Domain swapping reveals that the N-terminal domain of the sensor kinase KdpD in Escherichia coliis important for signaling
© Heermann et al; licensee BioMed Central Ltd. 2009
Received: 13 March 2009
Accepted: 09 July 2009
Published: 09 July 2009
The KdpD/KdpE two-component system of Escherichia coli regulates expression of the kdpFABC operon encoding the high affinity K+ transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp). It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of E. coli.
Full response to salt stress was only achieved with a chimera that contains UspC, probably due to unaffected scaffolding of the KdpD/KdpE signaling cascade by soluble UspC. Unexpectedly, chimeras containing either UspF or UspG not only prevented kdpFABC expression under salt stress but also under K+ limiting conditions, although these hybrid proteins exhibited kinase and phosphotransferase activities in vitro. These are the first KdpD derivatives that do not respond to K+ limitation due to alterations in the N-terminal domain. Analysis of the KdpD-Usp tertiary structure revealed that this domain has a net positively charged surface, while UspF and UspG are characterized by net negative surface charges.
The Usp domain within KdpD not only functions as a binding surface for the scaffold UspC, but it is also important for KdpD signaling. We propose that KdpD sensing/signaling involves alterations of electrostatic interactions between the large N- and C-terminal cytoplasmic domains.
K+ plays an important role in turgor maintenance in bacteria . KdpFABC is a high affinity K+ uptake system that serves as an emergency system to scavenge K+ when other transporters cannot sustain the cellular requirement for K+. The corresponding kdpFABC operon is under control of the two-component system KdpD/KdpE, which induces kdpFABC expression under K+ limiting conditions or under osmotic stress imposed by a salt [2, 3]. Upon stimulus perception, KdpD undergoes autophosphorylation and subsequently, the phosphoryl group is transferred to the cytoplasmic response regulator KdpE . Phosphorylated KdpE exhibits increased affinity for a 23-base pair sequence upstream of the canonical -35 and -10 regions of the kdpFABC promoter and triggers kdpFABC expression . The enzymatic activities of purified KdpD and KdpE were determined in vitro . All data known thus far indicate that KdpD does not sense a single specific parameter, but integrates the information of intracellular parameters imposed by K+ limitation or salt stress. The current working model proposes that KdpD perceives alterations in the intracellular K+ concentration, the ionic strength, and the ATP concentration as stimuli .
KdpD consists of a characteristic C-terminal transmitter domain, which is fused via a small linker region to the large N-terminal input domain. Several regions of the input domain have been identified as important for stimulus perception and integration. The four transmembrane domains (TM1-TM4) anchor the sensor kinase in the cytoplasmic membrane and separate the two large cytoplasmic domains from each other [7, 8]. The transmembrane helices TM2 and TM3 function as a type of clip and are responsible for the correct positioning of the large cytoplasmic domains relative to each other . We have previously shown a direct interaction between these KdpD cytoplasmic domains . The α-helix of TM4 extends from the membrane into the cytoplasm and encompasses a cluster of positively charged amino acids (R503-R511) that are mainly involved in stimulus perception, and has therefore been proposed as a K+ binding site by Altendorf and coworkers [10, 11]. This hypothesis is in accord with the finding that amino acid replacements resulting in K+-independent kdpFABC expression are located within TM4 and the adjacent region [11–13]. It was previously shown that the cluster of positively charged amino acids is important for modulation of the kinase and phosphatase activity, because individual replacements of these amino acids resulted in KdpD derivatives with either enhanced kinase and reduced phosphatase activity, or enhanced phosphatase and reduced kinase activity . Furthermore, a KdpD derivative lacking the cytoplasmic N-terminal region and the first two transmembrane domains of KdpD were able to respond to K+ limitation, which supports the assumption that the K+ binding site is located within this region .
The Usp superfamily encompasses an ancient and conserved group of proteins that are found in bacteria, archaea, fungi, flies, and plants (see  for review). Usp-containing organisms are usually equipped with several copies of usp genes. The usp genes encode either small Usp proteins (one Usp domain), larger versions with two Usp domains in tandem, or Usp domains integrated in multi-domain proteins . E. coli contains six Usp proteins that can be divided into two subfamilies on the basis of sequence similarities . Three of these proteins belong to the UspA subfamily (UspA, UspC, and UspD), and two to the UspFG subfamily (UspF and UspG). The UspE protein is a tandem-like protein consisting of two Usp domains. The UspE domain1 is more related to the UspA sub-family, whereas the domain2 is closer related to the UspFG sub-family. The intracellular copy number of UspA, UspC, UspD, and UspE increases upon stress conditions such as starvation, moderate heat stress, oxidative stress, and osmotic stress . UspG is induced under osmotic stress and has recently been shown to undergo autophosphorylation and autoadenylation . However, the exact functions of these small proteins are unclear.
