Team:Hamburg/Project/PezT

Project: PezT

UDP-GlnNAc-kinase system

The suicide mechanism is to be achieved by the UDP NAcGln kinase system. Here, the osmotic barrier is destabilized, leading to lysis of the cell, stopping the miRNA production and releasing the miRNA. The gene of the kinase is going to be right behind a heat-inducible promoter pQF50KgroE inserted into the pJT119b plasmid. By this design the successive switching between the different genes is possible.

Production

Both plasmids are to be established in various experiments in E. coli. The products / lysis of target cells is to be examined and verified. Finally, both expression vectors (including promoter) will be combined on one plasmid in E. coli. The organism is then assayed for its ability to produce the miRNA2911 and to autolyse.

PezT – bacterial self-destruction [1]

PezT (pneumococcal epsilon-zeta toxin) is part of a natural toxin-antitoxin system in procaryotes leading to cell death as a response for stress and is a mechanism of growth regulation. This mechanism is based on its kinase activity – phosphorylating uridine diphosphate acetylglucosamine (UNAG) (Fig. 1).

Figure 1: Enzymatic activity of PezT. [1]

UNAG is an ubiquitous molecule required from MurA for the first step of peptidoglycan biosynthesis. The phosphorylation from UNAG into UNAG-3P is not only leading to a dead-end metabolite, but also to a competitive inhibition of MurA, weakening the bacterial cell wall and causing osmotic instability (Fig. 2,3).

This process is lethal for fast dividing bacteria, though not for bacteria with a decreased proliferation (Fig. 3).

Figure 3: Stress induced self-destruction of bacteria caused by PezT. [1]

This kinase was expressed in E. coli leading to massive cell death in only ½ to 1 hour. Possible applications of PezT might be a biotechnological approach anterior harvesting or the development of new antibiotica based on the UNAG-3P-structure consisting the increasing resistance of pathogenic bacteria.

H. Mutschler, M. Gebhardt, R. L. Shoeman, A. Meinhart (2011) A Novel Mechanism of Programmed Cell Death in Bacteria by Toxin–Antitoxin Systems Corrupts Peptidoglycan Synthesis. PloS Biology Vol. 9: 1-12.