Team:Berkeley/Project

ToxR

ToxR is a characterized transmembrane transcription factor from Vibrio cholerae with active domains in both the periplasm and cytoplasm. Natively, it is involved in the transcription of a number of virulence factors, including the two subunits of the cholera toxin ctxAB and the TCP pilus. It is activated in trans by ToxS, a membrane-anchored periplasmic protein coded by the gene directly downstream of toxR. Active ToxS in the periplasm stabilizes the dimerization of the periplasmic domain of ToxR. The association transfers through the membrane, coupling the cytoplasmic domains of ToxR to form an active homodimer. The active ToxR drives transcription from the ctx promoter by binding to TTTGAT repeats and recruiting transcription machinery.

ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.

Stress Promoters

Due to the toxicity caused by toxR in E. coli, a method of regulating toxR expression to produce the maximum amount of toxR without killing the cell was needed. One method was to use a stress promoter that downregulated toxR gene expression whenever the cell became stressed, creating a feedback loop that allows for the highest level of toxR expression without adverse effects on the cell. In order to determine the appropriate stress promoter that would render toxR nonlethal to bacterial cells, a library approach was taken.

Using microarray results from Moen, et. al. (2009), 35 stress promoters that appeared to downregulate expression when under stress were selected. The region of the genome upstream of the ORF was isolated through PCR, creating a library of the 35 stress promoters. An rbs library was then assembled for each stress promoter. The resulting library of rbs and stress promoters was then transformed into reporter cells containing a Pctx.ffGFP plasmid. Pctx is induced only when toxR is expressed, resulting in fluorescence. Cells were assayed for colony size and fluorescence, indicating health and desired transcriptional function. The stress promoter rffGH was found to be the ideal promoter that allowed for expression of toxR while maintaining cell viability.

In a different approach, the stress promoters of interest were coupled to an ffGFP reporter gene, transformed into E. coli, and tested under stress conditions to determine if fluorescence decreased under any general stress condition. A variety of generalized stress conditions were tested. After exposing the cells to stress, the fluorescence was measured and four of the stress promoters were shown to have downregulated expression of ffGFP under cold conditions. Experiments by flow cytometry confirmed the difference in fluorescence between the stress promoters that downregulated expression under stress and the unresponsive promoters. rffGH was also found to downregulate ffGFP expression under cold stress, indicating that it is responsive to general stress in addition to regulating toxR stress.

ToxR Chimeras

Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA.(insert source) Fusion proteins or ToxR-based two-hybrid systems have previously been made for the detection of periplasmic and cytoplasmic protein-protein interactions in E. coli. (insert source) These two-hybrid systems were simply made to determine if two proteins interacted. There is a periplasmic domain that detects changes in the environment and a cytoplasmic domain that activates transcription. These domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by utilizing this dimerization-dependent transcriptional activation feature of the ToxR system.

We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the cytoplasmic domain of ToxR. The fused protein will dimerize only in the presense of a particular ligand. This will cause ToxR to dimerize and activate the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.

Our initial step was to validate that a ToxR based two-hybrid system could be expressed in E. Coli. We first truncated ToxR to eliminate the periplasmic domain because it is only the dimerization of the cytoplasmic domain that controls transcription. Next, we attached the constitutively dimerizing proteins IILK to these ToxR truncates such that the constitutively dimerizing proteins were located in the periplasm. However, the initial ToxR chimeras under the pBad promoter were toxic to the cell and could not be expressed. The picture to the right is a plate of our initial attempt to fuse the constitutively dimerizing proteins to ToxR. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well.

PICTURE OF DEAD CELLS. :(

This high toxicity is likely due to stress that overexpression of these transmembrane proteins cause on the cell membrane. To address this toxicity, we looked at microarray data and screened for stress promoters that showed downregulation upon stresses such as cold temperatures. The promoter rffGH demonstrated a negative feedback system, which allowed us to express the ToxR chimeras that were initially too toxic to be expressed.

The table to the left is fluorescence data demonstrating that IILK is dimerizing and driving ToxR to dimerize. There was just as much if not more fluorescent than our positive control. The results of this experiment demonstrate that ToxR can be expressed in E. coli. Furthermore, it demonstrates that dimerization of ToxR does activate transcrition off of the ctx promoter.

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