BoNTs derived? And what is, or was, the primary ecological role of BoNTs in the environment? In this article, we examine these questions in light of recent studies identifying homologs of BoNTs in the genomes of non-clostridial bacteria, including Weissella, Enterococcus and Chryseobacterium. Genomic and phylogenetic analysis of these more distantly related toxins suggests that they are derived from ancient toxin lineages that predate the evolution of BoNTs and are not limited to the Clostridium genus. We propose that BoNTs have therefore evolved from a precursor family of BoNT-like toxins, and ultimately from non-neurospecific toxins that cleaved different substrates (possibly non-neuronal SNAREs). Comparison of BoNTs with these related toxins reveals several unique molecular features that underlie the evolution of BoNT's unique function, including functional shifts involving all four domains, and gain of the BoNT gene cluster associated proteins. BoNTs then diversified to produce the existing serotypes, including TeNT, and underwent repeated substrate shifts from ancestral VAMP2 specificity to SNAP25 specificity at least three times in their history. Finally, similar to previous proposals, we suggest that one ecological role of BoNTs could be to create a paralytic phase in vertebrate decomposition, which provides a competitive advantage for necrophagous scavengers that in turn facilitate the spread of Clostridium botulinum and its toxin.

Comparative pathogenomics of Clostridium tetani.

Clostridium tetani and Clostridium botulinum produce two of the most potent neurotoxins known, tetanus neurotoxin and botulinum neurotoxin, respectively. Extensive biochemical and genetic investigation has been devoted to identifying and characterizing various C. botulinum strains. Less effort has been focused on studying C. tetani likely because recently sequenced strains of C. tetani show much less genetic diversity than C. botulinum strains and because widespread vaccination efforts have reduced the public health threat from tetanus. Our aim was to acquire genomic data on the U.S. vaccine strain of C. tetani to better understand its genetic relationship to previously published genomic data from European vaccine strains. We performed high throughput genomic sequence analysis on two wild-type and two vaccine C. tetani strains. Comparative genomic analysis was performed using these and previously published genomic data for seven other C. tetani strains. Our analysis focused on single nucleotide polymorphisms (SNP) and four distinct constituents of the mobile genome (mobilome): a hypervariable flagellar glycosylation island region, five conserved bacteriophage insertion regions, variations in three CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems, and a single plasmid. Intact type IA and IB CRISPR/Cas systems were within 10 of 11 strains. A type IIIA CRISPR/Cas system was present in two strains. Phage infection histories derived from CRISPR-Cas sequences indicate C. tetani encounters phages common among commensal gut bacteria and soil-borne organisms consistent with C. tetani distribution in nature. All vaccine strains form a clade distinct from currently sequenced wild type strains when considering variations in these mobile elements. SNP, flagellar glycosylation island, prophage content and CRISPR/Cas phylogenic histories provide tentative evidence suggesting vaccine and wild type strains share a common ancestor.

[Characteristics of Clostridium tetani and laboratory diagnosis of tetanus]. / Charakterystyka Clostridium tetani i laboratoryjna diagnostyka tezca.

The causative agent of tetanus is the obligate anaerobic bacterium--Clostridium tetani. These bacteria form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. Infection generally occurs through wound contamination. We can distinguish several forms of tetanus: generalized, local and neonatal. Diagnosis of tetanus is based primarily on the patient's clinical symptoms (muscle cramps, painful back muscle spasms, generalized contractions of the arcuate curvature of the body) as well as on microbiological diagnosis. This article is a brief review of C. tetani and diagnosis of infections caused by these organisms in humans.

Molecular characterization of Clostridium tetani strains by pulsed-field gel electrophoresis and colony PCR.

Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.

[Differentiation of the main species of Clostridium by gas chromatography]. / Differentsiatsiia osnovnykh vidov klostridii metodom gazovoi khromatografii.

For the first time in the USSR the properties of microorganisms of the genus Clostridium have been studied with the use of the gas-chromatographic techniques. The analysis of the quantitative and qualitative composition of extracellular alcohols and carboxylic acids in 99 museum and newly isolated strains of 18 Clostridium species has made it possible to classify these microorganisms with 7 sharply differing groups. The above techniques permit the classification of clostridia with one of the groups within 2 hours if the microbial cultures have been grown in glucose-containing peptone yeast medium.

Electrophoretic study of Clostridium species.

Polyacrylamide gel electrophoretic analysis of soluble cellular proteins (without sodium dodecyl sulfate) of 70 Clostridium species indicated that the procedure was readily applicable to the differentiation of species in the genus. The protein patterns correlated well with the available DNA homology data and with most accepted differential tests. Results indicated that several earlier names for species were synonyms of those of accepted species and that two accepted species may be synonymous.

Taxonomy of Clostridium tetani and related species.

Clostridium tetani and its related species C. tetanomorphum, C. cochlearium and C. lentoputrescens were examined for DNA-DNA homology and biochemical properties. Two distinctly different groups were included under the name of C. tetanomorphum: one was identical with C. cochlearium and the name C. tetanomorphum was applied to the other group with some amendment of biochemical properties. Comparison of the type strain of C. lentoputrescens with wild strains obtained from horse faeces indicated that the name C. lentoputrescens should be abolished as a later synonym of C. cochlearium. Liquefaction of gelatin and spore shape, which have been used as the important criteria for differentiation of C. tetani-related species, were genetically insignificant.

Taxonomy of the Clostridia: ribosomal ribonucleic acid homologies among the species.

rRNA homologies have been determined on reference strains representing 56 species of Clostridium. Competition experiments using tritium-labelled 23S rRNA were employed. The majority of the species had DNA with 27 to 28% guanine plus cytosine (%GC). These fell into rRNA homology groups I and II, which were well defined, and a third group which consisted of species which did not belong in groups I and II. Species whose DNA was 41 to 45% GC comprised a fourth group. Thirty species were placed into rRNA homology group I on the basis of having 50% or greater homology with Clostridium butyricum, C. perfringens, C. carnis, C. sporogenes, C. novyi or C. pasteurianum. Ten subgroups were delineated in homology group I. Species in each subgroup either had high homology with a particular reference species or a similar pattern of homologies to all of the reference organisms. The eleven species in rRNA homology group II had 69% or greater homology to C. lituseburense. Species in groups I and II had intergroup homologies of 20 to 40%. The six species in group II had very low homologies with groups I and II. Negligible homology also resulted when five of the species were tested against the sixth, C. ramosum. The five species having DNA with 41 to 45% GC were C. innocuum, C. sphenoides, C. indolis, C. barkeri and C. orotic um. Little rRNA homology was apparent between C. innocuum and the other high % GC species or with several Bacillus species having similar %GC DNA. Correlations between homology results and phenotypic characteristics are discussed.

Characterization of clostridia by gas chromatography. I. Differentiation of species by cellular fatty acids.

Fatty acids of 41 strains representing 13 species of Clostridium were extracted directly from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparisons of the resulting chromatograms for the presence and relative amounts of large major peaks allowed rapid differentiation of C. perfringens, C. sporogenes, and C. bifermentans from each other and from 10 other species. Each of the three former species possessed a different characteristic fatty acid methyl ester profile that was exhibited by all strains tested within the respective species. Culture age and growth media influenced the relative proportions of certain of the acids, but such differences did not limit species differentiation.