ERA

Download the full-sized PDF of Structural Characterization of Bacterial Antimicrobial PeptidesDownload the full-sized PDF

Analytics

Share

Permanent link (DOI): https://doi.org/10.7939/R3TB0Z372

Download

Export to: EndNote  |  Zotero  |  Mendeley

Communities

This file is in the following communities:

Graduate Studies and Research, Faculty of

Collections

This file is in the following collections:

Theses and Dissertations

Structural Characterization of Bacterial Antimicrobial Peptides Open Access

Descriptions

Other title
Subject/Keyword
peptide
bacteriocin
antimicrobial
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Lohans, Christopher T
Supervisor and department
Vederas, John (Chemistry)
Examining committee member and department
Lowary, Todd (Chemistry)
McMullen, Lynn (Agricultural, Food and Nutritional Sciences)
Cairo, Christopher (Chemistry)
van der Donk, Wilfred (Chemistry)
Harynuk, James (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2014-10-02T11:22:57Z
Graduation date
2014-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Paenibacillus polymyxa NRRL B-30509, Paenibacillus terrae NRRL B-30644 and P. polymyxa NRRL B-30507 were found to produce several bacteriocins and non-ribosomal peptides. All three strains produce tridecaptins, non-ribosomal lipopeptides antimicrobially active against food pathogen Campylobacter jejuni. Two of these strains also produce polymyxins, lipopeptides also active against Gram-negative bacteria. Highly cyclized lantibiotics paenicidins A and B were isolated from P. polymyxa NRRL B-30509 and P. terrae NRRL B-30644, respectively. The lanthionine (Lan) and methyllanthionine (MeLan) post-translational modifications of paenicidin A were characterized by a novel partial desulfurization strategy, revealing the connectivity of three interlocking thioether-containing rings. Biosynthetic gene clusters responsible for the production of the tridecaptins and paenicidins were identified in the genome sequences of these strains. Carnobacterium maltaromaticum C2 produces carnolysin, a novel two-component lantibiotic with homology to enterococcal cytolysin. The post-translational modifications of carnolysins A1′ and A2′ were characterized with NMR spectroscopy and tandem mass spectrometry, revealing the Lan and MeLan bridging patterns. Like cytolysin, carnolysin contains unusual LL-Lan and LL-MeLan stereoisomers in the corresponding positions. However, carnolysin also contains D-alanine and D-aminobutyrate residues not found in cytolysin. The carnolysin biosynthetic gene cluster was identified in the genome of C. maltaromaticum C2. Heterologous expression of carnolysin in Escherichia coli was achieved by expressing the carnolysin precursor peptides with lantibiotic synthetase CrnM and reductase CrnJ. Antimicrobially active products were obtained by proteolytically removing the N-terminal leader sequences of the carnolysins isolated from C. maltaromaticum C2. These digested peptides were active against an array of Gram-positive indicator organisms, while not demonstrating the hemolytic activity associated with cytolysin at the levels tested. Enterocin 7A and 7B are leaderless bacteriocins produced by Enterococcus faecalis 710C. The impact of solvent on the secondary structure of enterocin 7A was examined by circular dichroism spectroscopy, revealing a high degree of α-helicity in fully aqueous conditions. The solution structure of enterocin 7A was solved based on NMR spectroscopic data, demonstrating that this peptide consists primarily of three amphiphilic α-helices burying a hydrophobic core. The structures of enterocins 7A and 7B resembled a region of the circular bacteriocin carnocyclin A, potentially having implications regarding the mode of action of these leaderless bacteriocins. The cysteines involved in the N-terminal disulfide bridge of class IIa bacteriocin leucocin A were replaced with leucine residues to probe the impact of this substitution on structure. While this mutant peptide retained antimicrobial activity, consistent with similar leucocin A mutants. The solution structure of (C9L,C14L)-leucocin A in aqueous trifluoroethanol was studied by NMR spectroscopy. Instead of the N-terminal three-strand β-sheet found in wild-type leucocin A, the N-terminus of the mutant structure is α-helical. However, this may not represent the active conformation of this peptide, and the structure of the N-terminus may be influenced by the choice of solvent.
Language
English
DOI
doi:10.7939/R3TB0Z372
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication

File Details

Date Uploaded
Date Modified
2014-11-15T08:24:07.790+00:00
Audit Status
Audits have not yet been run on this file.
Characterization
File format: pdf (PDF/A)
Mime type: application/pdf
File size: 6908125
Last modified: 2015:10:12 17:16:10-06:00
Filename: Lohans_Christopher_T_201409_PhD.pdf
Original checksum: 797312bde8178ae3396fd9b6dc5a347e
Well formed: true
Valid: true
File title: Lohans - Full Thesis UNSCANNED MOST RECENT - WITH REFS - no highlights
File author: Chris
Page count: 270
Activity of users you follow
User Activity Date