Project description:The purpose of this study was to compare the gene content of several group II C. botulinum strains to determine the utility of DNA microarrays for subtyping this organism. Group II type B, E, and F C. botulinum strains were compared in a study to examine the gene content of a unique type E strain (CDC66177).
Project description:The purpose of this study was to compare the gene content of several group II C. botulinum strains to determine the utility of DNA microarrays for subtyping this organism. Group II type B, E, and F C. botulinum strains were compared in a study to examine the gene content of a unique type E strain (CDC66177). One replicate of genomic DNA isolated from 16 type E strains, 4 type B strains, and 2 type F strains were examined.
Project description:The purpose of this study was to determine the level of genomic content similarity among selected strains of Clostridium botuinum type F strains. In this study, strains were selected that had already been chacaterized by botulinum neurotoxin gene sequencing. Strains harboring bont/F1, bont/F4 and bont/F5 were compared.
Project description:Genomic DNA of 61 strains of proteolytic Clostridium botulinum or Clostridium sporogenes was subjected to analysis by DNA microarray.
Project description:The PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships. Clostridium botulinum produces botulinum neurotoxin (BoNT)and is classified as a “Category A” select agent. BoNT can be classified into seven serotypes designated A-G. There is considerable genetic variation within these serotypes, as demonstrated by the recognition of at least 47 subtypes. The most studied serotype, BoNT/A, has been found in a large and diverse group of clostridia, most of which express the subtype BoNT/A1. The BoNT/A1 producing C. botulinum strain ATCC 3502, used to obtain an initial annotated genome sequence, is not representative of the diverse clostridia group producing BoNT. Nearly 50% of C. botulinum strains producing BoNT/A1 have been shown to also encode unexpressed variants of BoNT/B with a distinct cluster arrangement. This nucleotide cluster is completely absent from the published genome sequence. In addition, a recently identified novel BoNT/A1 strain lacks the gene cluster seen in the genome sequence of ATCC 3502. Furthermore, a strain designated Hall A Hyper differs greatly from the sequenced strain as indicated by its ability to produce higher quantities of BoNT/A1. The genetic and phenotypic basis for this difference in BoNT expression is currently unknown, and the sequences of the BoNT gene and the cluster are identical in both strains. This observation supports the hypothesis that genes outside the toxin cluster are involved in the regulation and maturation of BoNT. The flow of genetic information within this group motivated us to identify novel genes for the purpose of creating a “species” DNA microarray to better understand the ancestral relationships among its members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based array), 20 diverse C. botulinum strains were selected for sequencing. Sequence information obtained from this project, and from other publicly available sources, led to the development of a comprehensive species microarray for C. botulinum group members. The availability of the C. botulinum species DNA microarray has allowed us to carry out a collaborative CGH genotyping project to validate this microarray as well as understand the phylogenomic relationships among members of C. botulinum group.
Project description:Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp) which carry 3,650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbours a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of C. difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.
Project description:In this study, we identified the O-glycosylation sites in deletion constructs of recombinant Clostridium botulinum flagellin (CbFla) expressed from the E. coli strain, EV136 (K1:K12 strains engineered by Prof. Eric Vimr’s group to accumulate CMP-sialic acid in the cytosol). The recombinant flagellins were expressed with or without co-expression of motility associated factor (Maf) (flagellin nonulosonic acid glycosyltransferase) in these strains, purified and subjected to MS/MS analysis at Taplin Biological Mass Spectrometry facility. We also studied the O-glycosylation of recombinant Clostridium botulinum flagellin (CbFla) co-expressed with Geobacillus kaustophilus Maf. We also studied the O-glycosylation of flagellin chimeras made by grafting mini-flagellin sequences on to an unrelated protein with an F-type lectin domain from Streptosporangium roseum SrNaFLD.
Project description:In this study, we identified the O-glycosylation sites in recombinant Clostridium botulinum flagellin (CbFla) and Geobacillus kaustophilus flagellins, GkFlaA1 and GkFlaA2, expressed from the E. coli strains, EV136 and EV240 (K1:K12 strains engineered by Prof. Eric Vimr’s group to accumulate CMP-sialic acid in the cytosol), EV36 (wildtype K1:K12 strain), and BL21(DE3) (laboratory strain used for protein expression using T7 RNA polymerase). The recombinant flagellins were expressed with or without co-expression of motility associated factor (Maf) (flagellin nonulosonic acid glycosyltransferase) in these strains, purified and subjected to MS/MS analysis at Taplin Biological Mass Spectrometry facility.