The degree of similarity of the Usp domain within KdpD (Fig. 1) varies among all known KdpD sequences. To elucidate the role of the Usp domain in KdpD for signaling, we used a "domain swapping" approach, wherein the E. coli KdpD-Usp domain was replaced with homologous domains or the six E. coli Usp proteins. These KdpD chimeras were characterized in vivo as well as in vitro.
"Domain swapping" of the Usp domain within KdpD
The N-terminal region of the cytoplasmic input domain containing the KdpD domain (pfam02702) is highly conserved , whereas the C-terminal region containing the Usp-domain (cd01987) (I253-P365) is less conserved (Fig. 1). The KdpD-Usp domain of other bacteria, for example Agrobacterium tumefaciens (KdpD/R249-D372), Streptomyces coelicolor (KdpD/R233-I354), Salmonella enterica serotype Typhimurium (KdpD/I253-P365), and Pseudomonas aeruginosa (KdpD/R248-R358) are characterized by different degrees of identity and similarity. The highest degree of sequence identity has the KdpD-Usp domain of S. enterica serotype Typhimurium compared to the corresponding E. coli domain (86% identity, 89% similarity). The other KdpD-Usp domains are less conserved (A. tumefaciens: 30% identity, 45% similarity; P. aeruginosa: 28% identity, 43% similarity; S. coelicolor: 25% identity, 42% similarity). The KdpD-Usp domain belongs to the UspA subfamily. Despite the lack of amino acid sequence identity, proteins of this (sub)family (UspA, UspC and UspD) are predicted to have a homologous tertiary structure which consists of four to five central β-sheets surrounded by four a-helices [19, 22]. To examine the specifics of the KdpD-Usp domain and its importance in KdpD signaling, we replaced amino acids L221-V358 of E. coli KdpD with the homologous KdpD-Usp domains of A. tumefaciens (L218-I371), S. enterica serotype Typhimurium (L221-V358), S. coelicolor (L202-V355), and P. aeruginosa (L218-Q361) as described in Methods, and designated the chimeras Agrocoli-KdpD, Salmocoli-KdpD, Streptocoli-KdpD, and Pseudocoli-KdpD (Fig. 2) . Furthermore, we exchanged the KdpD-Usp domain of E. coli with the six soluble Usp protein sequences of E. coli, yielding the chimeras KdpD-UspA, KdpD-UspC, KdpD-UspD, KdpD-UspE, KdpD-UspF, and KdpD-UspG (Fig. 2). Since the predicted tertiary structure of these domains is rather similar, we expected no major effects on the overall tertiary structure of KdpD.
The response of KdpD-Usp chimeras to salt stress
Usp proteins form homodimers and oligomers, thus it is conceivable that UspC interacts with KdpD-UspC and thereby facilitates scaffolding. Although the Salmonella KdpD-Usp domain has the highest degree of similarity to the E. coli KdpD-Usp-domain, scaffolding by UspC seemed to be abolished. The induction level supported by this chimera was comparable to wild-type KdpD in a ΔuspC mutant . Scaffolding might also be prevented in Agrocoli-KdpD. These data underline the importance of the KdpD-Usp domain for scaffolding the KdpD/KdpE signaling cascade under salt stress. The negative results obtained for all other KdpD chimeras might be explained by steric hindrance of the protein dynamics due to binding of other Usp proteins, major structural changes, or altered enzymatic activities.
The response of KdpD-Usp chimeras towards K+limitation
The enzymatic activities of the KdpD-Usp chimeras in vitro
Kinase-phosphotransferase to phosphatase ratios of the KdpD chimeras.
