Increasing the Scalability of Toxin-Intein Orthogonal Combinations

Abstract

Inteins are proteins embedded into host proteins from which they are excised in an autocatalytic reaction. Specifically, split inteins are separated into two independent fragments that reconstitute the host protein during the catalytic process. We recently developed a novel strategy for the specific killing of pathogenic and antibiotic resistant bacteria based on toxin-intein combinations. Bacterial type II toxin-antitoxin systems are protein modules in which the toxin can provoke cell death whereas the antitoxin inhibits toxin activity. Although our previous system was based on a split intein (iDnaE) and the CcdB toxin, we demonstrated that iDnaE is able to reconstitute four different toxins. To expand the applicability of our system by widening the repertoire of toxin-intein combinations for complex set-ups, we introduced a second intein, iDnaX, which was artificially split. We demonstrate that iDnaX is able to reconstitute the four toxins, and we manage to reduce its scar size to facilitate their use. In addition, we prove the orthogonality of both inteins (iDnaE and iDnaX) through a toxin reconstitution assay, thus opening the possibility for complex set-ups based on these toxin-intein modules. This could be used to develop specific antimicrobial and other biotechnological applications.

Keywords: bacterial killing; inteins; microbial synthetic biology; protein splicing; toxin−antitoxin systems.

Figure 1

Intein–toxin activity assay in vivo. Growth on LB media with the antibiotics needed to maintain the two plasmids, in two different conditions: repressive (1% glucose) or inducible (1 mM IPTG and 0.2% arabinose). Each plate has three different plasmid combinations: “N” with N-terminal fusion plasmid, “C” with the C-terminal fusion plasmid and “N + C” with both plasmids. For “N” and “C”, the bacteria also carries the empty partner plasmidic vector to allow bacteria to grow on the same media.

Figure 2

Orthogonality of toxins halves (a) and split inteins (b). E. coli spots on LB antibiotics and either 1% glucose or 1 mM IPTG and 0.2% arabinose, incubated at 37 °C overnight. Each spot corresponds to a bacteria harboring two plasmids: one carrying the N-terminal intein–toxin fusion listed in the first column, and the other plasmid with the C-terminal fusion as listed above the spots. (a) Orthogonality of toxin halves. In all tested fusions the intein is iDnaE, and the fused toxin is indicated for each combination of plasmids. (b) Orthogonality of intein halves. In all fusions the toxin is CcdB, and the fused intein half is indicated for each combination of plasmid. Spots are made from 10-fold serial dilutions.

Figure 3

Intein splicing kinetics. Spots from the four different combinations of ccdB-iDnaE and ccdB-iDnaX on LB media containing the necessary antibiotics and either glucose (upper panel) or IPTG + arabinose (bottom panel). Overnight cultures in glucose and antibiotics were diluted 1/1000 in media containing IPTG and arabinose, incubated at 37 °C and spotted from 10-fold serial dilutions at different times, as indicated in the figure.