Structural Study Of A Quorum Sensing Signal Synthetase AmbB

Antibiotics used in clinic achieve the purpose of anti-infection by killing microbes or inhibiting the growth of them. However, the overuse of them induces drug resistance. Bacteria can communicate with each other by quorum sensing (QS), which can release the autoinducers to control a number of bacterial virulence factors. The QS system has been regarded as a promising target for developing novel approaches to controlling bacterial infections, as it can not cause drug resistance due to the fact that it just suppresses the pathogenicity caused by QS instead of affecting their growth.Pseudomonas aeruginosa, an opportunistic pathogen, can cause respiratory tract infection or pneumonia. Three QS systems consisting of las, rhl and pqs that function in a hierarchical manner have been identified in P. aeruginosa. Among them, the las system controls the expression of the rhl and pqs genes, which is at the top of the regulatory networks. Evidence is emerging that las may be dispensable in activating downstream QS networks and expression of the virulence genes. Recent studies revealed that the nonribosomal peptide synthase AmbB was involved in the production of IQS, a potent new cell-cell communication signal in QS. IQS can modulate the pqs and rhl systems instead of las. Disruption of ambB impaires QS systems, resulting in a dramatic decrease of IQS and attenuates bacterial virulence. Given the importance of AmbB, in this thesis, I was to try to illustrate the synthesis mechanism of IQS through the structural study of AmbB.The results are shown as follows:1.I have cloned the AmbBc gene into different expression vectors, and obtained AmbBc with the purity of95%.2. Ten conditions have been screened out to grow crystals.3. A2.5A X-ray diffraction data set has been collected using synchrotron-radiation source.4. I have obtained the heavy-atom-derivatized cystals and selenomethionine-derivatized crystals.5. The structure of AmbBc has been solved by enhanced anomalous scattering signal through introducing extra selenomethionine via site-directed mutation stratagy.