Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under differentconditions

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and ( ii) to study the expression of five housekeeping genes which are involved i n nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reducta se [DBFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and p rotein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time q uantitative PCR. The detection limit of our method was 10 copies of RNA. Th e average intersample variability was 16%. A 10-fold increase in the expres sion of the hsp-60 gene was induced by exposure to a 10 degreesC heat shock (37 to 47 degreesC) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during t he mid-exponeatial-growth phase. Maximal gene expression was 110- to 300-fo ld higher than gene expression during stationary phase. This , indicates th at housekeeping metabolism is a very dynamic process that is extremely capa ble of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bact erial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).