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. 2005 Jul;187(14):4921-7.
doi: 10.1128/JB.187.14.4921-4927.2005.

An atypical KdpD homologue from the cyanobacterium Anabaena sp. strain L-31: cloning, in vivo expression, and interaction with Escherichia coli KdpD-CTD

Anand Ballal  1 , Marc Bramkamp, Hema Rajaram, Petra Zimmann, Shree Kumar Apte, Karlheinz Altendorf
Affiliations

An atypical KdpD homologue from the cyanobacterium Anabaena sp. strain L-31: cloning, in vivo expression, and interaction with Escherichia coli KdpD-CTD

Anand Ballal et al. J Bacteriol. 2005 Jul.

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.

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Figures

FIG. 1.
FIG. 1.
Location of Anabaena sp. strain L-31 kdp genes in p4AD3K and p2AD28K. The kdpB, kdpG, kdpC, and kdpD ORFs are shown by thick arrows. The p4AD3K insert contains the 3′ end of Anabaena kdpB, complete kdpG, kdpC, and 840 bp of the Anabaena kdpD gene. The p2AD28K insert shows a 974-bp overlap with the p4AD3K insert and contains the entire kdpD gene. Restriction sites: H, HindIII; C, ClaI; and E, EcoRV.
FIG. 2.
FIG. 2.
(A) Localization of Anabaena KdpD(6His) in E. coli cell fractions. Membranes and cytosolic fractions of BL21(DE3)/pLysS/pETDI were prepared as described in Materials and Methods. For comparison, the membrane pellet obtained after the ultracentrifugation step was resuspended (in lysis buffer) in a volume equal to that of the supernatant (cytosol). Lane 1, membrane vesicles (20 μl); lane 2, cytosol (20 μl). The samples were also electroblotted onto a nitrocellulose membrane and probed with the monoclonal anti-His antibody. Detection was carried out by a secondary anti-mouse immunoglobulin G coupled to alkaline phosphatase (lanes 3 and 4). Anabaena KdpD(His6) is indicated by an arrow. (B) Purification of Anabaena KdpD(6His) from E. coli membranes. Anabaena KdpD(6His) was solubilized with 1% Aminoxid WS35 from E. coli BL21(DE3)/pLysS/pETDI membranes and purified by affinity chromatography on an Ni-NTA matrix. The samples were resolved on an SDS-polyacrylamide (11%) gel and stained with Coomassie brilliant blue. Lane 1, solubilized membranes (10 μg); lane 2, unbound fraction (10 μg); lane 3, 10 mM imidazole wash; lane 4, 20 mM imidazole wash; lane 5, 40 mM imidazole wash; lane 6, 200 mM imidazole elution (5 μg). The position of Anabaena KdpD(His6) is indicated by an arrow.
FIG. 3.
FIG. 3.
(A) Localization of KdpD in Anabaena sp. strain L-31. The antiserum raised against purified KdpD(6His) was used to detect the KdpD protein from Anabaena sp. strain L-31. Anabaena sp. strain L-31 membrane (M) and cytosolic (C) fractions from cells grown in BG11K5 medium were prepared as described (1). About 200 μg of protein was loaded in lanes 2 to 5, while 150 ng of purified Anabaena KdpD(6His) was loaded in lane 1. Lanes 1 to 3 were probed with the Anabaena KdpD(6His) antiserum, while lanes 4 and 5 were probed with the control preimmune serum. (B) Effect of K+ limitation on kdpD expression in Anabaena sp. strain L-31. Anabaena sp. strain L-31 cells were grown in BG11K5 medium and then transferred to BG11 medium containing 0 mM or 5 mM K+. At the times indicated, the cells were harvested, membranes were isolated, 200 μg total membrane protein was electrophoresed, and Anabaena KdpD (shown by the arrow) was detected as before.
FIG. 4.
FIG. 4.
Detection of ATP binding by Anabaena KdpD(6His) by photoaffinity labeling with 8-azido-[α-32P]ATP. E. coli BL21(DE3)/pLysS everted membranes containing Anabaena KdpD(6His) (lanes 1 and 2) and purified cytosolic Anabaena KdpD(6His) (lanes 3 and 4) were labeled with 8-azido-[α-32P]ATP in the presence or absence of 2 mM ATP as indicated. After electrophoresis on 11% SDSpolyacrylamide gels, the radiolabeled proteins were detected by a PhosphorImager system (Molecular Dynamics). Anabaena KdpD(6His) is shown by an arrow.
FIG. 5.
FIG. 5.
(A) In vivo interaction of Anabaena KdpD(6His) with E. coli KdpD-CTD. E. coli TKW22692 transformed with pBScD1 or the empty vector was grown in K115 medium and then subjected to K+ limitation as indicated. Thirty minutes after K+ downshock, total protein was extracted from the cells and KdpC (arrow) was detected with the E. coli KdpC antiserum. Lanes 1 and 3, TKW22692/pBluescript; lanes 2 and 4, TKW22692/pBScD1. (B) In vitro interaction of Anabaena KdpD(6His) with E. coli KdpD-CTD. The kinase activity (black bars) and the phosphatase activity (grey bars) of E. coli KdpD-CTD in the presence and in the absence of Anabaena KdpD(6His) were determined. The data are presented as percentages of the initial rates relative to E. coli KdpD-CTD. The kinase reaction was initiated by addition of [γ-32P]ATP to everted membrane vesicles containing E. coli KdpD-CTD with or without Anabaena KdpD(6His). For phosphatase measurements, purified KdpE∼32P was added to the vesicles in the presence of 1 mM ATP-γ-S. For both assays, aliquots were removed at definite time points and added to SDS sample buffer to stop the reaction. The proteins were separated by SDS-PAGE, and the radiolabeled proteins were quantified with a PhosphorImager (Molecular Dynamics). For KdpD-CTD (100% values), the phosphorylation activity was 2.5 pmol KdpD∼P min−1 mg protein−1 and the phosphatase activity was 0.01 pmol of Pi released min−1 mg protein−1. (C) Coelution of Anabaena KdpD (6His) and E. coli KdpD-CTD. The E. coli TKR2000/pBADD1/pBD3/Δ12-395 cell lysate was used for coelution experiments as described in Materials and Methods. The elution fraction was resolved on SDS-PAGE and probed separately with E. coli KdpD (lane 1) and Anabaena KdpD(6His) (lane 2) antisera.
FIG. 6.
FIG. 6.
Organization of the kdp genes in different bacteria. The arrowhead indicates the direction of transcription. SK, sensor kinase; RR, response regulator.
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