Signal‐sensing mechanisms of the putative osmosensor KdpD in Escherichia coli

Abstract

The KdpD protein is a membrane-located sensory kinase (or signal transducer) critically involved in the regulation of the kdpABC operon that is responsible for a high-affinity transport system in Escherichia coli. In this study, a set of KdpD mutants, each resulting in a single amino acid substitution around the membrane-spanning regions of KdpD, was isolated. Amino acid substitutions in these KdpD mutants were located non-randomly, particularly within the C-terminal half of the membrane-spanning regions. This set of KdpD mutants exhibited altered transmembrane-signalling properties in response to external K+ and other stimuli. In particular, these mutants were found to be insensitive, if not completely, to the K+ signal. However, they were able to respond to other stimuli such as high-salt stress, as in the wild type. Therefore, in contrast to the wild type, the cells carrying these mutations exhibited high levels of the steady-state expression of kdp, regardless of external K+, provided that high concentrations of ionic solutes were supplemented to the cultures. More interestingly, the set of KdpD mutants could also respond to high concentrations of external non-ionic solutes such as sucrose and D-arabinose, thereby increasing substantially the steady-state expression of kdp in response to the medium osmolarity. Furthermore, it was found that certain chemicals, ethanol, chlorpromazine and procaine, could function as effectors for the KdpD mutants at relatively low concentrations in the media. Based on these findings, we have examined the primary signal(s) that regulates the function of KdpD. We propose here that KdpD can be considered to be an environmental sensor that exhibits sensing mechanisms in response to both the level of K+ and the physico-chemical state of the cytoplasmic membrane.

Characterization of transcriptional regulation of the kdp operon of Salmonella typhimurium

“…For reasons discussed above, this observation is inconsistent with the turgor control model of kdp (4). Our data are better explained by the model proposed for E. coli by Sugiura et al (16) in which the kdp operon is regulated by two antagonistic signals: high osmolality, which acts positively to induce the operon, and K ϩ , which functions negatively to repress it. How- a Strains were grown overnight in LB medium (3) plus 25 g of ampicillin per ml and then subcultured at a 1:100 dilution into K0 medium (5) containing 10 mM glucose, 25 g of ampicillin per ml, and the indicated concentration of K ϩ (added as KCl).…”

Tuning of the porin expression under anaerobic growth conditions by His‐to‐Asp cross‐phosphorelay through both the EnvZ‐osmosensor and ArcB‐anaerosensor in Escherichia coli

“…7B). KdpE is the potassium-sensing KdpDdependent response regulator which belongs to the OmpR family of response regulators (like ArcA) (Sugiura et al 1994), whereas UvrY is an orphan response regulator whose cognate sensor kinase has not yet been identi®ed (Mizuno 1997). In any case, ArcB was capable of phosphorylating OmpR, as was the case for ArcA, while neither KdpE nor UvrY was phosphorylated at all under these in vitro conditions (Fig.…”

“…The OmpR protein was puri®ed, as previously described ). The KdpE protein was also puri®ed, as previously described (Sugiura et al 1994). The UvrY protein was puri®ed in this study (details will be described elsewhere).…”

Truncation of Amino Acids 12–128 Causes Deregulation of the Phosphatase Activity of the Sensor Kinase KdpD of Escherichia coli

“…Cells were grown in TY medium (1% tryptone, 0.5% yeast extract) (11) or minimal medium (26) supplemented with NaCl and KCl as indicated. Cells were grown to midlogarithmic growth phase and harvested by centrifugation.…”

“…This model has been challenged by more recent findings (9,10) demonstrating that there is a difference in expression of kdp-FABC when the osmolarity of the medium is increased by a sugar or a salt. Analysis of mutant forms of KdpD that result in constitutive expression of kdpFABC independent of the K ϩ concentration of the medium but retain the ability to respond to changes in medium osmolarity led to the suggestion that KdpD senses two stimuli, decrease in turgor and K ϩ concentration (11).…”