MS analysis of modified tryptic peptides from flagellar proteins of C. difficile strains 630 and BI-1. The total mass of the glycopeptide was 3,160.24 Da (predicted peptide mass, 2,364.26 Da), which suggested modifications with two glycans of 398 Da. "The architecture of the murein (peptidoglycan) in gram-negative bacteria: vertical scaffold or horizontal layer (s)?.” Journal of bacteriology 186.18 (2004): 5978-5987. Intact mass analysis of C. difficile flagellins.The observed masses of flagellin proteins from C. difficile strains 630, BI-1, M7465, and M9349 were obtained by infusion into a QTOF2 MS (Table 1). Clostridium septicum is a gram positive, spore forming, obligate anaerobic bacterium.. Clostridium septicum can cause gas gangrene, but unlike other Clostridium species like Clostridium perfringens, no trauma is necessary at the site of the infection.It is thought that the infection is established by hematogenous spread from the gastrointestinal tract. Cell membranes containing stimuli receptors detect stimuli causing activation of the flagella. Accurate mass determination of the glycan oxonium ion and glycan-related fragment ions. The computer display is an overlay of the phase image where each cell is identified by a line running from its head to tail, colored either by cell identity (e.g., Edge by identity.avi) or by cell heading (Edge by heading.avi). The same specificity was demonstrated in the transformation through conjugation of C sporogenes with cytosine deaminase (CD) plasmid, whose product can cleave the pre-administered 5-flourocytosine (5-FC) into the cytotoxic 5-flourouracil (5-FU) for tumor lysis (15). This colonization process is an important first step in the disease process, whereby the organism penetrates the mucus layer and adheres to the underlying colonic epithelial cells, thereby facilitating the delivery of toxins to host cell receptors. (26), the group of genes comprising the flagellar glycosylation locus from QCD32g58 were not included in the microarray. 2D). Future work will be directed toward defining whether motility or the glycans play a role in colonization and in determining if there is a correlation between glycan structure and virulence of isolates. [18] Poehlein, Anja, et al. The current study provides the first example of a gram-positive anaerobe in which the glycosylation process is required for flagellar assembly. (A) The reconstructed molecular mass profile of FliC from C. difficile 630, showing two peaks at 33,559 and 33,160 Da. “Complete Genome Sequence of the Nonpathogenic Soil-Dwelling Bacterium Clostridium Sporogenes Strain NCIMB 10696.” Genome Announcements 3.4 (2015): e00942–15. In some cases, it has been demonstrated that the process of flagellar glycosylation has a role in both flagellar assembly and host-pathogen interactions (17). [7] Brunt, Jason et al. The fragments were cloned directly into pMTL007, and the targeted intron was named to indicate the site of insertion within the gene (base number) and orientation (sense [s] or antisense [a]). The C. difficile 630::fliCerm and C. difficile 630::0240erm strains no longer displayed a spreading phenotype in this motility agar, in contrast to the parent strain (Fig. PLOS Pathogens 10.9 (2014): e1004382. Top-down analyses of C. difficile flagellins.Previously, we have shown that examination of intact flagellar proteins by electrospray ionization MS allows the identification of labile-glycoprotein-associated ions, in particular, glycan-related ions (24). ETD was then performed using fluoranthene as the anionic reagent and with supplementary activation enabled. In one mechanism, MotI could function as a molecular brake by adding resistance between the stator and the rotor until flagellar rotation becomes locked (7–9, 16). Flagellin protein purification.Flagellin proteins were isolated using the following procedure. Electron microscopy.Copper grids (Electron Microscopy Sciences, Fort Washington, PA) were covered with Formvar film and coated with carbon. In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. We also show that glycosylation of the flagellin protein is required for proper assembly and consequent motility. None of the intact masses measured matched precisely the mass predicted from the translated gene sequence, suggesting that these flagellins were posttranslationally modified. All strains examined in this study, with the exception of QCD32g58, M7465, and M9349, do not appear to be clonally related. (C) C. difficile 630::0240 erm. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. Comparison of the observed protein intact mass of C. difficile 630 suggests that it corresponds to the flagellin protein FliC, bearing six or seven residues modified with a glycan of 398 Da. 12 terms. With the recent development of mutagenesis tools for clostridial species, it will now be possible to determine if the glycosylation process is also required for flagellar assembly in C. botulinum. Web. However, due to high risk of infection and toxicity after the spore germinated in tumor-bearing animal (8), bacterial treatment is not widely accepted. Lane 1, C. difficile 630; l ane 2, C. difficile BI-1; l ane 3, C. difficile BI-7; l ane 4, C. difficile QCD32g58; l ane 5, C. difficile M23257; l ane 6, C. difficile M46846; l ane 7, C. difficile M7465; l ane 8, C. difficile M9349; l ane 9, CM- 26; l ane 10, CM- 56; l ane 11, 06CD130. The resulting solution was infused in a hybrid quadrupole time of flight mass spectrometer (MS) (QTOF2; Waters, Beverly, MA) at a flow rate of 0.5 μl min−1. These top-down studies revealed marker ions characteristic of glycan modifications. TFP-dependent gliding motility has been seen in many Gram-negative bacteria but not in Gram-positive bacteria. However, a homolog