CbtA

CbeA-CbtA is one of the Escherichia coli TA systems, and the Toxin, CbtA, has been reported to inhibit the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ.(Fugure 1.)

Figure 1. The mechanism of CbeA and CbtA(type IV TA system).

Usage and Biology

The population and growth condition can be reflected by Abs600, so through the curve of Abs600-time, the effect of CbeA and CbtA towards bacteria can be displayed. Abs600 values were measured from 0 to 14 hours in two groups and the toxication curves were drawn as shown in Figure 2. The growth rate of vector group bacteria after adding L-Arabinose sped up for a short time and then slowed down. At last, the trend was consistent with that without L-Arabinose. However, TA group, with the addition of L-Arabinose, showed the same OD and Abs600 values from the 4th hour for a while. After that, Abs600 values displayed a little increase.

Figure 2. (A) Different concentrations of L-Arabinose were added into vector group and the growth curve was drawn to made sure the effect of L-Arabinose towards bacteria. According to the results, the addition of L-Arabinose would first slightly enhance and then suppress the growth. However, this effect would disappear after a while. (B) Different concentrations of L-Arabinose were added into TA group and the growth curve was drawn to made sure the effect of CbtA(induced by L-Arabinose) towards bacteria. According to the results, with the addition of L-Arabinose the growth would be stagnated at first and then recover.

Besides, after culturing for 14 hrs, difference in turbidities of bacteria in different groups was visible.(Figure 3.)

Figure 3. (A) and (B) The two figures were taken to compare the turbidities of TA group after 14 hours od culturing, and the difference could be told by eyes.

Characterization:

Through agarose gel electrophoresis, the plasmid we constructed was verified.(figure 4.) We chose DL 10000 as the marker and used BBa_K864402 as the control. Eight of the lanes contained pSB1C3-CbtA monoclones and Eight of the lanes contained pSB1C3-CbeA monoclones. After that, we sent them for sequencing and the results showed that the sequences were right.

Figure 4. The result of agarose gel electrophoresis.

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A toxin-antitoxin system is a set of two or more closely linked genes that together encode both a "toxin" protein and a corresponding "antitoxin". Toxin-antitoxin systems are widely distributed in prokaryotes, and organisms often have them in multiple copies. [1]

In nature, toxins and antitoxins form a stable complex inhibiting the toxin activity under normal growth conditions. In case of cellular damage or stress conditions, antitoxins are degraded, allowing the free toxin to bind its cellular target. [2]

In type IV TA systems, the antitoxin does not inhibit the toxin through direct binding but neutralizes its toxicity by stabilizing the toxin target proteins. CbtA, as a representative toxin, aimed at MreB and FtsZ, two major cytoskeleton proteins in Escherichia coli.

By the way, MreB and FtsZ are the homologues of eukaryotic actin and tubulin respectively Those two proteins are important for cell growth and division. MreB is responsible for maintaining the cell shape, polarity ,as well as orienting cell wall synthesis. FtsZ forms a ring structure at the mid-cell and functions as a scaffold during the assembly of divisomes, which is a multi-protein complex and essential for cell division. [3]

Therefore, CbtA holds the advantages of controllable toxic and less effects to the growing environment during co-culture.

References: [1] Fozo, E. M., Makarova, K. S., Shabalina, S. A., Yutin, N., Koonin, E. V., & Storz, G. (2010). Abundance of type I toxin–antitoxin systems in bacteria: searches for new candidates and discovery of novel families. Nucleic Acids Research, 38(11), 3743–3759. doi:10.1093/nar/gkq054
[2] Allocati, N., Masulli, M., Di Ilio, C., & De Laurenzi, V. (2015). Die for the community: an overview of programmed cell death in bacteria. Cell Death & Disease, 6(1), e1609–e1609. doi:10.1038/cddis.2014.570
[3] Masuda, H., Tan, Q., Awano, N., Wu, K.-P., & Inouye, M. (2012). YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli. Molecular Microbiology, 84(5), 979–989. doi:10.1111/j.1365-2958.2012.08068.x