On the role of penicillin-binding protein SpoVD in endospore cortex assembly
(2015)- Abstract
- Bacteria of the genera Bacillus and Clostridium can form endospores as a strategy to survive unfavourable environmental conditions. Endospore formation involves synthesis of cortex, a thick layer of modified peptidoglycan that surrounds the spore. This layer is required for heat resistance of the spore and mutant spores lacking the cortex layer can be identified by a simple heat shock assay.
B. subtilis SpoVD is a class B, high molecular weight, penicillin-binding protein (PBP) essential for spore cortex peptidoglycan synthesis. The exact role of the protein in cortex assembly is nknown but it most likely catalyses the formation of cross-links between glycan strands in nascent peptidoglycan. SpoVD deficient strains produce heat... (More) - Bacteria of the genera Bacillus and Clostridium can form endospores as a strategy to survive unfavourable environmental conditions. Endospore formation involves synthesis of cortex, a thick layer of modified peptidoglycan that surrounds the spore. This layer is required for heat resistance of the spore and mutant spores lacking the cortex layer can be identified by a simple heat shock assay.
B. subtilis SpoVD is a class B, high molecular weight, penicillin-binding protein (PBP) essential for spore cortex peptidoglycan synthesis. The exact role of the protein in cortex assembly is nknown but it most likely catalyses the formation of cross-links between glycan strands in nascent peptidoglycan. SpoVD deficient strains produce heat sensitive spores without cortex layer. Two conserved cysteine residues (Cys332 and Cys351) in the transpeptidase domain of SpoVD seem important for activity of the enzyme. They can form an intramolecular disulfide bond and this is catalysed by the membrane-bound thiol-disulfide oxidoreductase BdbD. The disulfide bond in SpoVD is located close to the transpeptidase active site and blocks the function of the protein. The bond is broken by the action of StoA, a sporulation-specific membrane-bound thiol-disulfide oxidoreductase. Based on these findings a thiol-based redox switch regulation of SpoVD activity was proposed in 2010.
The aim of this PhD project was to elucidate the function of SpoVD in cortex synthesis and to find out the physiological role of the proposed switch and the two cysteine residues in SpoVD. In depth investigation of the process of cortex assembly contribute to our understanding of peptidoglycan synthesis in general. This is of considerable medicinal interest since, e.g., bacterial cell wall synthesis is an effective target for many antibiotics in clinical use, such as penicillins and cephalosporins and eventual new drugs.
I demonstrate, by the use of a constructed SpoVD active site mutant strain, that synthesis of cortex explicitly depends on the transpeptidase activity of the protein. I show that the C-terminal PASTA domain of SpoVD is not important for the function of the protein in cortex synthesis. My results from in vitro experiments with several isolated protein variants strengthen the view that SpoVD is a specific target for StoA. My findings, supported by data available in the literature, indicate that the two cysteine residues in SpoVD affect the dynamics of the transpeptidase domain. Finally, I
propose a revised model for the function of BdbD and StoA in modulation of the redox state of SpoVD, where BdbD and StoA are suggested to act (mainly) during the folding of newly synthesised SpoVD. (Less) - Abstract (Swedish)
- Popular Abstract in English
An endospore, which can be formed by some bacteria, is a dormant and extremely endurable state which helps the cell to survive long periods of harsh environmental conditions. The penicillin-binding protein SpoVD in Bacillus subtilis is essential for the synthesis of the endospore cortex, a protective layer consisting of modified peptidoglycan. In depth investigation of the process of cortex assembly contributes to our understanding of peptidoglycan synthesis in general. Peptidoglycan is a major component of the bacterial cell wall, a structure that determines cell shape and is crucial for cell viability. The findings presented in this PhD thesis in microbiology is of considerable medicinal... (More) - Popular Abstract in English
An endospore, which can be formed by some bacteria, is a dormant and extremely endurable state which helps the cell to survive long periods of harsh environmental conditions. The penicillin-binding protein SpoVD in Bacillus subtilis is essential for the synthesis of the endospore cortex, a protective layer consisting of modified peptidoglycan. In depth investigation of the process of cortex assembly contributes to our understanding of peptidoglycan synthesis in general. Peptidoglycan is a major component of the bacterial cell wall, a structure that determines cell shape and is crucial for cell viability. The findings presented in this PhD thesis in microbiology is of considerable medicinal interest, because several major human pathogens form endospores and peptidoglycan synthesis is an effective target for many antibiotics in clinical use (e.g. penicillins and cephalosporins) and eventual new drugs. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4882583
- author
- Bukowska-Faniband, Ewa LU
- supervisor
- opponent
-
- Dr Cava, Felipe, Department of Molecular Biology, Umeå University, Sweden
- organization
- publishing date
- 2015
- type
- Thesis
- publication status
- published
- subject
- keywords
- sporulation, cortex, Bacillus subtilis, penicillin-binding proteins, SpoVD, peptidoglycan
- pages
- 181 pages
- publisher
- Department of Biology, Lund University
- defense location
- Biology Lecture Hall, Sölvegatan 35, Lund
- defense date
- 2015-01-30 10:00:00
- ISBN
- 978-91-7623-225-5
- 978-91-7623-226-2
- language
- English
- LU publication?