of CD0240 (with >80% identity) which lies proximal to the fliC structural gene is present in all strains (GT1). Molecular masses (in kDa) are shown to the left of the gel. No evidence for a glycan oxonium ion at m/z 399 was observed for this flagellin sample. S-layer protein extraction.Partially purified S-layer was obtained from C. difficile cells by resuspending growth from a single BHI plate (24 h) in 500 μl of 0.2 M glycine buffer, pH 2.2. A 7-μl aliquot of each fraction was retained, and the remainder was immediately evaporated to dryness and stored at −20°C. Motility screening of fliC and CD0240 mutant strains.Each strain was grown overnight on supplemented BHI agar containing Erm. Vol. "Isolation and characterization of bacteriophages active on Clostridium sporogenes.” Journal of bacteriology 87.2 (1964): 408-415. While prevention through antibiotic stewardship and optimal management of disease is the most obvious strategy currently used, there is a great need for alternate methods of treatment. “Combination Bacteriolytic Therapy for the Treatment of Experimental Tumors.” Proceedings of the National Academy of Sciences of the United States of America 98.26 (2001): 15155–15160. Accurate mass determination of the glycan oxonium and glycan-related fragment ions was achieved using a number of neighboring peptide fragment ions as internal mass standards. Importantly, recurrence rates following treatment can be as high as 35% irrespective of the drug used in initial treatment (10, 35). In addition, PCRs with Erm-specific primers were used to confirm splicing of the retrotransposition-activated selectable marker. MS/MS spectra were acquired automatically on doubly, triply, and quadruply charged ions. If a fresh culture of bacteria is available, microscopy is the most accurate way to determine bacterial motility, and ‘hanging drop method’ is a commonly used microscopic technique. While the flagellin of Clostridium botulinum was shown to be glycosylated, the role of the glycosylation process in assembly and motility had not been investigated. Five of the seven sites of glycosylation were identified in this manner for C. difficile 630 FliC as S141, S174, T183, S188, and S205, and three of these sites were confirmed as sites of glycosylation in seven additional strains (Table 1). Similar second-generation ion spectra were recorded for multiply charged protein ions from C. difficile strains M9349 and M7465, with product ion spectra similar to those observed for MS/MS of multiply charged protein ions from strain BI-1 (data not shown). 26 terms. Edited by Kristina Platt, Phuong Nguyen, Sakib Hossain, students of Jennifer Talbot for BI 311 General Microbiology, 2014, Boston University. While nutrition is more commonly supplied by amino acid fermentation, glucose fermentation, where glucose is broken down into ethanol and carbon dioxide, can be utilized as a secondary source of nutrition. Malassezia furfur/Pityriasis Versicolor. Insertion of the TargeTron Erm resistance marker into either fliC or CD0240 was confirmed by PCR using primers flanking the gene of interest and with primers specific to the TargeTron Erm resistance marker (data not shown). This process of flagellar glycosylation has also been shown to be required for motility in a number of other bacterial pathogens, including Campylobacter jejuni, Helicobacter pylori, and Aeromonas caviae, which are all known to colonize the gastrointestinal tract. We extracted this band from the gel, and peptide MS/MS analysis confirmed that this protein was the product of the fliC gene. C. sporogenes exhibit motility using flagella (3). Further MS/MS spectra of the glycan-associated fragment ions were collected. Selenocysteine has a lower reduction potential than cysteine, therefore, it is preferred over cysteine in proteins involved in antioxidant activity (19). Spores of C. sporogenes target the hypoxic core of a tumor and can cause the tumor to lyse as a reaction to simple reproduction and expansion of the bacteria. Start studying Microbiology lecture 12 - Motility. No fragment ions corresponding to peptide type y or b ions were observed, suggesting that the observed fragment ions were more typical of those observed for glycan fragmentation. Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen which can cause serious diseases in immunocompromised patients (eg. "Glucose metabolism in Clostridium sporogenes and Clostridium sticklandii bacteria.” Mikrobiologiia 52.6 (1983): 869. PMC. Méd. Researchers have attempted to overcome this limitation by heat-inactivating C.sporogenes to produce its non-viable derivative before administration to a tumor culture. “Functional Characterisation of Germinant Receptors in Clostridium Botulinum and Clostridium Sporogenes Presents Novel Insights into Spore Germination Systems.” Ed. Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews. Glycopeptide-containing HPLC fractions were infused at 1 μl min−1 into the electrospray ionization source of an LTQ XL linear ion trap MS (Thermo Fisher Scientific, Ottawa, ON, Canada) capable of performing ETD. Collision-induced dissociation MS/MS analysis was performed on the glycopeptide ions to confirm their identity. Web. Sequence archive. 5A). Many of the MS/MS spectra could be readily assigned to unmodified flagellin tryptic peptides. In Table 2 spectra of the unknown fragment ion at m/z 524.1 364.1... Pairs is also been suggested that motility is a Type of social motility allowing the migration of highly differentiated cells. ] Liu, S. C., et al screening of FliC from strain QCD32g58 failed to contain any major species... Karbownik, Małgorzata, et al it was possible to infer the peptide MS/MS spectrum of the selectable! 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