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°C heat shock (37 to 47°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).In recent years, coagulase-negative staphylococci (CNS) have emerged as major pathogens that are mainly associated with indwelling or implanted foreign body infections (22,29,30,33). Their impact on public health is enormous (24, 31). It remains enigmatic why these normally innocent skin saprophytes become virulent in association with indwelling foreign bodies (17). CNS infections seem to be the result of a complex interaction between bacterium-related factors, host-related factors, and foreign body-related-factors. Genes involved in cell accumulation (13,33,34) and in initial adhesion (12) are presumed virulence factors in initial foreign body colonization and biofilm formation. A state of bacterial dormancy and a suppressed housekeeping metabolism are hypothesized to contribute to the persistent nature of foreign body-related CNS infections (5,25,26).For study of the pathogenesis of infectious diseases, researchers have access to a rapidly growing amount of genetic information. However, the exact links between the information encoded in the genome and the final virulence and housekeeping behavior of bacteria remain unclear. Methods to unravel these links are mutagenesis and the study of gene expression. Mutagenesis is a valuable phenotypical assay (1). However, mutations in important genes may lead to only minor phenotypical changes or to lethal mutants. In these cases it is not possible to draw firm conclusions on the role...

The role of σ B in persistence of Staphylococcus epidermidis foreign body infection

“…Gene quantification was done on a 2 ml sample (purified cDNA or gDNA), 12.5 ml 26 Taqman PCR master mix (PE Applied Biosystems), 0.9 mM of each primer and 0.2 mM of the probe in a final volume of 25 ml. The thermal cycling conditions were as previously published (Vandecasteele et al, 2001). To allow gene quantification, a standard dilution of a known quantity of the plasmid was included in each run.…”

“…The remaining 100 ml sterile distilled water purified with the RNeasy Mini kit (Qiagen) according to the manufacturers' instructions. Reverse transcriptase reaction was performed as described earlier (Vandecasteele et al, 2001).…”

“…Genomic DNA (gDNA) and RNA extraction from planktonic and sessile bacteria in vitro and in vivo were performed as described earlier (Vandecasteele et al, 2001). Briefly, 1 ml bacterial suspension (planktonic) was pelleted for 5 min at 12 000g at Knobloch et al (2004) rsbU : : erm derivate from S. epidermidis 8400 derived by phage transduction 8400rsbUVW Knobloch et al (2004) rsbUVW : : erm derivate from S. epidermidis 8400 derived by phage transduction 8400rsbUVWsigB Knobloch et al (2004) rsbUVWsigB : : erm derivate from S. epidermidis 8400 derived by phage transduction 1457 Mack et al (1992) Isolate from infected central venous catheter 1457rsbU Knobloch et al (2004) rsbU : : erm derivate from S. epidermidis 1457 derived by allelic gene replacement 1457rsbUVW Knobloch et al (2004) rsbUVW : : erm derivate from S. epidermidis 1457 derived by allelic gene replacement 1457rsbUVWsigB Knobloch et al (2004) rsbUVWsigB : : erm derivate from S. epidermidis 1457 derived by allelic gene replacement V. Pintens and others 4 uC.…”

Size-fraction partitioning of community gene transcription and nitrogen metabolism in a marine oxygen minimum zone

“…N gene transcript abundances were normalized based on gene length and expressed as a proportion of the abundance of transcripts matching the gene encoding RNA polymerase subunit B (rpoB), as has been done in studies of diverse bacteria (for example, Schumann et al, 2010;Ceja-Navarro et al, 2014;Dalsgaard et al, 2014;Eldholm et al, 2014). Although rpoB expression can vary (Vandecasteele et al, 2001), rpoB appears to be one of the more stably expressed housekeeping genes (Sue et al, 2004;Sihto et al, 2014). Furthermore, it has been shown that rpoB can be a proxy of bulk mRNA transcription level for bacteria (Milohanic et al, 2003;Sue et al, 2004)-rpoB-normalized values therefore reflect transcription of a target gene relative to a housekeeping gene under a given condition/sample.…”

Article

“…En effet, la variation du C T pour ces gènes entre la condition témoin et les conditions de phase stationnaire est la plus faible. Il faut souligner qu'une variation physiologique est toujours présente entre les phases exponentielle et stationnaire [20]. Pour la suite des expériences, le gène codant la D-lactate déshydrogénase (ldhD) a été retenu.…”