Characterizing desk as a potential in vivo reporter for membrane-thickness in Bacillus subtilis
(2021) MOBN02 20211Degree Projects in Molecular Biology
- Abstract
- Antimicrobial resistance (AMR) is a growing problem worldwide as more drug-resistant bacteria are identified. However, resistance is comparably rare against antibiotics that target the cytoplasmic membrane. Membrane thickness is an important factor for the mode of action of these antibiotics, and difficult to study. Thus, constructing a biosensor to measure membrane thickness in vivo is of great interest. The ubiquitous soil bacterium and Gram-positive model organism Bacillus subtilis has a way of sensing membrane thickness through the des operon. The aim of this project was to construct fluorescence and luminescence reporters fused to the des promoter, as well as fluorescent protein fusions to DesK and its minimal sensor domain, MSDesK,... (More)
- Antimicrobial resistance (AMR) is a growing problem worldwide as more drug-resistant bacteria are identified. However, resistance is comparably rare against antibiotics that target the cytoplasmic membrane. Membrane thickness is an important factor for the mode of action of these antibiotics, and difficult to study. Thus, constructing a biosensor to measure membrane thickness in vivo is of great interest. The ubiquitous soil bacterium and Gram-positive model organism Bacillus subtilis has a way of sensing membrane thickness through the des operon. The aim of this project was to construct fluorescence and luminescence reporters fused to the des promoter, as well as fluorescent protein fusions to DesK and its minimal sensor domain, MSDesK, under both a xylose-inducible and the native promoter. The resulting reporters were tested as a proof of concept to observe changes in signal intensity and localization in response to temperature shifts and antibiotic treatment.
We explored three potential avenues for our membrane thickness reporter, a Pdes luciferase strain (Pdes-Fluc) and three GFP fusion strains with either the native Pdes promoter (Pdes-desK-msfgfp and Pdes-MSdesK-msfgfp) or the xylose-inducible promoter (Pxyl-desK-msfgfp). The native promoter GFP fusion stains showed changes in GFP intensity, when treated with 4 °C cold shock and daptomycin. The inducible promoter showed changes in DesK localization when treated with 4 °C cold shock, 50 °C heat shock, and the antimicrobial peptide cWFW. In the future, in addition to further characterization of this DesK reporter, it would be desirable to diversify the tools available to estimate membrane thickness in vivo. Overall, DesK is an interesting candidate for a membrane thickness and fluidity reporter as it can respond to changes in membrane fluidity. (Less) - Popular Abstract
- Creating a ruler for membrane thickness in bacteria
Antimicrobial resistance is a growing problem worldwide as more and more drug-resistant bacteria are identified. In the last 20 years, we have not developed any new antibiotic class that can used for treatment. This problem affects everyone as common infections now become life-threatening. However, resistance is comparably rare in antibiotics that target the bacteria’s cell membrane, because they attack bacteria quickly and in many different points, which makes it hard for bacteria to create defenses. To better understand how these antibiotics work we need to see how they interact with the bacterial cell membrane.
To study what happens to the bacteria’s membrane, when it is attacked... (More) - Creating a ruler for membrane thickness in bacteria
Antimicrobial resistance is a growing problem worldwide as more and more drug-resistant bacteria are identified. In the last 20 years, we have not developed any new antibiotic class that can used for treatment. This problem affects everyone as common infections now become life-threatening. However, resistance is comparably rare in antibiotics that target the bacteria’s cell membrane, because they attack bacteria quickly and in many different points, which makes it hard for bacteria to create defenses. To better understand how these antibiotics work we need to see how they interact with the bacterial cell membrane.
To study what happens to the bacteria’s membrane, when it is attacked by antibiotics, we chose Bacillus subtilis as a model organism. While we have tools to measure different characteristics of the membrane, it is very hard to measure how thick the membrane is. The aim of our project is to create a ruler that will go into the membrane to measure its thickness. To do this, we created a special copy of a B. subtilis protein, called DesK, with a green-fluorescent tag, made with a protein called GFP. DesK should identify thicker spots on the membrane and the GFP tag will let us see the protein location in the bacteria under the microscope. To turn on our ruler, we made two different on-switches. The first switch (Pdes) will turn on, when the bacteria need it, and the second switch (Pxyl) we turn on on-demand with a compound called xylose. The combination of switch and protein is called a reporter. We tested our reporters to see, if they could detect changes in membrane thickness and if we could use them to find the specific spots on the membrane where the thickness changed.
