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|Title:||Design and application of cyclic-di-GMP biosensors.||Authors:||Ho, Chun Loong.||Keywords:||DRNTU::Science::Biological sciences::Biochemistry||Issue Date:||2013||Source:||Ho, C. L. (2013). Design and application of cyclic-di-GMP biosensors. Doctoral thesis, Nanyang Technological University, Singapore.||Abstract:||Cyclic‐di‐GMP is an important bacterial secondary messenger molecule that regulates motility, virulence and biofilm formation in many pathogenic bacteria. This messenger molecule is synthesized from cellular GTP by diguanylate cyclase (DGC) and hydrolyzed by phosphodiesterases (PDE) to form 5‐pGpG and GMP. In this dissertation, I have designed biosensors that can report the in vitro and in vivo changes of c‐di‐GMP concentration that can be used for elucidating the function of DGC and PDE proteins in the regulation of c‐di‐GMP that modulates the biofilm formation. The in vitro biosensor was designed using the catalytically inactive EAL domain of FimX (EALFimX) that binds to c‐di‐GMP at sub‐micromolar concentrations. Out of 6 different mutants designed for fluorescent dye labeled biosensor, the mutant Q484C‐MDCC showed to be the best performer. When titrated with c‐di‐GMP, this biosensor protein shown a disassociation constant (Kd) of 172 nM with a decrease in fluorescence by 46%. This biosensor was demonstrated to be highly specific to c‐di‐GMP and is able to report c‐di‐GMP concentration changes in real time. The biosensor was used to determine the steady‐state kinetics of AxDGC2 and RocR. Screening of nucleotide library revealed that AxDGC2 is inhibited by Rp‐GTP‐α‐S and GDP. The IC50 value of Ca2+ as an inhibitor of RocR was calculated to be at 141 μM. This demonstrates that the Q484C‐MDCC was able to report changes in c‐di‐GMP concentrations resulting from DGC and PDE activities and can be used to screen for inhibitors effective against these catalytically active proteins. The FRET in vivo biosensors were designed by flanking PilZ domain proteins, VCA0042 and MrkH with fluorescent proteins mCerulean and mVenus. By measuring the FRET activity, we were able to observe cellular fluctuations of c‐di‐GMP when bacteria cells expressing these sensors were treated with biofilm triggering antibiotic and biofilm dispersing factors. The treatment of E. coli Bl21(DE3) and wild type UTI89 with antibiotics that target cell wall synthesis and ribosomal activity at sub‐MIC shown to up‐regulate c‐di‐GMP, whilst treatment with biofilm dispersal factors causes a down‐regulation of c‐di‐GMP levels. It was also observed that when these E. coli cells were engulfed by murine macrophage cells, the antimicrobial agents secreted for digestion of these xiv bacterial cells down‐regulates the c‐di‐GMP concentrations. This proves that the FRET biosensors are capable of reporting changes in c‐di‐GMP concentrations in E. coli cells and can be used for testing different drugs to promote or disperse biofilms. The polyketide synthase (PKS) are a group of multi-domain proteins involved in synthesizing the various polyketide chains that makes up the majority of drugs today. We have described the optimization of the expression conditions for 2 groups of these large sized proteins. These proteins are the SalA-SalB hybrid system and the PKS8 from Salinospora tropica and Saccharopolyspora erythraea respectively.||URI:||http://hdl.handle.net/10356/55277||Fulltext Permission:||restricted||Fulltext Availability:||With Fulltext|
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