Difference between revisions of "Team:BIT-China/Killing Mechanisms"

Toxin-antitoxin (TA) systems have been abundantly found on plasmids and chromosomes of free-living prokaryotes and genes encoding a toxin and its cognate antitoxin are generally contiguous. Toxins are stable proteins that inhibit one of essential cellular processes, such as DNA replication, translation, peptidoglycan synthesis or cell division.
To date, TA systems are classified into five types according to the nature and the function of antitoxins.
In type I and type III TA systems, the antitoxins are small noncoding RNAs. Type I antitoxins bind to the cognate toxin mRNAs to inhibit their translation initiation or to induce their degradation, while type III antitoxin inactivate their cognate toxins through protein-RNA interactions.
Type II, IV, and V antitoxins are proteins. Type II is the best-characterized TA systems where antitoxins neutralize the harmful effects of the cognate toxins through direct binding. In type IV and V TA systems, only one example has been identified, respectively. The type IV antitoxin, CbeA, antagonizes the activity of CbtA toxin by interfering with the binding of CbtA to its target cytoskeleton proteins, MreB and FtsZ. In type V TA system, GhoS antitoxin itself is an endoribonuclease, which specifically cleaves GhoT toxin mRNA to block its expression.
Our study has focused on the lethal efficiency of different toxin proteins.

Regulations of MazF toxin in E. coli MazF-MazE TA system

Escherichia coli MazF, which belongs to the MazEF family with its cognate antitoxin MazE, is one of the best-characterized toxins. Under stressful environments, MazF specifically cleaves cellular RNAs at ACA sites irrespective of the ribosome, serving as a post-transcriptional regulator.

Interestingly, MazF homologues are well-conserved in the prokaryotic domain. Additionally, they cleave discrete RNA sites based on recognition length and sequences. Therefore, MazF homologues are thought to play diverse biological roles; indeed, they have been implicated in programmed cell death, dormancy, phage abortive infection, and pathogenicity.

Regulations of hokD toxin in E. coli hok/sok TA system

The hok/sok system of the E. coli plasmid R1 was originally discovered in a screen for a locus that mediates efficient plasmid stabilization by killing plasmid-free cells. Sok antitoxin RNA is encoded by plasmids, represses the synthesis of a small, hydrophobic protein (Hok) that kills the host cell by damaging the bacterial cell membrane.
These loci code for three small genes that in hok/sok have been denoted hok (host killing), sok (suppression of killing) and mok (modulation of killing).27 The hok gene encodes a highly toxic transmembrane protein of 52 amino acids that irreversibly damages the cell membrane, and is thus lethal to host cells.

The mok reading frame overlaps extensively with hok, and is required for expression and regulation of hok translation. The sok gene specifies a small cis-acting antisense RNA of 64 nucleotides that is complementary to the hok mRNA leader region. Sok RNA is quite labile (half-life is about 30 sec), but is constitutively expressed from a relatively strong promoter. In contrast, hok mRNA is very stable (half-life is about 20 min)34 and is constitutively expressed from a relatively weak promoter. Genetic analyses showed that Sok RNA inhibits translation of the mok reading frame and that translation of hok is coupled to the translation of mok.31 Consequently, Sok RNA indirectly inhibits translation of hok by preventing mok translation.35 Because Sok RNA is very unstable and is quickly degraded when the R1 plasmid is lost from the cell, the more stable hok mRNA is translated and cells increase plasmid maintenance by killing plasmid-free cells. This plasmid stabilization system is as a result of a phenotype called post-segregational killing (PSK).

In terms of hok/sok gene family. The chromosome of E. coli K-12 contains six hok/sok homologous loci (A, B, C, D, E and X). The hokD gene, formerly known as relF, is encoded by the third gene of the relBEF operon. This gene does not contain upstream regulatory elements and lacks the sok gene. Thus, the hokD locus probably constitutes an evolutionary relic of an ancient hok-homolog.

Since we just need a killer gene to realize the suicide procedure, only the toxin protein is needed.
In early stage of our project, according to the document literature, considering protein stability, dose relationship, accessibility and theoretical lethal efficiency or some other factors, mazF (theoretical lethal efficiency reaches to 95%)and hokD(has a relatively clear quantitative relation with Lon protease which can hydrolyse hokD’s corresponding antitoxin) are chosen as our candidates of killer gene from the frequently-used TA system.
After constructing the circuits of killer device and testing the efficiency of mazF and hokD controlled by pBAD, which can be induced by arabinose, mazF finally become our final choice for having better lethal effect and will be applied in our final circuit.