Filamentous cell formation by overexpression of yneA

Bacillus subtilis cell division is dependent on FtsZ. FtsZ forms a 30 subunit ring at the midpoint of the cell and contracts.

YneA indirectly stops the formation of the FtsZ ring. In nature, yneA is expressed during SOS response, allowing the cell to repair DNA damage before continuing with the division cycle.

It is hypothesized that YneA acts through unknown transmembrane proteins to inhibit FtsZ ring formation; we call these unknown components "Blackbox proteins".

By expressing YneA and therefore inhibiting FtsZ ring formation, cells will grow filamentous.

Part

Our IPTG-inducible filamentous cell formation part puts yneA under the control of the strongly LacI-repressible promoter that we designed, hyperspankoid. In the presence of LacI, induction with IPTG will result in a filamentous cell phenotype.

The part has no terminator, allowing for transcriptional fusion with gfp and visualisation under the microscope.

This is part BBa_K302012 on the parts registry.

Computational model

	We wrote a computational model of our filamentous cell system in SBML and simulated it in COPASI to help us verify our part's behaviour before we built it. The graph on the left shows that FtsZ ring formation is low when yneA is highly expressed.

	Visualisation of the model's biochemical network in CellDesigner.

Downloads:

• SBML-shorthand
• SBML

Cloning strategy

yneA cloning strategy

Characterisation

We integrated our part into the Bacillus subtilis 168 chromosome at amyE (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at amyE destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.

The part was co-transcribed with gfp fluorescent marker by transcriptional fusion after the yneA coding sequence.

We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate lacI into the Bacillus subtilis 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.

Normal Bacillus subtilis 168

Filamentous cells

Filamentous cells showing GFP signal

Filamentous cells (integrated at amyE)

Filamentous cells showing GFP signal(integrated at amyE)

Graphs

Table 1:

Figure 1:

Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.

Figure1: shows statistics for populations of cells overexpression of the yneA construct (ΔamyE:pSpac(hy)-oid::yneA(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control Bacillus subtilis 168. pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)

with inhibition cell lengths are comparable to Bacillus subtilis 168 at 0μM IPTG and longer with IPTG induction.

Figure 2:

Figure2: Bacillus subtilis 168 cells (left),Bacillus subtilis expressing yneA(centre) and Bacillus subtilis overexpressing yneA(right)

The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length.

Figure 3:

Figure 3 shows the percentage of cells at different lengths (μm) uninduced

Figure 4:

Figure 4:Bacillus subtilis 168 cells (left) and non-induced cells (right)

Figure 5:

Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG

Figure 6:

Figure 6: Bacillus subtilis 168 cells (left) and cells induced at 0.2mM IPTG (right)

Figure 7:

Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG

Figure 8:

Figure 8: Bacillus subtilis 168 cells (left) and cells induced at 1mM IPTG(right)

Research

Initial Research

References

Kawai, Y., Moriya, S., & Ogasawara, N. (2003). "Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis". Molecular microbiology, 47(4), 1113-22.

Mo, A.H. & Burkholder, W.F., (2010). "YneA , an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis , Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity" ᰔ †. Society, 192(12), 3159-3173.