Miticka, H., Rezuchova, B., Homerova, D., Roberts, M. and Kormanec, J. (2004) Identification of nucleotides critical for activity of the sigma(E)-dependent rpoEp3 promoter in Salmonella enterica serovar Typhimurium. FEMS Microbiology Letters, 238(1), pp. 227-233. (doi: 10.1111/j.1574-6968.2004.tb09760.x)
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Publisher's URL: http://dx.doi.org/10.1111/j.1574-6968.2004.tb09760.x
Abstract
We previously described a two-plasmid system for the identification of promoters recognized by <i>Salmonella enteric</i> serovar Typhimurium (<i>S</i> . Typhimurium) σ<sup>E</sup>. The <i>S</i> . Typhimurium σ<sup>E</sup>-dependent <i>rpoEp3</i> promoter was active in the <i>E. coli</i> two-plasmid system only after arabinose-induced expression of <i>S</i> . Typhimurium <i>rpoE</i> . In the present study, we have exploited this two-plasmid system for the identification of nucleotides critical for activity of the <i>rpoEp3</i> promoter. A library of randomly mutated DNA fragments containing the <i>rpoEp3</i> promoter was cloned upstream of a <i>lacZα</i> reporter gene and screened for activity in the presence of <i>S</i> . Typhimurium σ<sup>E</sup>. The clones exhibiting reduced LacZ activity were sequenced to identify the mutations. The activity of the mutated <i>rpoEp3</i> promoters were studied further using a luciferase-based promoter-probe plasmid. All of the important nucleotides of the <i>rpoEp3</i> promoter (in capital) were located in the −35 (ggAActt) and −10 (TctaA) regions. The critical nucleotides were also the most conserved in known σ<sup>E</sup>-dependent promoters. The study also revealed the importance of the 16-bp spacing between −10 and −35 region, as reducing the spacing to 15-bp greatly reduced activity of the promoter. This method should be generally applicable for the identification of important nucleotides in the cognate promoters of other σ factors.
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Roberts, Professor Mark |
| Authors: | Miticka, H., Rezuchova, B., Homerova, D., Roberts, M., and Kormanec, J. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine |
| Journal Name: | FEMS Microbiology Letters |
| Publisher: | Wiley |
| ISSN: | 0378-1097 |
| ISSN (Online): | 1574-6968 |
| Published Online: | 22 June 2004 |
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