X. fastidiosa uses quorum sensing molecules to communicate among species in a cell density-dependent manner. The quorum sensing molecule X. fastidiosa uses is part of the family of diffusible signaling factors (DSF) . DSF molecules are typically 2-cis unsaturated fatty acids, produced by a variety of unrelated species like Burkholderia cenocepacia and Pseudomonas aeruginosa, but also by X. fastidiosa . The PDB will be a related species to X. fastidiosa, namely Xanthomonas arboricola, which also uses DSF to communicate among species .
Our proposed system was not able to generate the expected fluorescent signal, at least optically, in none of the tested conditions, independently on the distribution of the several elements, the types of DSF, and its respective concentrations (1, 5 and 10 µM). On the other hand, the high copy number plasmid pSB1C3 showed GFP expression in both induced and uninduced states (Fig. 4). Alternatively, different approaches were performed to determine the functionality of the riboswitch part by part.
First, the TMD and the complete RpfCch protein were both fused to GFP. This to observe whether the RpfCch proteins were being located in the membrane. Results were visualized with fluorescence microscopy and demonstrated the successful transcription, translation and transport of RpfCch to the membrane (Figure 5).
Figure 5: Fluorescent microscopy pictures of the RpfCch (TMD) - GFP fusion. (A-C) Negative control of a strain not expressing GFP. (D-F) Images from a strain constitutively expressing GFP. (G) Image of the RpfCch-sfGFP fusion (BBa_K3286206). (H-I) Image of the RpfCch(TMD)-sfGFP fusions(BBa_K3286207).
The second strategy aimed to test whether GFP translation was inhibited by the Vc2 riboswitch. RNA isolation and cDNA synthesis were performed. cDNA was used as a template in PCR for GFP amplification. Successfully, GFP was transcribed when the Vc2 riboswitch was present in both pSEVA23 and pSB1C3 (Fig. 6). The optical fluorescence can be visualized in Fig. 7.
Third Approach arrow_downward
In the third strategy, we wanted to test overexpression of the phosphodiesterase PdeH . Constitutive expression of this PdeH would lead to lower levels of c-di-GMP . Possibly resulting in GFP translation due to riboswitch alleviation. Unfortunately, the sequencing results of this construct were incorrect due to an insertion of around 30-40 nucleotides between the RBS and the start codon, this will have a strong negative impact on enzyme production. This part was dropped due to time constraints.