| Summary: | 博士 === 國立交通大學 === 生物科技學系 === 100 === E. coli has developed pleiotropic regulator for gene expression, such as cyclic AMP (cAMP)-cAMP receptor protein (Crp) complex. Glucose is generally regarded as a carbon source to modulate cyclic AMP (cAMP) regulated genes. In the present study, we found that the stability of ompA mRNA was reduced in Escherichia coli when glucose was present in the LB medium. This effect was associated with the low level of cAMP induced by the glucose. The evidence was further confirmed in an E. coli mutant lacking of cAMP. Northern blot and Western blot analyses revealed that the host factor I (Hfq) (both mRNA and protein) levels were down-regulated in the presence of cAMP, in which we showed that complex formation between cAMP receptor protein (Crp) and cAMP plays a role in binding to a specific promoter region of hfq (between -112 and -87). The finding was substantiated in vivo by an hfq-deficent mutant transformed with an exogenous hfq gene containing the promoter. These results demonstrated that the repression of hfq expression by cAMP leaded to a cascade regulation of cAMP and Hfq on the stability of ompA mRNA. Additional experiment showed that cAMP also increased the stability of fur mRNA. Taken together, these results suggested that the repression of Hfq by cAMP may contribute to others post-transcriptional regulations in E. coli.
The regulation of fumarase (fumA, B, C) genes was dependent on growth rate in transcription and post-transcription. Our results showed that cAMP and Crp were an activator for fumA and fumC genes expressions under various oxygen using fumA–lacZ and fumC–lacZ fusion strains. Further experiments also showed glucose catabolite repression on fumA and fumC genes, but no effect on fumB gene. Subsequently, we examined the effect of fumA expression in transcription, post-transcription and translation level by growth rate regulation. In continuous culture, the amount of the fumA mRNA was reduced with increasing growth rate. The fumA mRNA was more stable when growth rate was increased. The contrary results between amount and stability of fumA mRNA caused the same amount of FumA protein with different growth rates. This finding may provide a reference value for the future industrial application for the preparation of recombinant proteins.
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