UniversityFacultyInstitute
Prof. Dr. Marc Bramkamp

Prof. Dr. Marc Bramkamp

tel: 0221 / 470 64 72

fax: 0221 / 470 50 91

email: marc.bramkamp@uni-koeln.de



Cell division in Bacilli and Corynebacteria

M. Bramkamp, C. Donovan, B. Sieger, J. Bach, P. Sawant, L. Brant, A. Wittmann, A. Gronewold



The pictures on this page are taken from recent presentations or lectures. Please feel free to contact us for further information.


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Scientific Background

Cytokinesis is a prerequisite for cellular life. Therefore, it is not surprising that the process of cell division is tightly controlled and intimately linked to other key cellular processes such as genome replication. Understanding the molecular mechanisms behind cytokinesis revealed fascinating insights in the subcellular organization of eukaryotic and prokaryotic cells.
Our lab is devoted to understand how Gram positive, rod-shaped bacteria, such as Bacillus subtilis and Corynebacterium glutamicum, are accomplishing cell division and how this process is regulated in time and space.

Bacillus subtilis

Rod-shaped bacteria often divide with high precision at midcell to produce two equally sized daughter cells. The positioning of the division machinery in Escherichia coli and Bacillus subtilis is spatially regulated by two inhibitory systems, the nucleoid occlusion and the Min system. The current models suggest that the target of the inhibitory mechanism is the cytoskeletal element FtsZ and that the concerted action of nucleoid occlusion and Min are necessary for correct placement of the division machinery. However, recent advances show that at least the Min system also ensures that division occurs only once in a cell cycle and might act downstream of FtsZ assembly. We have recently identified a new division site selection protein in B. subtilis, MinJ. MinJ serves as a bridge between DivIVA and MinD. Strikingly, we observed that MinJ localization is dynamic and one central function of the Min system is that it acts to prevent re-initiation of cytokinesis at the sites of division rather than simply preventing the formation of new septa close to the cell poles. MinJ interacts with several membrane integral division proteins and seems to couple the inhibitory effect of MinCD to the membrane-integral parts of the divisome.

Cell division in <i>Bacillus subtilis</i>

Fig 1. Cell division in Bacillus subtilis.
Cell division in Bacillus subtilis is studied using light and electron microscopy. Several staining techniques and the use of fluorecently labeled proteins enable us to detect subcellular localization of proteins and compartment specific gene expression. (A) Cell division starts with polymerization of FtsZ (green) between the segregated nucleoids (blue). The cell membrane is stained with nile red (red). (B) Expression of GFP under control of a sF specific promoter within the prespore compartment. The prespore is formed by an asymetric cell division close to one pole of the cell. (C) Transmission EM image of a dividing B. subtilis cell. Note the inwardly growing cell wall, which is typical for Gram positive bacteria. (D) Sites of active peptidoglycan synthesis were stained with a fluorescent antibiotic. Dividing cells synthesize the majority of peptidoglycan at the division site.

Cell division in <i>Bacillus subtilis</i>

Fig. 2. Cell division in B. subtilis.
Step I
: Cell division is initiated by polymerization of the tubulin homologue FtsZ (Z) into a ring structure. The actin homologue FtsA (A) is associated with the Z-ring, while the membrane bound EzrA (Ez) protein might establish a connection of the ring with the cytoplasmic membrane. Step II: Subsequently, the membrane spanning proteins FtsL (L), DivIC (C), DivIB (B), PBP2b, and the integral protein FtsW are recruited to the cytokinetic ring. After assembly of the full divisiome, the two daughter cells are separated by an inwardly growing cell wall, following the constricting Z-ring. The cytoplasmic domain of FtsL and perhaps other division proteins couple the peptidoglycan synthesis machinery to the constricting cytokinetic ring. Step III: At the end of constriction, the peptidoglycan synthesis is stopped, probably by regulated intramembrane proteolysis (RIP). The regulatory protein FtsL is cleaved by a membrane integral metalloprotease (YluC) and then degraded. Step IV: After removal of the key regulator FtsL the divisome is disassembled and cell division is completed. While key regulators FtsL and DivIC are proteolytically degraded, other division proteins may diffuse from the division site.

