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1) Product Images from "Comparative pathogenomics of Clostridium tetani"
Article Title: Comparative pathogenomics of Clostridium tetani
Journal: PLoS ONE
Figure Legend Snippet: Electron microscopy of bacteriophages isolated from C . tetani . Representative transmission electron microscopic (TEM) images of UV-induced bacteriophages isolated from C . tetani strains C2, ATCC 19406, ATCC 453, and ATCC 9441. Distinct morphological features are evident in isolated bacteriophage, e.g. contractile tail for ATCC 453 and long, flexible tails for ATCC 19406. Note the presence of striations of phage tails observed for ATCC 453. Empty phage heads, lacking tails were observed in phage isolates from ATCC 9441. Scale bar = 50 nm. See S1 Appendix for detailed measurements.
Techniques Used: Electron Microscopy, Isolation, Transmission Assay, Transmission Electron Microscopy
Figure Legend Snippet: FGI is a mobile element in C . tetani . Annotation and alignment of the FGI genomic region across sequenced C . tetani strains. The FGI contains at least 80 predicted proteins that include the flagellar structural and polysaccharide processing enzymes in the sialic-acid like biosynthetic pathway (legionaminic acid and pseudaminic acid). Aligned regions that encompass fla flagellin genes (shown in blue, CTC01691—CTC01715) span 30 kb between 1,801,310–1,829,794 ( C . tetani E88 reference). Shaded regions define the boundaries of highly conserved sequences across strains, e.g. fla CTC01691 –predicted protein adjacent to CheY for strains ATCC 453/454, ATCC 9441, C2/E88, and GTC-14772. See shaded region that are conserved between GTC-14772 and 12124569/184.08 (predicted proteins 1–20). Clusters of orthologous genes and moderate conservation with known FGI from the genus Clostridium ( C . botulinum , C . carboxyvidorans , C . lundense , C . sporogenes , and C . tunisiense ) are labelled. Predicted Y1 transposases (green) not conserved in French strains 184.08 and 12124569, contain a predicted group IIc intron (element 22) and reverse transcriptase (element 23). Predicted glycosylation enzymes (red), transporters (magenta), CheY two-component system (yellow), and proteins of unknown function (black) are shown.
Figure Legend Snippet: CRIPSR spacer diversity and conservation. CRISPR/Cas arrays are organized into two primary arrays, I-A (A) and I-B (B) (see Figs 1 and 2 ) with low homology across all 11 sequenced strains. CRISPR/Cas arrays were organized into a single large array (I-A, array 1) or shorter arrays (I-B, arrays 2–9) consisting of a conserved leader sequence (triangle) and repeating alternating units of linkers and spacers (rectangles), color-coded based on conservation across C . tetani strains. Self-targeting spacers present in strains C2 and ATCC 9441 are shown (diamond) as well as spacers targeting identified C . tetani phages. CRISPR/Cas arrays present in vaccine strain C2 and ATCC 9441 were most similar among the oldest spacers at the tailing ends of both arrays. CRISPR/Cas spacers in GTC-14772 were least similar to other C . tetani strains and included an additional array that mapped to a 141kb contig with an incomplete complement of CRISPR/Cas proteins and phage-like proteins. CRISPR/Cas proteins were immediately upstream of the leader sequence for I-A, and distributed throughout the array for I-B. A CRISPR/Cas type III-A array was identified upstream of a single array (asterisk) in ATCC 453/454. A functional set of CRISPR/Cas proteins was absent in strain 184.08 despite the presence of 5 spacers and a distinct leader sequence. See S3 Table for CRISPR/Cas proteins.
Techniques Used: CRISPR, Sequencing, Functional Assay
Figure Legend Snippet: Genomic organization of sigK intervening element in C . tetani . Shown is a schematic representation of the genetic organization of prophage genomes identified in all C . tetani strains at insertion site A ( Fig 1 ). The identified prophage resembles a Bacillus subtilis phage DNA-like sigK intervening (skin) element. Predicted ORFs are represented as arrows in their respective orientation covering chromosomal location 1,135,168–1,192,484 nt in E88 reference strain. Conserved 5’- and 3’-flanking sequences (light grey), phage attachment sites attL and attR including the disrupted sporulation sigma factor, sigK (orange) are shown. Functional modules were assigned based on annotation and genomic organization for DNA packaging (magneta), capsid morphogenesis (blue), tail morphogenesis (light blue), lysis (red), lysogeny (pink), and DNA replication, transcription, and gene regulation (light green). Lysogenic phages were most highly conserved in genes associated with lysogeny and phage replication. Phage like-proteins (green) and hypothetical proteins (black) are shown as well. Predicted head-tail joining proteins (blue) and phage tail proteins (light blue) could only be identified in ATCC 453 (regions 39, 40 and 45–59) and ATCC 9441 (regions 48–50 and 55–72). Abbreviations: attachment sites attL and attR ; XkdT , baseplate protein; DNA-h , DNA helicase; DNA-pol , DNA polymerase; HMP , head morphogenesis protein; MCP , major capsid protein; PP , portal protein; RO , replisome organizer; SR , serine recombinase; TFP , tail fiber/collar protein; XkdK , tail sheath protein; TTMP , tail tape measure protein; TerS and TerL , terminase small and large subunit; virE , virulence factor E.
Techniques Used: Functional Assay, Lysis
Figure Legend Snippet: Distribution and frequency of SNPs in C . tetani . (A-D) Venn diagram of SNPs present in wild and vaccine strains of C . tetani . Strain E88 was used as a reference for determining SNP calls. Venn diagrams for strain sub-groups: (A) Harvard-derived vaccine strains C2, CN655 and Strain A [ 13 ], and ATCC 19406; very few SNPs were identified for all 4 strains, (B) Strains sequenced in the present study: C2, ATCC 19406, ATCC 9441, and ATCC 453, (C) and (D) Venn diagram of wild strains, ATCC 453, ATCC 9441, GTC-14772, 184.08 and 12124569. Strains 184.08 and 12124569. (E) Frequency distribution plot of SNP density (#SNPs/20 kb) and location along the E88 reference genome (chromosomal location). Regions displaying greater SNP frequency were associated with mobile genetic elements and indicated with arrows corresponding to regions identified in Fig 1 : prophage insertions (A—E), CRISPR/Cas arrays (I-A and I-B), and FGI.
Techniques Used: Derivative Assay, CRISPR
Figure Legend Snippet: BLAST analysis of C . tetani draft genome sequences. C . tetani strain genomes were compared by pairwise BLAST using BRIG against Strain E88 (2.8 Mb, GenBank accession number NC_004557). Genomes shown include (from innermost to outermost ring) C2, ATCC 19406, Strain A, CN655, GTC-14772, 184.08, ATCC 9441, ATCC 453, ATCC 454, and 12124569 (See Methods for BioSample and nucleotide accession numbers). Genomic regions with
Figure Legend Snippet: C . tetani motility on blood agar. Shown are representative examples of strain-specific motility and swarming phenotype in C2 and ATCC 9441 strains of C . tetani . Strains were plated onto blood agar plates and incubated for 48 hrs. Swarming behavior, exemplified by large spreading colonies is observed only in wild strain ATCC 9441. Strain C2 forms small, compact filamentous colonies (white arrow in magnified view). Strain ATCC 9441 forms large swarming colonies with hemolysis in the center, and a migrating boundary (white arrow in magnified view). Compare illustrations depicting filamentous form (top) to an irregular filiform margin (bottom).
Techniques Used: Incubation