An Atypical KdpD Homologue from the Cyanobacterium Anabaena sp. Strain L-31: Cloning, In Vivo Expression, and Interaction with Escherichia coli KdpD-CTD

Abstract

ABSTRACT The kdpFABC operon of Escherichia coli , coding for the high-affinity K + transport system KdpFABC, is transcriptionally regulated by the products of the adjacently located kdpDE genes. The KdpD protein is a membrane-bound sensor kinase consisting of a large N-terminal domain and a C-terminal transmitter domain interconnected by four transmembrane segments (the transmembrane segments together with the C-terminal transmitter domain of KdpD are referred to as CTD), while KdpE is a cytosolic response regulator. We have cloned and sequenced the kdp operon from a nitrogen-fixing, filamentous cyanobacterium, Anabaena sp. strain L-31 (GenBank accession. number AF213466 ). The kdpABC genes are similar in size to those of E. coli , but the kdpD gene is short (coding only for 365 amino acids), showing homology only to the N-terminal domain of E. coli KdpD. A kdpE -like gene is absent in the vicinity of this operon. Anabaena KdpD with six C-terminal histidines was overproduced in E. coli and purified by Ni 2+ -nitrilotriacetic acid affinity chromatography. With antisera raised against the purified Anabaena KdpD, the protein was detected in Anabaena sp. strain L-31 membranes. The membrane-associated or soluble form of the Anabaena KdpD(6His) could be photoaffinity labeled with the ATP analog 8-azido-ATP, indicating the presence of an ATP binding site. The coproduction of Anabaena KdpD with E. coli KdpD-CTD decreased E. coli kdpFABC expression in response to K + limitation in vivo relative to the wild-type KdpD-CTD protein. In vitro experiments revealed that the kinase activity of the E. coli KdpD-CTD was unaffected, but its phosphatase activity increased in the presence of Anabaena KdpD(6His). To our knowledge this is the first report where a heterologous N-terminal domain ( Anabaena KdpD) is shown to affect in trans KdpD-CTD ( E. coli ) activity, which is just opposite to that observed for the KdpD-N-terminal domain of E. coli .