| Summary: | 碩士 === 國立交通大學 === 生物科技學系 === 104 === Acid resistance is a general response for enterobacteria to protect themselves from the damages by gastric acid. We have previously reported that deletion of the rcsB gene which coding for the response regulator of the two-component system Rcs (regulation of capsule synthesis) decreased the survivals of Klebsiella pneumoniae CG43 upon acid treatment. Two sets of acid fitness island (AFI) homologue were discovered in the genome of K. pneumoniae CG43, and the deletion of yfdX gene as well as of the AFI-I genes hdeB, hdeD and hdeB1, or of the AFI-II genes hdeA, hdeD1, and hdeB2, also have negative effects on the acid survivals. Here, we report using electrophoresis mobility shift assay (EMSA) to demonstrate that the recombinant RcsB could bind the biotin-labeled yfdX promoter, and either the non-labeled promoter DNA of yfdX, hdeB1, hdeA and hdeD1B2 inhibited the binding phenomenon, with different affinities. This suggested a direct regulation at the transcriptional level of RcsB on the expression of yfdX, hdeB1, hdeA and hdeD1B2. Besides RcsB, we also investigated the regulatory role of histone-like nucleoid protein H-NS and the RNA chaperone Hfq, both shown significant impacts on virulence and stress responses of other bacteria, in the acid stress response. In K. pneumoniae CG43, two homologous HNS coding genes were identified and namely hns-1 and hns-2. Loss of hns-1 decreased the acid survival while hns-2 deletion showed no apparent effect. Promotor activity analysis revealed that the hns-1 deletion decreased the promoter activity of yfdX but increased the expression of hdeA and hdeD1B2 when the bacteria cultured at pH 5 statically. Furthermore, deletion of hfq or fur significantly reduced the acid survival and furryhB had increased the acid survival when compared to fur. This suggested a positive regulatory role of Hfq and Fur but a negative role of the small RNA RyhB on the acid stress response. Western blot analysis revealed that the deletion of hfq blocked the acid-inducible expression of YfdX, while deletion of fur had no apparent effect but fur and ryhB double deletion significantly enhanced the YfdX expression at weak acid environment (pH 5). Deletion of rcsB had no effect on the expression of Hfq at either pH 5, pH 6 or pH 7, suggesting the RcsB regulation on the YfdX expression is independent of Hfq. Finally, qRT-PCR analysis further confirmed that RcsB and Hfq positively influenced the expression of yfdX, hdeB and hdeA, however, Fur and RyhB exerted different regulations on the expression of yfdX and the hde genes.
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