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Cell division in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Gram-negative bacterium Escherichia coli is a model system to describe the biochemistry and cell biology of cell division in bacteria. This process can be divided into three major steps. The first step involves the replication of the DNA, followed by an elongation step in which the cells become twice as long. In the last step the elongated cell constricts in the middle and the two daughter cells are separated. The cell division process in E. coli has been extensively studied for at least 50 years and a lot is known, however many details are still vague. New proteins involved in the process continue to be identified and the number of these proteins as well as the interactions among them are not yet fully known. It is therefore not completely understood how the contraction proceeds to form two daughter cells. In this thesis, I have carried out experiments that contribute to our understanding of cell division in E. coli. Using fluorescence microscopy I show that the contraction of the inner membrane in dividing E. coli proceeds in a linear fashion and that the periplasm closes after the cytoplasm. I have also analyzed the oligomeric state of two proteins involved in the cell division and I show that the early cell division protein ZipA can dimerize. This could explain how this protein can bundle FtsZ protofilaments, as it could bridge two protofilaments. Penicillin-binding protein 5 (PBP5) has been found to localize to the septum and it has been suggested to be connected to cell division. I have found that PBP5 forms a homo-oligomeric complex, most likely a dimer. The dimer can be modeled in a back-to-back conformation with the catalytic domains being flexible. This allows PBP5 to reach for pentapeptides of the peptidoglycan at different distances from the membrane. An understanding of the mechanisms used by the cell division proteins and their protein: protein interactions can be a first step towards determining new antibiotic targets.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2011. , 61 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-62908ISBN: 978-91-7447-339-1 (print)OAI: oai:DiVA.org:su-62908DiVA: diva2:446223
Public defence
2011-11-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript. Available from: 2011-10-27 Created: 2011-10-04 Last updated: 2011-10-18Bibliographically approved
List of papers
1. Estimating Z-ring radius and contraction in dividing Escherichia coli
Open this publication in new window or tab >>Estimating Z-ring radius and contraction in dividing Escherichia coli
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2010 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 76, no 1, 151-158 p.Article in journal (Refereed) Published
Abstract [en]

We present a fluorescence recovery after photobleaching-based method for monitoring the progression of septal Z-ring contraction in dividing Escherichia coli cells. In a large number of cells undergoing division, we irreversibly bleached cytosolically expressed Enhanced Green Fluorescent Protein on one side of the septal invagination and followed the fluorescence relaxation on both sides of the septum. Since the relaxation time depends on the cross-sectional area of the septum, it can be used to determine the septal radius r. Assuming that the fraction of the observed cells with r-values in a given interval reflects the duration of that interval in the division process we could derive an approximate time-course for the contraction event, as a population average. By applying the method repeatedly on individual cells, the contraction process was also followed in real time. On a population average level, our data are best described by a linear contraction process in time. However, on the single cell level the contraction processes display a complex behaviour, with varying levels of activity. The proposed approach provides a simple yet versatile method for studying Z-ring contraction in vivo, and will help to elucidate its underlying mechanisms.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62904 (URN)10.1111/j.1365-2958.2010.07087.x (DOI)
Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2017-12-08Bibliographically approved
2. Sequential closure of the cytoplasm then periplasm during cell division in Escherichia coli
Open this publication in new window or tab >>Sequential closure of the cytoplasm then periplasm during cell division in Escherichia coli
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2012 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 194, no 3, 584-586 p.Article in journal (Refereed) Published
Abstract [en]

To visualize the latter stages of cell division in live Escherichia coli, we have carried out Fluorescence Recovery After Photobleaching (FRAP) on 121 cells expressing cytoplasmic GFP and periplasmic mCherry. Our data show conclusively, that the cytoplasm is sealed prior to the periplasm during the division event.

Keyword
cell division, Escherichia coli, periplasm, FRAP
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62906 (URN)10.1128/JB.06091-11 (DOI)000299309200005 ()22101847 (PubMedID)
Note
5Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2017-12-08Bibliographically approved
3. Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli
Open this publication in new window or tab >>Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli
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2011 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 9, 1520-1529 p.Article in journal (Refereed) Published
Abstract [en]

Penicillin-binding protein 5 (PBP5) is a DD-carboxypeptidase, which cleaves the terminal D-alanine from the muramyl pentapeptide in the peptidoglycan layer of Escherichia coli and other bacteria. In doing so, it varies the substrates for transpeptidation and plays a key role in maintaining cell shape. In this study, we have analyzed the oligomeric state of PBP5 in detergent and in its native environment, the inner membrane. Both approaches indicate that PBP5 exists as a homo-oligomeric complex, most likely as a homo-dimer. As the crystal structure of the soluble domain of PBP5 (i.e., lacking the membrane anchor) shows a monomer, we used our experimental data to generate a model of the homo-dimer. This model extends our understanding of PBP5 function as it suggests how PBP5 can interact with the peptidoglycan layer. It suggests that the stem domains interact and the catalytic domains have freedom to move from the position observed in the crystal structure. This would allow the catalytic domain to have access to pentapeptides at different distances from the membrane.

Keyword
membrane protein, homo-oligomer, penicillin-binding protein, peptidoglycan biogenesis
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62902 (URN)10.1002/pro.677 (DOI)000294383900006 ()
Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2017-12-08Bibliographically approved
4. The Escherichia coli cell division protein ZipA forms homo-dimers prior to its association with FtsZ
Open this publication in new window or tab >>The Escherichia coli cell division protein ZipA forms homo-dimers prior to its association with FtsZ
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62907 (URN)
Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2011-10-11Bibliographically approved

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