Analysis of the Neurotoxin Complex Genes in Clostridium botulinum A1-A4 and B1 Strains: BoNT/A3, /Ba4 and /B1 Clusters Are Located within Plasmids

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

Plastid DNA variation in Prunus serotina var. serotina (Rosaceae), a North American tree invading Europe

Black cherry (Prunus serotina) is a tree from North America, where it is often used for economical purposes, whereas it is widespread and invasive in Europe. Plastid DNA variation was Wrst investigated in both its native and invasive ranges using microsatellite loci and sequences of three intergenic spacers (trnT-trnL, trnD-trnT and trnS-trnG). This analysis was focused on P. serotina var. serotina, with the inclusion of samples of closely related taxa. Length variation at a microsatellite locus (ccmp5) and a few sequence polymorphisms were identi- Wed among P. serotina samples. Four new primer pairs were then designed to speciWcally amplify variable regions and a combination of Wve markers was Wnally proposed for phylogeographic studies in P. serotina. These loci allow identiWcation of six chlorotypes in P. serotina var. serotina, which may be particularly useful to depict the maternal origins of European invasive population

Production of “super-males” of asparagus by anther culture and its detection with SSR-ESTs

Anther culture is used to develop asparagus “super-male” (di-haploids) in asparagus, which can be used to develop “all-male” varieties, by crossing them with suitable females; their progenies will be formed only by males which is advantageous for producers. This report describe a new anther culture protocol adapted to “Morado de Huétor”, a Spanish tetraploid landrace, and studied the different factors involved in callus proliferation success from anther explants such as the microspore development stage, or the type of stress used to induce the symmetric division of the microspores, to obtain a high success rate (90 %). For plantlets regenerates from anther culture (PRACs) regeneration we develop a proliferation media supplemented with a combination of pCPA and BA able to induce callus proliferation and plantlet regeneration in the same step in a 50 % of calli, simplifying the procedure. The high percentage of heterozygous male recovery, originated from somatic cells, is an important problem in the anther culture, and to elucidate the origin of PRACs we have combined different tools: ploidy analysis, characterization with the linked sex-marker Asp1-T7 and with EST-SRRs. We can establish that 50 % of PRACs obtained in this work were regenerated from diploid microspores of “Morado de Huétor”, regenerating diploid, di-diploid and tetra-diploid plantlets. The di-diploids males (MMmm) would generate a ratio male:female of 5:1 (83.3 %) and the tetra-diploid males (MMMMmmmm) a ratio male:female of 69:1 (98.6 %), so the tetra-diploid males could be considered “super-males” and be used to develop “all-male” varieties of “Morado de Huétor”.Fil: Regalado González, Jose Javier. Instituto de Horticultura Subtropical y Mediterránea La Mayora; España. Consejo Superior de Investigaciones Científicas; España. Universidad de Málaga; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carmona Martín, E.. Instituto de Horticultura Subtropical y Mediterránea La Mayora; España. Consejo Superior de Investigaciones Científicas; España. Universidad de Málaga; EspañaFil: Madrid, E.. Instituto de Agricultura Sostenible; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Moreno, R.. Universidad de Córdoba; EspañaFil: Gil, J.. Universidad de Córdoba; EspañaFil: Encina, C. L.. Instituto de Horticultura Subtropical y Mediterránea La Mayora; España. Consejo Superior de Investigaciones Científicas; España. Universidad de Málaga; Españ