Stimulus perception by the KdpD/KdpE two-component system of Escherichia coli is still controversial with respect to the nature of the stimulus that is perceived by the sensor kinase KdpD. Limiting potassium concentrations in the medium or high osmolality leads to KdpD/KdpE signal transduction, resulting in kdpFABC expression. It has been hypothesized that changes in turgor are sensed by KdpD through alterations in the physical state of the cytoplasmic membrane. However, in this study the quantitative determination of expression levels of the kdpFABC operon revealed that the system responds very effectively to K ؉ -limiting conditions in the medium but barely and to various degrees to salt and sugar stress. Since the current view of stimulus perception calls for mainly intracellular parameters, which might be sensed by KdpD, we set out to test the cytoplasmic concentrations of ATP, K ؉ , Na ؉ , glutamate, proline, glycine, trehalose, putrescine, and spermidine under K ؉ -limiting conditions. As a first result, the determination of the cytoplasmic volume, which is a prerequisite for such measurements, revealed that a transient shrinkage of the cytoplasmic volume, which is indicative of a reduction in turgor, occurred only under osmotic upshift but not under K ؉ -limiting conditions. Furthermore, the intracellular ATP concentration significantly increased under osmotic upshift, whereas only a slight increase occurred after a potassium downshift. Finally, the cytoplasmic K ؉ concentration rose severalfold only after an osmotic upshock. For the first time, these data indicate that stimulus perception by KdpD correlates neither with changes in the cytoplasmic volume nor with changes in the intracellular ATP or K ؉ concentration or those of the other solutes tested. In conclusion, we propose that a reduction in turgor cannot be the stimulus for KdpD.

We developed a real-time RT-PCR method for the quantification of dissimilatory (bi)sulphite reductase (DSR) mRNA in Desulfobacterium autotrophicum cells. The amount of DSR mRNA was determined relative to the amount of 16S rRNA at different growth conditions during transition from exponential to stationary phase: sulphate respiration with lactate, thiosulphate respiration with lactate, sulphate respiration with H2 and pyruvate fermentation. The dsr gene was expressed constitutively, although DSR mRNA content per-cell varied under different growth conditions. The maximum DSR mRNA per-cell content was 2.0 to 4.1-fold higher during sulphate or thiosulphate respiration than during pyruvate fermentation. After transfer of a pyruvate-fermenting culture into sulphate-rich medium, upregulation of the DSR mRNA content was observed. Irrespective of the mode of metabolism the per-cell DSR mRNA content changed significantly during growth (up to 310-fold from the early to the late exponential phase during respiration with thiosulphate). The maximum DSR mRNA per-cell contents correlated with cell-specific sulphate reduction rates for all experiments. Environmental applications for the quantification of DSR mRNA are discussed.

The histidine kinase/response regulator system EnvZ/ OmpR of Escherichia coli regulates transcription of the genes ompF and ompC, encoding two porins of the outer membrane. Although the total amount of OmpF and OmpC remains constant, the relative levels of the two proteins fluctuate in a reciprocal manner depending on medium osmolality. The membrane-anchored sensor EnvZ somehow monitors changes in environmental osmolality. To characterize the nature of the stimulus perceived by EnvZ, this protein was overproduced, purified, and reconstituted into proteoliposomes. Autokinase activity of purified and reconstituted EnvZ was stimulated by an increase of the K ؉ concentration. Rb