On the role of penicillin-binding protein SpoVD in endospore cortex assembly

Ewa Bukowska-Faniband

On the role of penicillin-binding protein SpoVD in endospore cortex assembly

Ewa Bukowska-Faniband

Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)

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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.

Originalspråk	engelska
Kvalifikation	Doktor
Tilldelande institution
Handledare	Hederstedt, Lars, handledare
Tilldelningsdatum	2015 jan. 30
Förlag	Department of Biology, Lund University
ISBN (tryckt)	978-91-7623-225-5
ISBN (elektroniskt)	978-91-7623-226-2
Status	Published - 2015

Bibliografisk information

Defence details

Date: 2015-01-30
Time: 10:00
Place: Biology Lecture Hall, Sölvegatan 35, Lund

External reviewer(s)

Name: Cava, Felipe
Title: Dr
Affiliation: Department of Molecular Biology, Umeå University, Sweden

---

Ämnesklassifikation (UKÄ)

Biologiska vetenskaper

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@phdthesis{5eed6a75c9e94e34ba50599c284aae8b,

title = "On the role of penicillin-binding protein SpoVD in endospore cortex assembly",

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 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.",

keywords = "sporulation, cortex, Bacillus subtilis, penicillin-binding proteins, SpoVD, peptidoglycan",

author = "Ewa Bukowska-Faniband",

note = "Defence details Date: 2015-01-30 Time: 10:00 Place: Biology Lecture Hall, S{\"o}lvegatan 35, Lund External reviewer(s) Name: Cava, Felipe Title: Dr Affiliation: Department of Molecular Biology, Ume{\aa} University, Sweden ---",

year = "2015",

language = "English",

isbn = "978-91-7623-225-5",

publisher = "Department of Biology, Lund University",

type = "Doctoral Thesis (compilation)",

}

TY - THES

T1 - On the role of penicillin-binding protein SpoVD in endospore cortex assembly

AU - Bukowska-Faniband, Ewa

N1 - Defence details Date: 2015-01-30 Time: 10:00 Place: Biology Lecture Hall, Sölvegatan 35, Lund External reviewer(s) Name: Cava, Felipe Title: Dr Affiliation: Department of Molecular Biology, Umeå University, Sweden ---

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

KW - sporulation

KW - cortex

KW - Bacillus subtilis

KW - penicillin-binding proteins

KW - SpoVD

KW - peptidoglycan

M3 - Doctoral Thesis (compilation)

SN - 978-91-7623-225-5

PB - Department of Biology, Lund University

ER -

On the role of penicillin-binding protein SpoVD in endospore cortex assembly

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