Molecular tweezer tensile strength test.

Initial research: Filamentous Cells

We have chosen to produce a filamentous cell phenotype. Our Initial research lead us to look at the genes and systems listed below. We eventually decided on overexpressing the yneA because we think it will produce the best phenotype and only requires we control one gene. Other advantages include its small gene size.

Filamentous cells genes list

• yneA (Transcribed with yneB, ynzC, is an analogue of sulA in E.coli) * our biobrick is designed to over express this gene reducing cell division possibly by inhibiting FtsZ ring formation or constriction.
• dinR (Homologue of lexA in E.coli transcribed in the opposite direction)
• ftsZ (Involved in the recruitment of other proteins to the divisisome for cytokinesis, strangely over expression results in disruption of Zring formation as well as reduced expression)
• secA (Involved in the secretion of extracellular proteins and the insertion of transmembrane proteins)
• recA (Involved in SOS response removing the repressor DinR (LexA))
• wpr and epr produce extracellular proteases that cleave the signal peptide/transmembrane domain of YneA
• ezr produces a protein which sequesters FtsZ monomer by binding its C terminal domain and also inhibits GTP binding; however overexpression does not result in filamentation.
• min C,D ,J and divIVA prevent polar cell division .
• Positive regulators of FtsZ: ftsA, zapA, zipA, ftsL and divIC
• Inhibitors of Daughter cell separation: lytC,D,E,F and cwlS *Chains rather than filaments, yneA is also reported to increase the time spent in chains well into the stationary phase of bacterial growth.

Alternative system: MinCDJ

In B.subtilis cell division occurs precisely mid cell via the formation of the FtsZ ring (tubulin homologue), through Noc (Nucleoid occlusion: prevents division over nucleoids) and the min system (well described in E.coli), which prevents division taking place at the ends of the cell (poles).

Main function of Min system to prevent mini cell formation and ensuring only one cell division occurs per cell cycle.

Min’s role is not just in the inhibition of FtsZ. FtsZ recruits other components causing synthesis of the new wall and cell invagination.

Cell division is regulated spatially and temporally.

Min C inhibits FtsZ ring formation; Min C interacts with Min D via its C-terminal domain. Min C inhibits lateral interaction between the filaments.

Min D is a membrane associated ATPase. (Min E ensures high concentrations of MinCD at the poles in E.coli, no Min E homologue in B.subtilis! Instead DivIVA acts as a topological factor).

Min C in B.subtilis is responsible for localisation (shown using GFP) showed a primary site of localisation at the site of active division challenging the original model for the role of Min.

Min J was discovered linking Min D to DivIVA and therefore necessary for localisation.

Over expression of Min D in the absence of Min J causes lethal filamentation.

Min C sequence

http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=2858550&to=2859298&strand=true&report=graph&content=5

http://www.ncbi.nlm.nih.gov/gene/937500

MinD sequence

http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=2857736&to=2858622&strand=true&report=graph&content=5

http://www.ncbi.nlm.nih.gov/gene/937499

MinJ sequence

http://www.ncbi.nlm.nih.gov/nuccore/NC_000964.3?from=3620287&to=3621598&strand=true&report=graph&content=5

http://www.ncbi.nlm.nih.gov/gene/936668

van Baarle, S., & Bramkamp, M. (2010). The MinCDJ system in Bacillus subtilis prevents minicell formation by promoting divisome disassembly. PloS one, 5(3), e9850. doi: 10.1371/journal.pone.0009850.

Bramkamp, M. & van Baarle, S., 2009. Division site selection in rod-shaped bacteria. Current opinion in microbiology, 12(6), 683-8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19884039.