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Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

ABSTRACT: Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although they carry several putative beta-lactamase genes. Instead, we found that the megaplasmids are essential for growth of the neurotoxigenic C. butyricum type E strains at the relatively low temperature of 15°C, and are also relevant for growth of strains under limiting pH and salinity conditions, as well as under favorable environmental conditions. Moreover, the presence of the megaplasmids was associated with increased transcript levels of the gene encoding BoNT/E in the C. butyricum type E strains, indicating that the megaplasmids likely contain transcriptional regulators. However, the levels of BoNT/E in the supernatants of the cured and uncured strains were similar after 24 and 48 h culture, suggesting that expression of BoNT/E in the C. butyricum type E strains is not ultimately controlled by the megaplasmids. Together, our results reveal that the C. butyricum type E megaplasmids exert pleiotropic effects on the growth of their microbial hosts under optimal and limiting environmental conditions, and also highlight the possibility of original regulatory mechanisms controlling the expression of BoNT/E.

SUBMITTER: Scalfaro C

PROVIDER: S-EPMC4766289 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

Scalfaro Concetta C Iacobino Angelo A Grande Laura L Morabito Stefano S Franciosa Giovanna G

Frontiers in microbiology 20160225

Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although the ...[more]

PMID: 26941734

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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:Clostridium 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.Chromosome 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.The 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.

Project description:We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question.

Project description:Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P) and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human pathogen.

Dataset Information

Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

Publications

Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

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