Localization of a division protein in B. subtilis

Fig. 3. Localization of a division protein in B. subtilis.
A new cell division protein in B. subtilis localizes to the division site (A). In absence of FtsZ polymers (FtsZ polymerization was inhibited by an antibiotic compound) the division protein is dispersed throughout the cell (B), showing the dependence on the FtsZ polymer.

Corynebacterium glutamicum

Corynebacterium glutamicum is a soil-dwelling Gram positive bacterium. It belongs to the high GC content Gram positives, also called Actinobacteria. C. glutamicum is capable of secreting amino acids such as L-glutamate and L-lysine. Because of the high industrial importance of these amino acids as food flavor enhancer and food additives C. glutamicum is an intensively studied organism. Likewise, C. glutamicum is a model organism for other members of the suborder Corynebacterianeae. This suborder includes important pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, and C. diphtheriae. The Corynebacterianeae are classified by characteristic cell wall components that give unique characteristics to the cell wall. Although the Corynebacterianeae have a thick peptidoglycan layer like other Gram positive bacteria, they have a second permeability barrier formed by a bilayer of mycolic acids on the cell surface. Mycoloyl residues are covalently liked to arabinogalactan, another cell wall component of Corynebacterianeae. The cell morphology of Corynebacterianeae is diverse. The name giving “coryne-form” (club-shaped) is often observed, however, cells can form as classical rods or cocci, depending on growth conditions. Unlike other Gram poitive bacteria members of the Actinobacteria have little or no cylindrical cell wall growth. These cells only grow at the division sites and cell poles. Because of this interesting morphology we are studying how C. glutamicum manages to divide and how the cell shape is achieved. This is of interest since the Corynebacterianeae lack a MinCD system, which positions the cytokinetic ring in many other bacteria. This suborder of the Actinobacteria also lacks actin-like cytoskeletal elements, which are involved in cell shape determination and chromosome segregation in various bacteria. Our research is currently focused on the mechanisms involved in septum placement and pole formation.

Cell division in <i>Corynebacterium glutamicum</i>

Fig. 4. (A) Dividing Corynebacterium glutamicum cells.
A characteristic division phenotype of Corynebacteriaceae is the “V-shaped” division. The division occurs after the nucleoids (stained with DAPI, blue) are segregated. The pole determining protein DivIVA is visualized using a DivIVA-GFP fusion protein (second copy under native promoter). DivIVA localizes to the cell poles (GFP fluorescence is colored in green).
(B) Sites of cell wall synthesis in C. glutamicum.
Staining with a fluorescent vancomycin derivative indicates the areas of peptidoglycan synthesis. In C. glutamicum cell wall synthesis occurs at the poles and the division site.

Cell division in <i>Corynebacterium glutamicum</i>

Figure 5. (A) Subcellular localization of ParA and PldP in C. glutamicum.
Subcellular localization of ParA and PldP was analyzed using strains were the native alleles have been replaced by cfp fusion genes. Shown are phase contrast images (Phase), membrane stain (Membrane), DNA stain with Hoechst dye (DNA), CFP fluorescence (CFP, false colored in green), and a merge image of membrane stain, DNA stain and CFP fluorescence (Overlay). ParA-CFP localization is shown in the upper panel. Characteristic polar foci of ParA-CF are indicated by arrows in the CFP channel. PldP localization in is shown in the middle panel. The arrow points to midcell localization of PldP. Scale bars are 2 µm.
(B) The origin of replication and ParB are localized to the cell poles in C. glutamicum (upper panel). Localization of ParB-CFP (false colored in green) in C. glutamicum (lower panel).
A strain expressing YFP-TetR grown in MMI medium were analyzed microscopically. A compilation of cells showing YFP-TetR foci is shown in the upper panel. DAPI staining is depicted in blue and YFP fluorescence is shown in yellow (ori). The lower panel (ParB) shows wild type cells after immuno-fluorescence staining with polyclonal antibodies against ParB. The scale bars are 2 µm.

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AIMS

We use the prevailing model organisms B. subtilis and C. glutamicum to unravel principal mechanisms of division site selection and subcellular organization of rod-shaped bacteria. These fundamental questions in bacterial cell biology are tackled with state-of-the art techniques such as live cell imaging, in vitro reconstitution and protein-protein or protein-DNA interaction studies.


