K+ and ionic strength directly influence the autophosphorylation activity of the putative turgor sensor KdpD of Escherichia coli

The membrane-bound histidine kinase KdpD is a putative turgor sensor that r egulates, together with the response regulator KdpE, the expression of the Kdp-FABC operon coding for the high affinity K+-uptake system KdpFABC of Es cherichia coil. To elucidate the nature of the primary stimulus for KdpD, w e developed an in vitro assay based on right-side-out membrane vesicles. Co nditions were varied inside and outside of the vesicles, and KdpD autophosp horylation activity was tested. It was shown that an increase of the ionic strength inside the vesicles was accompanied by an increase of the autophos phorylation activity of KdpD with ATP. However, K+ at concentrations higher than 1 mM inhibited KdpD autophosphorylation activity. This KC specific ef fect was not observed with KdpD-Arg-511 --> Gln, a KdpD derivative, which c auses K+-independent kdpFABC expression. When the osmolality outside the ve sicles was increased, autophosphorylation activity of KdpD was stimulated, whereby salts were more effective than sugars. Treatment of the vesicles wi th amphipathic compounds did not affect KdpD autophosphorylation activity. Based on these results it is proposed that changes of intracellular paramet ers elicited by K+ limitation or osmotic upshock directly influence KdpD au tophosphorylation activity, whereby K+ has an inhibitory and ionic strength a stimulatory effect.