A chimeric Anabaena/ Escherichia coli KdpD protein (Anacoli KdpD) functionally interacts with E. coli KdpE and activates kdp expression in E. coli.
The kdpFABC operon, coding for a high-affinity K(+)-translocating P-type ATPase, is expressed in Escherichia coli as a backup system during K(+) starvation or an increase in medium osmolality. Expression of the operon is regulated by the membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE. From a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31, a kdpDgene was cloned (GenBank accession no. AF213466) which codes for a KdpD protein (365 amino acids) that lacks both the transmembrane segments and C-terminal transmitter domain and thus is shorter than E. coli KdpD. A chimeric kdpD gene was constructed and expressed in E. coli coding for a protein (Anacoli KdpD), in which the first 365 amino acids of E. coli KdpD were replaced by those from Anabaena KdpD. In everted membrane vesicles, this chimeric Anacoli KdpD protein exhibited activities, such as autophosphorylation, transphosphorylation and ATP-dependent dephosphorylation of E. coli KdpE, which closely resemble those of the E. coli wild-type KdpD. Cells of E. coli synthesizing Anacoli KdpD expressed kdpFABC in response to K(+) limitation and osmotic upshock. The data demonstrate that Anabaena KdpD can interact with the E. coliKdpD C-terminal domain resulting in a protein that is functional in vitro as well as in vivo.
Cell Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India.
Anand
A
Ballal
Ralf
R
Heermann
Kirsten
K
Jung
Michael
M
Gassel
Kumar
K
Apte
Karlheinz
K
Altendorf
D017033:Anabaena / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D009154:Mutation; D009876:Operon; D009994:Osmolar Concentration; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C075615:kdpE protein, E coli; C489580:KdpD protein, E coli; D011494:Protein Kinases; C075616:kdpD protein, Bacteria; D010744:Phosphoric Monoester Hydrolases; D011188:Potassium