Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity

Carbon Nanotubes by a CVD Method. Part I: Synthesis and Characterization of the (Mg, Fe)O Catalysts

The controlled synthesis of carbon nanotubes by chemical vapor deposition requires tailored and wellcharacterized catalyst materials. We attempted to synthesize Mg1-xFexO oxide solid solutions by the combustion route, with the aim of performing a detailed investigation of the influence of the synthesis conditions (nitrate/urea ratio and the iron content) on the valency and distribution of the iron ions and phases. Notably, characterization of the catalyst materials is performed using 57Fe Mo¨ssbauer spectroscopy, X-ray diffraction, and electron microscopy. Several iron species are detected including Fe2+ ions substituting for Mg2+ in the MgO lattice, Fe3+ ions dispersed in the octahedral sites of MgO, different clusters of Fe3+ ions, and MgFe2O4-like nanoparticles. The dispersion of these species and the microstructure of the oxides are discussed. Powders markedly different from one another that may serve as model systems for further study are identified. The formation of carbon nanotubes upon reduction in a H2/CH4 gas atmosphere of the selected powders is reported in a companion paper

Genetic characterization of type A enterotoxigenic Clostridium perfringens strains

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains. © 2009 Deguchi et al

Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum

Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids

Grain-boundary chemistry and weak-link behavior of polycrystalline YBa[subscript 2]Cu[subscript 4]O[subscript 8]

Magnetic-susceptibility and grain-boundary-chemistry data were obtained on dense YBa[subscript 2]Cu[subscript 4]O[subscript 8] (Y1:2:4) samples. Transmission-electron microscopy showed that the grain boundaries were free of second phases and the dislocation density was low. Cation content, oxygen composition, and hole density were determined by the combined techniques of nanoprobe energy-dispersive x-ray spectroscopy and electron-energy-loss spectroscopy. A total of 25 pairs of grains was analyzed and the results indicated that grain boundary and bulk compositions do not differ. The relative orientations of the crystallites were determined, and the results show that a wide variety of misorientations was sampled. Almost all of the grain boundaries were fully oxygenated and there are no hole deficiencies, but the magnetic susceptibility measurements showed that the material is granular weak linked. These results indicate that Y 1:2:4 is a material in which clean, stoichiometric boundaries still form weak links