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Current Cell Division Team (January 2012)

Current Team

From left to right: Lili Brant, Martijn Snelling Berg, Juri Bach, Ailisa Blum, Boris Sieger, Catriona Donovan, Marc Bramkamp, Prachi Sawant, Anja Gronewold, Gabi Sitek, Anja Wittmann

Prof. Dr. Marc Bramkamp
Group leader
0221 - 470 6472
marc.bramkamp@uni-koeln.de

Anja Wittmann
Technician
0221 - 470 6472
a200430@uni-koeln.de

Gabi Sitek
General Staff
0221 - 470 6472
gsitek@uni-koeln.de

Catriona Donovan (M. Sc.)
PhD Student
Cell division in C. glutamicum
0221 - 470 6472
cdonovan@uni-koeln.de

Boris Sieger (Dipl. Chem.)
PhD Student
Cell wall synthesis in C. glutamicum
0221 - 470 6472
b.sieger@uni-koeln.de

Juri Bach (M. Sc.)
PhD Student
Membrane dynamics in B. subtilis
0221 - 470 6472
juribach@googlemail.com

Prachi Sawant (M. Sc.)
PhD Student
Membrane dynamics in B. subtilis
0221 - 470 6472
prachisawant9@gmail.com

Lilija Brant (B. Sc.)
Master Student
Cell division in B. subtilis
0221 - 470 6472
lbrant@smail.uni-koeln.de

Anja Gronewold (B. Sc.)
SHK
Cell division in B. subtilis
0221 - 470 6472
agronewo@smail.uni-koeln.de

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Alumni

name

has left the group in

topic

degree

Lisa Renfordt

2011

Cytokinesis in C. glutamicum

B. Sc.

Dr. Inga Wadenpohl

2011

Cytokinesis in B. subtilis

PhD

Yvonne Merkler

2011

Sporulation in B. subtilis

B. Sc.

Katja Nagler

2010

Sporulation in B. subtilis

B. Sc.

Sabatini Jacob

2010

Cytokinesis in C. glutamicum

B. Sc.

Nina Ebert

2010

Cytokinesis in B. subtilis

M. Sc.

Frank Bürmann

2009

Cytokinesis in B. subtilis

Dipl. Biol.

Dr. Suey van Baarle

2009

Cytokinesis in B. subtilis

PhD

Joana Mehlmann

2009

Cytokinesis in B. subtilis

B. Sc.

Dr. Astrid Schwaiger

2009

Cytokinesis in C. glutamicum

PhD

Sabah Elouelji

2008

Cytokinesis in C. glutamicum

Dipl. Biol.

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Funding

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Cooperation

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Selected Publications

  • A synthetic E. coli system identifies a conserved origin tethering factor in Actinobacteria.
    Catriona Donovan, Boris Sieger, Reinhard Krämer, Marc Bramkamp.
    Mol Microbiol. 2012 Feb 17. doi: 10.1111/j.1365-2958.2012.08011.x


  • A bacterial dynamin-like protein mediating nucleotide-independent membrane fusion.
    Bürmann F, Ebert N, van Baarle S, Bramkamp M.
    Mol Microbiol. 2011 Mar;79(5):1294-304. doi: 10.1111/j.1365-2958.2011.07523.x.


  • DivIC stabilizes FtsL against RasP cleavage.
    Wadenpohl I, Bramkamp M.
    J Bacteriol. 2010 Jul;192(19):5260-5263.


  • Subcellular localization and characterization of the ParAB system from Corynebacterium glutamicum.
    Donovan C, Schwaiger A, Krämer R, Bramkamp M.
    J Bacteriol. 2010 Jul;192(13):3441-51.


  • The MinCDJ system in Bacillus subtilis prevents minicell formation by promoting divisome disassembly.
    van Baarle S, Bramkamp M.
    PLoS One. 2010 Mar 24;5(3):e9850.


  • The putative Bacillus subtilis L,D-transpeptidase YciB is a lipoprotein that localizes to the cell poles in a divisome-dependent manner.
    Bramkamp M.
    Arch Microbiol. 2010 Jan;192(1):57-68. Epub 2009 Dec 16.


  • Division site selection in rod-shaped bacteria.
    Bramkamp M, van Baarle S.
    Curr Opin Microbiol. 2009 Dec;12(6):683-8. Epub 2009 Nov 1.


  • Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for non-essentiality and for phosphorylation of OdhI and FtsZ by multiple kinases.
    Schultz C, Niebisch A, Schwaiger A, Viets U, Metzger S, Bramkamp M, Bott M.
    Mol Microbiol. 2009 Nov;74(3):724-41. Epub 2009 Sep 28


  • Characterization and subcellular localization of a bacterial flotillin homologue.
    Donovan C, Bramkamp M.
    Microbiology. 2009 Jun;155(Pt 6):1786-99. Epub 2009 Apr 21.


  • A novel component of the division-site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD.
    Bramkamp M, Emmins R, Weston L, Donovan C, Daniel RA, Errington J.
    Mol Microbiol. 2008 Dec;70(6):1556-69. Epub 2008 Oct 23.


  • Population Heterogeneity in Corynebacterium glutamicum ATCC 13032 caused by prophage CGP3.
    Frunzke J, Bramkamp M, Schweitzer JE, Bott M.
    J Bacteriol. 2008 Jul;190(14):5111-9. Epub 2008 May 16.


  • Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex from Escherichia coli (Review).
    Bramkamp M, Altendorf K, Greie JC.
    Mol Membr Biol. 2007 Sep-Dec;24(5-6):375-86.


  • Regulated intramembrane proteolysis of FtsL protein and the control of cell division in Bacillus subtilis.
    Bramkamp M, Weston L, Daniel RA, Errington J.
    Mol Microbiol. 2006 Oct;62(2):580-91.


  • Prokaryotic Kdp-ATPase: recent insights into the structure and function of KdpB.
    Haupt M, Bramkamp M, Coles M, Kessler H, Altendorf K.
    J Mol Microbiol Biotechnol. 2005;10(2-4):120-31.


  • The holo-form of the nucleotide binding domain of the KdpFABC complex from Escherichia coli reveals a new binding mode.
    Haupt M, Bramkamp M, Heller M, Coles M, Deckers-Hebestreit G, Herkenhoff-Hesselmann B, Altendorf K, Kessler H.
    J Biol Chem. 2006 Apr 7;281(14):9641-9.


  • An atypical KdpD homologue from the cyanobacterium Anabaena sp. strain L-31: cloning, in vivo expression, and interaction with Escherichia coli KdpD-CTD.
    Ballal A, Bramkamp M, Rajaram H, Zimmann P, Apte SK, Altendorf K.
    J Bacteriol. 2005 Jul;187(14):4921-7.


  • Single amino acid substitution in the putative transmembrane helix V in KdpB of the KdpFABC complex of Escherichia coli uncouples ATPase activity and ion transport.
    Bramkamp M, Altendorf K.
    Biochemistry. 2005 Jun 14;44(23):8260-6.


  • Functional modules of KdpB, the catalytic subunit of the Kdp-ATPase from Escherichia coli.
    Bramkamp M, Altendorf K.
    Biochemistry. 2004 Sep 28;43(38):12289-96.


  • Inter-domain motions of the N-domain of the KdpFABC complex, a P-type ATPase, are not driven by ATP-induced conformational changes.
    Haupt M, Bramkamp M, Coles M, Altendorf K, Kessler H.
    J Mol Biol. 2004 Oct 1;342(5):1547-58.


  • Amino acid substitutions in putative selectivity filter regions III and IV in KdpA alter ion selectivity of the KdpFABC complex from Escherichia coli.
    Bertrand J, Altendorf K, Bramkamp M.
    J Bacteriol. 2004 Aug;186(16):5519-22.


  • 1H, 13C and 15N resonance assignment of the nucleotide binding domain of KdpB from Escherichia coli.
    Haupt M, Coles M, Truffault V, Bramkamp M, Altendorf K, Kessler H.
    J Biomol NMR. 2004 Jul;29(3):437-8.


  • FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli.
    Bramkamp M, Gassel M, Altendorf K.
    Biochemistry. 2004 Apr 20;43(15):4559-67.


  • Mutational analysis of charged residues in the putative KdpB-TM5 domain of the Kdp-ATPase of Escherichia coli.
    Bramkamp M, Altendorf K.
    Ann N Y Acad Sci. 2003 Apr;986:351-3.


  • The Methanocaldococcus jannaschii protein Mj0968 is not a P-type ATPase.
    Bramkamp M, Gassel M, Herkenhoff-Hesselmann B, Bertrand J, Altendorf K.
    FEBS Lett. 2003 May 22;543(1-3):31-6.


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