Functional study of domains of class B penicillin-binding proteins 4b and SpoVD in Bacillus subtilis
(2017) MOBT01 20162Degree Projects in Molecular Biology
- Popular Abstract
- How are penicillin-binding proteins sorted in cells?
A bacterial cell wall can be regarded as a brick building, in which the bricks and the cement hold the building together, provide structure for the building, protect its contents, and allow only certain substances to enter. Many antibiotics target proteins that function in cell wall morphogenesis (the process of building the brick wall in this analogy) in bacteria. However, since bacteria develop resistance to these antibiotics, new drugs need to be developed. The soil bacterium Bacillus subtilis is a good experimental model for research on cell wall peptidoglycan synthesis.
Peptidoglycan is a polymer which is composed of polysaccharide and peptide chains. If we imagine... (More) - How are penicillin-binding proteins sorted in cells?
A bacterial cell wall can be regarded as a brick building, in which the bricks and the cement hold the building together, provide structure for the building, protect its contents, and allow only certain substances to enter. Many antibiotics target proteins that function in cell wall morphogenesis (the process of building the brick wall in this analogy) in bacteria. However, since bacteria develop resistance to these antibiotics, new drugs need to be developed. The soil bacterium Bacillus subtilis is a good experimental model for research on cell wall peptidoglycan synthesis.
Peptidoglycan is a polymer which is composed of polysaccharide and peptide chains. If we imagine peptidoglycan as a “brick arrangement in a building”, then a glycan chain would be one layer of those “bricks”. The different layers of glycan chains in a peptidoglycan polymer are held together by branches of amino acids. These branches of amino acids are analogous to the “cement” that binds together the layers of bricks in a building. The process of cross-linking (connecting) the glycan chains in a peptidoglycan polymer is called “transpeptidation” and is carried out by so called class B high molecular weight (HMW) penicillin-binding proteins (PBPs). B. subtilis can form endospores which are metabolically dormant and extremely heat resistant. The cortex layer in a B. subtilis spore is made of peptidoglycan and essential for spore heat resistance. It serves as model system for studies on peptidoglycan synthesis.
SpoVD is a sporulation-specific class B HMW PBP with an essential role in B. subtilis spore cortex synthesis. SpoVD-deficient mutants form heat-sensitive spores that lack the cortex layer. PBP4b is a protein whose physiological function is unknown, i.e. PBP4b deficient cells sporulate normally. The close similarity between the two PBPs suggest that they may have overlapping activities. Both proteins have an N-terminal transmembrane anchor, a domain of unknown function, and a transpeptidase domain. SpoVD, in addition, contains one PASTA domain at the C-terminal end. The domains of a protein are analogous to the parts of a doll: the “foot” represents transmembrane domain, the “body” represents the domain with unknown function, and the “head” represents transpeptidase domain (and PASTA).
In this study, the function of the SpoVD domains and the role of PBP4b in B. subtilis were studied. This was done by analyzing the properties of various PBP4b/SpoVD protein chimeras (containing mixed PBP4b and SpoVD domains) in strains lacking the genes for these proteins.
My work shows that chimeric proteins are unable to compensate for the lack of SpoVD. Several possible reasons for this negative result were ruled out, leading me to focus on the subcellular localization of the proteins by using the fluorescence signal of Green Fluorescent Protein (GFP) and fluorescence microscopy. During sporulation, GFP-PBP4b and GFP-SpoVD localized differently in the cell. The protein chimeras containing the transmembrane domain of PBP4b followed the GFP-PBP4b localization pattern, whereas those with the transmembrane domain of SpoVD followed the GFP-SpoVD localization pattern. These results indicate that the transmembrane domain determines the subcellular localization of SpoVD and PBP4b.
The physiological role of PBP4b remains unknown. The role of the transmembrane domain opens the way for new studies into the mechanism of how SpoVD is targeted to the forespore membrane. Furthermore, this study has contributed in understanding the extreme properties of spores, the resistance to antibiotics, and the development of new drugs.
Master’s Degree Project in Molecular Biology, Molecular Genetics and Biotechnology 60 credits 2017
Department of Biology, Lund University
Advisors: Dr. Ewa Bukowska-Faniband and Professor Lars Hederstedt
Microbiology group, Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8925278
- author
- Sidarta, Margareth
- supervisor
- organization
- course
- MOBT01 20162
- year
- 2017
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 8925278
- date added to LUP
- 2017-09-11 12:08:39
- date last changed
- 2017-09-11 12:08:39
@misc{8925278,
author = {{Sidarta, Margareth}},
language = {{eng}},
note = {{Student Paper}},
title = {{Functional study of domains of class B penicillin-binding proteins 4b and SpoVD in Bacillus subtilis}},
year = {{2017}},
}