Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

Abstract

Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.Several methods for the quantification of transcripts in different organisms are available. These techniques differ largely in the amounts of time and expense required for data acquisition. In general, these methods can be grouped into those that target single transcripts and those that target whole transcriptomes. Northern hybridization has the disadvantage of a rather low level of sensitivity, limited quantification potential, and a large time commitment but is comparatively inexpensive. On the other hand, global and quantitative assays like transcriptome microarray analysis call for major investments in equipment and disposables, aside from limitations in view of quantitative data evaluation/expression analysis. For the quantification of single transcripts, reverse transcription (RT)-PCR is currently the method of choice. RT-PCR can be performed as a quantitative or semiquantitative assay depending on whether absolute transcript numbers are required or whether relative expression levels are investigated.A major drawback of RT-PCR is the necessity of purified high-quality RNA that is virtually free o...

Compartment and signal-specific codependence in the transcriptional control of Salmonella periplasmic copper homeostasis

“…A mutant lacking CueP has an increased cellular copper load (15) and delayed copper-induced expression compared with the other Cue components (Fig. S1A), resembling the expression kinetics of the Escherichia coli cus locus (20). In addition, and unlike the other genes of the Cue regulon, its induction by copper is observed only in neutral conditions (pH 7.0), and not in acidic conditions (pH 5.5) (Fig.…”

Survival of Escherichia coli Cells on Solid Copper Surfaces Is Increased by Glutathione

“…This may indicate that Cu I is more toxic than Cu II ; however, no uptake system was known for (28). On the other hand, upregulation of copA expression from background expression to about 350 copies per cell took only 2 min after addition of Cu II to the cells (32), and this regulatory event is performed by the MerR-type regulator CueR, which is usually constantly bound to its operator on the DNA (62). Thus, a Cu I import pathway should exist.…”

“…To address the question of whether DNA damage is involved in killing of E. coli cells on copper surfaces, the dps gene was deleted in wild-type E. coli strain W3310 and various mutants carrying deletions in genes (copA, cueO, cusCFBA; gshA and gshB for synthesis of glutathione [GSH] via gamma-glutamyl-cysteine) that are involved in copper tolerance (32,37,38,44,57). RT-PCR controls were done in all experiments to demonstrate the absence or presence of the gene transcripts in the respective mutants (data not shown).…”

Escherichia coli stress response systems and their reaction to terahertz radiation

“…В среднем количество молекул CusR при стрессе от избытка ионов меди меняется мало и состав-ляет 50-100 молекул на клетку. Таким образом, система CusRS участвует в обеспечении устойчивости к ионам в анаэробных условиях, где происходит увеличение их токсичности, но только в дополнение к эффлюксу меди при помощи CopA, экспрессия которого в анаэробных условиях также увеличивается (Outten et al, 2001;Thieme et al, 2008).…”