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Fluorescence Studies of Cell Division in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In Escherichia coli the cell division is carried out by a large dynamic protein complex called the divisome. The divisome assembles in a two-step manner starting with the localization of the eukaryotic tubulin homologue FtsZ to the midcell. Together with other early arriving proteins FtsZ form an intermediate structure called the Z-ring. After a considerable time lag the divisome maturates fully by recruiting several other late arriving proteins before it starts to constrict the cell envelope that ultimately will lead to cytokinesis and the formation of two identical daughter cells. Despite of being objectives of extensive study over the last decades, understanding of the exact molecular roles of many of the divisome proteins is still lacking and to date there is very limited knowledge of the disassembly process of the divisome. In this thesis I have used various fluorescence microscopy based methods to better characterize the role of FtsZ and other divisome proteins during the final stages of the cell division. I have shown that FtsZ disassembles from the divisome prior to inner membrane closure indicating that it is not the force generator during this final step of division that it is widely thought to be. I have also shown that the disassembly of the divisome is a multistep process in which the proteins that arrive in the second step of divisome assembly also remain at the division septum longer than those proteins that arrive in the first step. These findings add new important information regarding the cell division and together they provide a more complete picture of this event that ultimately may lead to more efficient identification of novel antibiotic targets.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2014. , 84 p.
Keyword [en]
E. coli, Cell division, FtsZ, Fluorescence microscopy, FRAP
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-102619ISBN: 978-91-7447-852-5 (print)OAI: oai:DiVA.org:su-102619DiVA: diva2:711789
Public defence
2014-06-03, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius Väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2014-05-12 Created: 2014-04-11 Last updated: 2014-05-13Bibliographically approved
List of papers
1. 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
2. Disassembly of the divisome in Escherichia coli: evidence that ftsz dissociates before compartmentalisation
Open this publication in new window or tab >>Disassembly of the divisome in Escherichia coli: evidence that ftsz dissociates before compartmentalisation
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2014 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 92, no 1, 1-9 p.Article in journal (Refereed) Published
Abstract [en]

In most bacteria cell division is mediated by a protein super-complex called the divisome that co-ordinates the constriction and scission of the cell envelope. FtsZ is the first of the divisome proteins to accumulate at the division site and is widely thought to function as a force generator that constricts the cell envelope. In this study we have used a combination of confocal fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) to determine if divisome proteins are present at the septum at the time of cytoplasmic compartmentalization in Escherichia coli. Our data suggest that many are, but that FtsZ and ZapA disassemble before the cytoplasm is sealed by constriction of the inner membrane. This observation implies that FtsZ cannot be a force generator during the final stage(s) of envelope constriction in E. coli.

Keyword
FtsZ, Cell division, FRAP, Microscopy
National Category
Microbiology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-102615 (URN)10.1111/mmi.12534 (DOI)000334331300001 ()
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2017-12-05Bibliographically approved
3. ZipA and FtsA disassemble from the divisome prior to FtsQ, FtsL and FtsI during cell division in Escherichia coli
Open this publication in new window or tab >>ZipA and FtsA disassemble from the divisome prior to FtsQ, FtsL and FtsI during cell division in Escherichia coli
(English)Manuscript (preprint) (Other academic)
Keyword
Divisome, Cell division, E. coli
National Category
Microbiology Biophysics
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-102618 (URN)
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2014-04-11Bibliographically approved
4. Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli
Open this publication in new window or tab >>Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli
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2012 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 21, no 10, 1571-1576 p.Article in journal (Refereed) Published
Abstract [en]

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.

Keyword
topology, membrane protein, split-GFP, inner membrane
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-82120 (URN)10.1002/pro.2131 (DOI)000308994300015 ()
Note

AuthorCount:6;

Available from: 2012-11-09 Created: 2012-11-08 Last updated: 2017-12-07Bibliographically approved

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