We found that the first switch (Pdes) was turned on, when the bacteria were in an ice bath, a physical stress that makes their membrane thicker, but we could not see in the microscope, where these spots occurred. We got the same results, when we used an antibiotic (daptomycin) that makes the membrane rigid and thicker. This tells us that our reporter can measure when the membrane thickens, but not where. We tested the second switch (Pxyl) with shifts in temperature and antibiotics in order to see where these changes occurred. We saw that when the bacteria were in an ice bath or in a heat bath (50 °C), our reporter showed up strongly in some spots. Indicating that it identifies both membrane thickening and thinning. When we treated our bacteria with antibiotics, we found that daptomycin had no effect on this ruler. We were curious to see if our ruler was protecting the bacteria against the antibiotic but found that it was more likely that the xylose we used to turn on our ruler is protecting the bacteria. We then tested a different antibiotic (cWFW) and found that it shifted our ruler to thinner membrane spots and depleted it from the thicker spots. This shows that our ruler changes location in reaction to shifts in temperature and antibiotic treatment, possibly pointing to thinner spots were the ruler concentrates. An un expected discovery, since we expected it to identify thicker spots.
Further research is needed with our DesK ruler to corroborate our findings, and to create more tools to estimate the membrane thickness in bacteria. We have tools to measure other membrane characteristics and dyes that let us see differences in areas of the membrane, which let us draw equivalences to membrane thickness. However, having different rulers for membrane thickness will allow for further better understanding on how bacteria grow and divide, and how antibiotics work. The ruler we made here is a promising step in this direction.
Master’s Degree Project in Molecular Biology, Molecular Genetics, and Biotechnology, 45 credits 2021
Department of Biology, Lund University
Advisors: Michaela Wenzel and Margareth Sidarta
Division of Chemical Biology, Department of Biology and Biological Engineering, Chalmers University of Technology (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9067457
- author
- Monsalve Iguiniz, Anuntxi
- supervisor
- organization
- course
- MOBN02 20211
- year
- 2021
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9067457
- date added to LUP
- 2021-10-29 15:45:59
- date last changed
- 2021-10-29 15:45:59
@misc{9067457,
abstract = {{Antimicrobial resistance (AMR) is a growing problem worldwide as more drug-resistant bacteria are identified. However, resistance is comparably rare against antibiotics that target the cytoplasmic membrane. Membrane thickness is an important factor for the mode of action of these antibiotics, and difficult to study. Thus, constructing a biosensor to measure membrane thickness in vivo is of great interest. The ubiquitous soil bacterium and Gram-positive model organism Bacillus subtilis has a way of sensing membrane thickness through the des operon. The aim of this project was to construct fluorescence and luminescence reporters fused to the des promoter, as well as fluorescent protein fusions to DesK and its minimal sensor domain, MSDesK, under both a xylose-inducible and the native promoter. The resulting reporters were tested as a proof of concept to observe changes in signal intensity and localization in response to temperature shifts and antibiotic treatment.
We explored three potential avenues for our membrane thickness reporter, a Pdes luciferase strain (Pdes-Fluc) and three GFP fusion strains with either the native Pdes promoter (Pdes-desK-msfgfp and Pdes-MSdesK-msfgfp) or the xylose-inducible promoter (Pxyl-desK-msfgfp). The native promoter GFP fusion stains showed changes in GFP intensity, when treated with 4 °C cold shock and daptomycin. The inducible promoter showed changes in DesK localization when treated with 4 °C cold shock, 50 °C heat shock, and the antimicrobial peptide cWFW. In the future, in addition to further characterization of this DesK reporter, it would be desirable to diversify the tools available to estimate membrane thickness in vivo. Overall, DesK is an interesting candidate for a membrane thickness and fluidity reporter as it can respond to changes in membrane fluidity.}},
author = {{Monsalve Iguiniz, Anuntxi}},
language = {{eng}},
note = {{Student Paper}},
title = {{Characterizing desk as a potential in vivo reporter for membrane-thickness in Bacillus subtilis}},
year = {{2021}},
}