Designing the Transfer of the Type Six Secretion System into E. coli

Using a fosmid to transfer the T6SS genes into E. coli

The T6SS is comprised of 23 genes across several operons. Capturing and moving these genes via standard restriction digest cloning was determined to be impractical. We discovered that the sequence of the P. aeruginosa strain (PAO1) we were using was solved using fosmids (essentially large plasmids). We were able to locate these fosmids and were very excited to find that one of the fosmids contained all of the necessary T6SS genes organized nicely in two divergent operons. We successfully transferred the fosmid into E. coli. It was not clear, however, whether or not the genes would be expressed in E. coli, since the promoters that controlled the expression of the T6SS genes on the fosmid were P. aeruginosa' genes. In order to verify expression of the T6SS genes we performed a Western blot using antibodies against Fha1, which is a critical protein of the secretion system and a reporter for T6SS activity.

Obtained Fosmid Contains T6SS Genes Organized into Two Divergent Operons

Testing the native Pseudomonas aeriginosa promoter in E. coli

To determine whether the T6SS genes from the fosmid were being expressed from the native P. aeriginosa promoter, the fosmid was tranferred into E. coli, and a Western blot was performed on cell extracts for Fha1, one of the proteins critical for T6SS activity. As expected, the Western blot showed no bands (shown below), indicating that E. coli was probably not transcribing the T6SS units from the native P. aeriginosa promoter. To allow expression of these genes in E. coli, the promoter was engineered to be a bidirectional T7 promoter, which would promote transcription in both the 5' and 3' direction. T7 promoters are well characterized, and known to be highly robust for expression in E. coli.

Expression of T6SS Components from the Fosmid Occurs in P. aeruginosa but Not in E. coli

Toxin/ Antitoxin System

Regulating the Toxin and Antitoxin

We chose to separate the toxin/antitoxin (Tse2/Tsi2) regulation from the regulation of the T6SS, since controlling all of these components would be limited by the complexity of the T6SS, and also the massive amount of protein production. The purpose of generating novel Tse2/Tsi2 regulatory circuits is to activate the killing activity of our E. coli antibiotic only when necessary. If our probiotic system were constitutively producing Tse2, this would adversely affect natural gut flora. In addition, natural gut flora might develop resistance to Tse2 and pass this resistance to potential Gram-negative pathogens. As a BioBrick, the Tse2Tsi2 circuit is modular, and therefore promoters and other regulatory components can be swapped out. For example, one could ingest the probiotic, have it exist in the gut, but only induce its killing behavior by ingesting some user-defined signal, such as arabinose, which would activate expression of the toxin, which would then travel through the already-present T6SS into Gram-negative targets. In addition, one could envision a scenario in which it would be advantageous to separately regulate Tsi2, to kill the probiotic when it is no longer useful by preventing Tsi2 expression, causing cell suicide due to the production of Tse2. For our purpose in this project, we decided to make a probiotic that produced the Tse2 toxin only when the probiotic detected the presence of a pathogen.

Design of our Inducible Tse2/Tsi2 system

Activating Tse2 production when a pathogen is present requires a promoter that is inducible by some molecular stimulus unique to a specific pathogen. In addition, the expression of Tsi2 would need to be constituitive, or induced by the same stimulus that induced Tse2 expression. As a proof-of-concept, this project uses the LuxR-pLux transcription factor- promoter system from Vibrio fischeri to regulate expression of the Tse2-Tsi2 locus. V. fischeri excretes 3OC6HSL, a small membrane permeable molecule(hereafter referred to as HSL). HSL binds to LuxR, changing the conformation of LuxR, which then induces the pLux promoter. Since V. fischeri also produces HSL, expression from the pLux promoter is linked to cell density. This is referred to as quorum sensing. Quorum sensing is found in many pathogenic species, making the use of the pLux-LuxR system a good proof-of concept. When our probiotic detects a gram-negative pathogen-specific molecule (modeled by HSL), transcription is induced by an inducible promoter (modeled by pLux). This leads to expression of Tse2 (a toxic protein) and Tsi2 (its antitoxin). The T6SS then attacks the pathogen, puncturing the cell wall. Tse2 is then secreted into the gram negative pathogen, killing the pathogen. This system could easily be modified to target a wide range of gram-negative pathogens by just changing the regulation of the Tse2/Tsi2 locus.

Diagram of the Tse2/Tsi2 HSL inducible circuit

The Tse2/Tsi2, toxin/antitoxin, system has a relatively simple circuit design. Tse2 and Tsi2 are present in one operon (as in Pseudomonas aeruginosa) and are regulated by the pLux promoter. The LuxR transcriptional factor is constituitively expressed because no tetR is present to repress the production of LuxR. When HSl is present it binds to LuxR resulting in the induction of Tse2 and Tsi2 production. The pTet, LuxR, and pLux region of the construct is present in part [http://partsregistry.org/Part:BBa_F2620 F2620]. This made the construction of the circuit considerably easier.