The Corynebacterianeae are classified by characteristic cell wall components that give unique characteristics to the cell wall. Although 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.
Figure 1: Dividing Corynebacterium glutamicum cells. (A) 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) 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.
A vital prerequisite for production of viable offsprings is the correct duplication and segregation of genetic material. We have identified the chromosomal partitioning proteins ParAB as important chromosome segregation machinery in C. glutamicum (Figure 2). New results from our lab show that chromosomes are tethered in a DivIVA dependent manner to the cell poles (Figure 2B). Further, we have identified a ParA-like ATPase, PldP, as division site selection protein. Current research focuses on the connection between chromosome segregation and cytokinesis and the molecular functions of the ParAB system as well as the PldP protein.
Figure 2: Subcellular localization of ParA and PldP in C. glutamicum. (A) 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-CFP 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. (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. Scale bars are 2 µm.
Apical Cell Growth
Unlike other rod-shaped bacteria, Actinobacteria including Corynebacterium and Mycobacterium grow by apical insertion of peptidoglycan. Again, the DivIVA protein plays a crucial role in recruiting the cell wall synthesis machinery to the cell poles. Currently, we investigate which components are used to build the apical growth machinery. Our research focuses here on the putative Lipid II flippases RodA and FtsW.
Figure 3: Apical cell growth in C. glutamicum. At the cell poles Lipid II synthesis is mediated by several genes encoding Mra and Mur proteins. Subsequently, Lipid II is flipped across the membrane by the Lipid II flippase RodA or FtsW, respectively. Finally, the cell wall precursors are incoorporated at the outer membrane surface by the Penicillin-binding proteins (Pbp).