Development of a green fluorescent protein based reporter gene system in Staphylococcus epidermidis
Abstract number: 1134_03_165
Franke G.C., Dobinsky S., Horstkotte M.A., Knobloch J.K.-M., Mack D., Rohde H.
Based on its biofilm forming capacity Staphylococcus epidermidis is the most frequent cause of foreign-body related infections. Several specific factors mediating primary attachment and accumulation have been characterized. In order to analyze the concerted action of these factors methods for in situ expression analysis are demanded. The aim of the present study was to establish a Green Fluorescent Protein (GFP) based reporter gene system in Staphylococcus epidermidis.
Therefore we cloned gfpmut3.1 (Clontech) into pASI under the control of a xylose-inducible promotor and introduced the resulting construct into S. epidermidis 1457. However, under xylose induction no significant increase of fluorescence intensity compared to the un-induced control was detected. This finding may be attributed to inefficient translation initiation at the natural Shine-Delgarno (SD) sequence. Indeed, by coupling different SD-sequences from well-characterized S. epidermidis genes in front of the gfp start codon the crucial impact of this element for gfp translation initiation was demonstrated: whereas a strong fluorescence signal was obtained in presence of the hld SD sequence, almost no signal was detected with the sarA SD sequence. The influence of different SD sequences on transcription and translation of gfpmut3.1 was also analysed in real-time transcription and semi-quantitative western blotting experiments. In all constructs investigated an almost identical gfp transcription level was found regardless of the SD sequence present. In contrast, western blot analysis using an anti-GFP antibody revealed huge differences in GFP amounts that corresponded to the respective quantitative fluorescence signal. The calculated half-life in S. epidermidis 1457 is 6.9 h. Importantly, expression of gfpmut3.1 did not interfere with the biofilm-positive phenotype of this strain.
In conclusion, using an appropriate SD sequence for optimal translation initiation gfpmut3.1 offers an attractive system for monitoring gene expression in S. epidermidis.
|Session name:||XXIst ISTH Congress|
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