| Summary: | 碩士 === 國立交通大學 === 分子醫學與生物工程研究所 === 104 === The envelope stress two component system CpxAR, which is composed of the sensor kinase CpxA and the cognate response regulator CpxR, allows bacteria monitor the ever changing environments and respond with proper adjustments. The known cpx activation signals include alkaline pH, osmotic pressure, temperature, metals, alterations of membrane composition and misfolded proteins. We have previously reported that deleting cpxAR gene from Klebsiella pneumoniae CG43S3 significantly enhanced the expression of type 1 and type 3 fimbriae. A comparative transcriptomic analysis of CG43S3, CG43S3ΔcpxAR, CG43S3ΔcpxR by RNA sequencing and qRT-PCR were subsequently employed to understand how Cpx system affects the type 3 fimbriae expression. In this thesis, we firstly analyzed the cpxAR and cpxP promoter activity and found that activities could be repressed by zinc ion and hydrogen peroxide, but had no apparent response to the addition of urate. The deletion of cpxAR increased the sensitivity to antibiotics and the defects could be complemented by introducing a expression plasmid into the mutant which further confirmed that CpxAR may play an important role in the resistance to antibiotic. Next, after analysis and intergration of the aforementioned transcriptome data, we selected three potential members of the CpxAR regulatory pathways for further study. One is yfiN which coding for diguanylate cyclase (DGC), the second is rcsA that encodes the major transcription regulator for the capsular polysaccharides synthesis, the third is mrkH which is an activator for the expression of type 3 fimbriae. Promoter activity analysis revealed that the deletion of cpxAR increased the expression of yfiRNB and EMSA analysis indicated that the recombinant CpxR was able to specificity bind to the yfiRNB promoter. Furthermore, we have found that the rcsA deletion increased the production of MrkA (the major pilin of type 3 fimbriae) as assessed using western blot analysis, and the deletion also increased the cellulose production. Moreover, the rcsA deletion effects could be restored by transforming a YjcC expression plasmid into the mutant, and bioinformatic analysis revealed that the putative promoter region of yjcC and ycdT contain RcsAB binding sequences. These results suggest the RcsA-mediated regulation is through controlling the c-di-GMP levels thereafter affecting the production of type 3 fimbriae and cellulose. At last, deletion of mrkH from ΔcpxAR or ΔyjcC decreased the cellulose production, and MrkH binding elements could be identified on the puatative promoter of six c-di-GMP level modulators D364_24605, D364_06025, D364_08130, yoaD, D364_14405 and D364_13985, implying a MrkH dependent expression of these genes. In addition, western blot analysis showed that MrkH expression could only be detected in ΔcpxARΔyjcC. Taken together, we speculate that CpxAR regulates the activity of YfiN, RcsA, and MrkH thereby influencing the c-di-GMP levels and eventually affects the type 3 fimbriae expression and cellulose production.
|
|---|
