Understanding the role of non-protein-coding ribonucleic acids in prokaryotes

Jimenez Diaz, Manuel
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University of Delaware
Previous acid stress studies in Clostridium acetobutylicum and Streptococcus mutans were applied in Escherichia coli to further understand the mechanisms of acid tolerance and the effect of non-protein-coding RNA (ncRNA) in prokaryotes. The strain overexpressing the 4.5S ncRNA, as well as the intergenic upstream 16S ncRNA sequences with a low copy plasmid, pACYC184, in E. coli Top10 F cells. These cells were grown to staitionary phase under the following acid stresses: lactic acid, acetic acid, and butyric acid. The cells were grown in a minimal medium containing succinate as the main carbon source and in LB medium. Optical density was measured at various time points to assess the growth of the strains. In most cases the data indicates that the aforementioned RNA sequences did not provide acid tolerance to E. coli. It was evident that the Ffh protein when overexpressed in high copy numbers was lethal to the E. coli strains. A similar phenomenon was observed when the intergenic 16S and the ffs sequences with natural promoters and terminators were transformed into E. coli. The Ffh protein was also intended to be cloned in the lower copy number plasmid pACYC184 but similar patterns of cell toxicity were also observed. The quantitative reverse transcription PCR assays was not sensitive enough to detect the short ncRNAs thus it is uncertain whether the constructs containing the lac promoter overexpressed the ncRNAs.