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Examining the structure, function and mode of action of bacteriocins from lactic acid bacteria Open Access

Descriptions

Other title
Subject/Keyword
carnocyclin A
bacteriocins
lactic acid bacteria
type IIa bacteriocins
immunity protein
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Martin-Visscher, Leah A.
Supervisor and department
Vederas, John (Chemistry)
Examining committee member and department
Bundle, David (Chemistry)
Burkart, Michael (Chemistry & Biochemistry)
Klassen, John (Chemistry)
Cairo, Christopher (Chemistry)
Uludag, Hasan (Chemical and Materials Engineering)
Department
Department of Chemistry
Specialization

Date accepted
2010-01-25T21:47:49Z
Graduation date
2010-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Carnocyclin A (CclA) is a remarkably stable, potent bacteriocin produced by Carnobacterium maltaromaticum UAL307. Elucidation of the amino acid and genetic sequences revealed that CclA is a circular bacteriocin. Preliminary structural studies (dynamic light scattering, NMR, circular dichroism, stereochemical analysis) indicated that CclA is monomeric and alpha-helical in aqueous conditions and composed of L-residues. The 3D structure of [13C,15N]CclA was solved by NMR, revealing a compact arrangement of four helices. To examine the structure of the precursor peptide (pCclA) several fusion proteins were constructed and overexpressed; however, pCclA could not be isolated. To investigate the requirements for cyclization, several internally hexahistidine-tagged (His6) pCclA mutants were constructed. Expression conditions are underway. PisI was heterologously expressed and confirmed to impart protection against piscicolin 126 (PisA). Labeled and unlabeled PisA and PisI were purified following overexpression as maltose-binding protein fusions (MalE-fusions) and Factor Xa cleavage. NMR studies indicated that PisI and PisA do not physically interact. The 3D structure of PisI was solved by NMR, confirming that the four-helix bundle is a conserved motif for the immunity proteins of type IIa bacteriocins. The putative receptor proteins for these bacteriocins were cloned and overexpressed as His6-fusion proteins. Experiments are underway to optimize the expression and purification of these membrane proteins. The peptidase domain of the ABC-transporter protein (CbnTP) for carnobacteriocin B2 (CbnB2) was overexpressed as a His6-fusion protein. Active protease could not be purified from inclusion bodies, but was obtained as soluble protein following low-temperature overexpression. The CbnB2 precursor pCbnB2 (and a truncated derivative pCbnB2-RP) was purified following overexpression as a MalE-fusion and Factor Xa cleavage. pCbnB2 was incubated with CbnTP and MALDI-TOF and activity testing confirmed that CbnTP cleaved the leader peptide from pCbnB2. Five CysSer CbnTP mutants were constructed. Crystallographic studies of CbnTP are underway. Six bacteriocins (nisin, gallidermin, lacticin 3147, CclA, PisA, enterocin 710C) were tested against Gram-negative bacteria (E. coli DH5α, Pseudomonas aeruginosa ATCC 14207, Salmonella typhimurium ATCC 23564) in the absence and presence of EDTA. PisA and lacticin 3147 exhibited minimal activity, whereas the other bacteriocins killed at least one strain, in the presence of EDTA.
Language
English
DOI
doi:10.7939/R31S4M
Rights
License granted by Leah Martin-Visscher (lamartin@ualberta.ca) on 2010-01-25T13:58:28Z (GMT): 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 the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein 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.
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