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

Abstract

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 in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein 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 quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) 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 the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable 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 bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).

FIG. 1

Gene expression levels after a heat shock of 10°C given for 10 min. Shaded bars, heat-shocked culture (47°C); open bars, controls (37°C). Each bar represents the number of copies of mRNA per CFU; that value is also given below the bar graph.

FIG. 2

Effect of glucose challenge in a stationary-phase culture. Shaded bars, gene expression in a stationary-phase culture after a glucose challenge (final concentration, 5%) for 10 min. Open bars, expression levels of the same culture without glucose challenge. Each bar represents the number of copies of mRNA per CFU; that value is also given below the bar graph.

FIG. 3

Expression of housekeeping genes during the exponential- and stationary-growth phases in vitro. Gene expression is rescaled as a percentage with respect to the measurement at 45 min. The left y axis, dots, and solid line represent the rescaled expression of each gene. Each open square, referring to the right y axis, represents the ln of CFU at a given time after inoculation. Dashed line, growth curve.