- yes
- id
- 5eed6a75-c9e9-4e34-ba50-599c284aae8b (old id 4882583)
- date added to LUP
- 2016-04-04 11:44:39
- date last changed
- 2020-04-29 12:31:43
@phdthesis{5eed6a75-c9e9-4e34-ba50-599c284aae8b,
abstract = {{Bacteria of the genera Bacillus and Clostridium can form endospores as a strategy to survive unfavourable environmental conditions. Endospore formation involves synthesis of cortex, a thick layer of modified peptidoglycan that surrounds the spore. This layer is required for heat resistance of the spore and mutant spores lacking the cortex layer can be identified by a simple heat shock assay.<br/><br>
B. subtilis SpoVD is a class B, high molecular weight, penicillin-binding protein (PBP) essential for spore cortex peptidoglycan synthesis. The exact role of the protein in cortex assembly is nknown but it most likely catalyses the formation of cross-links between glycan strands in nascent peptidoglycan. SpoVD deficient strains produce heat sensitive spores without cortex layer. Two conserved cysteine residues (Cys332 and Cys351) in the transpeptidase domain of SpoVD seem important for activity of the enzyme. They can form an intramolecular disulfide bond and this is catalysed by the membrane-bound thiol-disulfide oxidoreductase BdbD. The disulfide bond in SpoVD is located close to the transpeptidase active site and blocks the function of the protein. The bond is broken by the action of StoA, a sporulation-specific membrane-bound thiol-disulfide oxidoreductase. Based on these findings a thiol-based redox switch regulation of SpoVD activity was proposed in 2010.<br/><br>
The aim of this PhD project was to elucidate the function of SpoVD in cortex synthesis and to find out the physiological role of the proposed switch and the two cysteine residues in SpoVD. In depth investigation of the process of cortex assembly contribute to our understanding of peptidoglycan synthesis in general. This is of considerable medicinal interest since, e.g., bacterial cell wall synthesis is an effective target for many antibiotics in clinical use, such as penicillins and cephalosporins and eventual new drugs.<br/><br>
I demonstrate, by the use of a constructed SpoVD active site mutant strain, that synthesis of cortex explicitly depends on the transpeptidase activity of the protein. I show that the C-terminal PASTA domain of SpoVD is not important for the function of the protein in cortex synthesis. My results from in vitro experiments with several isolated protein variants strengthen the view that SpoVD is a specific target for StoA. My findings, supported by data available in the literature, indicate that the two cysteine residues in SpoVD affect the dynamics of the transpeptidase domain. Finally, I<br/><br>
propose a revised model for the function of BdbD and StoA in modulation of the redox state of SpoVD, where BdbD and StoA are suggested to act (mainly) during the folding of newly synthesised SpoVD.}},
author = {{Bukowska-Faniband, Ewa}},
isbn = {{978-91-7623-225-5}},
keywords = {{sporulation; cortex; Bacillus subtilis; penicillin-binding proteins; SpoVD; peptidoglycan}},
language = {{eng}},
publisher = {{Department of Biology, Lund University}},
school = {{Lund University}},
title = {{On the role of penicillin-binding protein SpoVD in endospore cortex assembly}},
url = {{https://lup.lub.lu.se/search/files/5844847/4882594.pdf}},
year = {{2015}},
}