| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 104 === Proteus mirabilis, a Gram-negative, facultative anaerobic bacteria, is one of the most common cause of urinary tract infections, especially in patients with indwelling catheters or structural abnormalities of the urinary tract. The bacteria have developed diverse response systems to survive in the harsh and changing conditions. Many Hfq-binding small RNAs have been found to play roles in stress responses in E. coli and S. typhimurium. The Spot42 RNA, a 109 nucleotides long, Hfq-dependent small non-coding RNA, has been reported to block the translation of galK gene in E. coli under growth in the presence of glucose and regulate over twenty-two genes associated with uptake and catabolism of non-favored carbon sources. On the other hand, Spot42 was also found to participate in bacterial acid resistance and upregulate the general stress response regulator, RpoS. But the connection between Spot42 and the bacterial stress responses is still poorly understood. In this study, we constructed spf deletion mutant to investigate the effect of Proteus mirabilis Spot42 on adaption to stress responses and the regulatory mechanisms. We found that spf mutation decreaced acidic (pH=3) and oxidative stress resistance (30 mM H2O2), the survival in macrophage, and the ability to invade and adhere to NTUB1 and A498 cells compared with wild-type strain. In addition, we also noticed that spf has nothing to do with cell growth, swarming motility, biofilm formation and the sensitivity to antibiotics. In the aspect of gene regulation, we confirmed that CRP-cAMP binds to spf promoter region and negatively controls the expression of Spot42 in P. mirabilis by EMSA and realtime PCR respectively. Further, the realtime PCR data indicates that Spot42 would positively regulate the expression of rpoS mRNA level. We used bioinformatic tools to predict RNA-RNA interactions, and also found out the binding site of Spot42 on rpoS mRNA. The realtime PCR data also demonstrates that CRP negatively regulates the expression of rpoS mRNA level. In summury, our data suggest that Spot42 is important in P. mirabilis stress resistance by either directly or indirectly modulating the expression of RpoS.
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