Kinase-phosphotransferase to phosphatase ratio
However production of phosphorylated KdpE should be possible in combination with the likewise decreased kinase-phosphotransferase activities. In summary, replacing the KdpD-Usp domain influences the enzymatic activities of KdpD, explaining altered kdpFABC expression patterns in some KdpD chimeras. Importantly, KdpD-UspF and KdpD-UspG are rare examples of KdpD derivatives that lost sensing capabilities in vivo, but exhibited kinase, phosphotransferase, and phosphatase activity in vitro.
UspF and UspG differ in surface charge from the E. coliKdpD-Usp domain
The N-terminal input domain of the KdpD sensor kinase contains a domain that belongs to the universal stress protein family [18, 19]. This domain has been characterized as an interaction site for the soluble UspC protein. Moreover, binding of UspC scaffolds the KdpD/KdpE signaling cascade under salt stress . Since the mechanistic significance of the KdpD-Usp domain for signaling was still unclear, we employed a "domain swapping" approach to replace this domain with four homologous domains or the six soluble Usp proteins. As shown earlier,  and corroborated here (Fig. 7), the tertiary structure of all inserted domains is very similar, although the degree of amino acid identity is rather low. In general, we have hypothesized three different mechanisms of how Usp domain swapping could affect KdpD/KdpE signaling: (i) UspC scaffolding under salt stress is increased/abolished due to affinity alterations of the inserted domains towards UspC, (ii) the enzymatic activities of the KdpD chimeras are altered, and (iii) the protein dynamics of the sensor are altered. Interestingly, we generated chimeras covering all these possibilities.
Scaffolding under salt stress was only observed when UspC was inserted into KdpD. In contrast, all other domains prevented scaffolding by UspC. It should be noted that the KdpD-Usp domain sequences differ among bacteria, and also the set of available soluble Usp proteins within these bacteria is variable. A. tumefaciens has three usp homologues (atu0496, atu0904, and atu1730), S. coelicolor has eleven usp homologues (sco0172, sco0178, sco0167, sco0180, sco0181, sco0198, sco0200, sco0937, sco7156, sco7247, and sco7299), P. aeruginosa has seven (pa1753, pa1789, pa3017, pa3309, pa4328, pa4352, and pa5027), and S. enterica serotype Typhimurium has six homologues similar to E. coli (uspA, uspC, uspD, uspE, uspF, and uspG). With the exception of S. enterica, none of these organisms has a uspC homologue, suggesting that KdpD/KdpE scaffolding either does not exist in these bacteria, or it is mediated by other Usp proteins. This leads to the conclusion that UspC is the specific scaffolding protein for KdpD/KdpE in E. coli.
Although all chimeras exhibited enzymatic activity, the ratio between kinase-phosphotransferase to phosphatase activity was shifted in some chimeras. In Pseudocoli-KdpD, the ratio was shifted towards the phosphatase activity, producing a significantly lower expression level than wild-type KdpD. Likewise, KdpD-UspC and Streptocoli-Usp had increased kinase-phosphotransferase to phosphatase ratios and were characterized by significantly higher induction values compared to wild-type KdpD.
The Usp domain within KdpD is important for proper KdpD/KdpE signaling. Alterations within this domain can completely prevent the response towards K+ limitation as well as salt stress. The KdpD-Usp domain surface contains numerous positively charged amino acids. Electrostatic repulsion and attraction between the N-terminal and C-terminal domain are supposed to be important for KdpD (de)activation. Therefore, the KdpD-Usp domain not only functions as a binding surface for the native scaffold UspC, but also seems to be crucial for internal KdpD signaling, shifting the protein from an "OFF" into an "ON" state.
[γ32-P]ATP and NAP-5 gel filtration columns were purchased from Amersham GE Healthcare. Goat anti-(rabbit IgG)-alkaline phosphatase was purchased from Biomol. All other reagents were reagent grade and obtained from commercial sources.
Bacterial strains and plasmids
E. coli strain JM 109 [recA1 endA1 gyrA96 thi hsdR17 supE44λrelA1 Δ(lac-proAB)/F'traD36 proA+B+lacI q lacZΔM15]  was used as carrier for the plasmids described. E. coli strain TKR2000 [ΔkdpFABCDE trkA405 trkD1 atp706]  containing different variants of plasmid pPV5-3 encoding the different KdpD-Usp derivatives (see below) was used for expression of the kdp-usp derivatives from the tac promoter. E. coli strain HAK006 [ΔkdpABCD Δ(lac-pro) ara thi]  carrying a kdpFABC promoter/operator-lacZ fusion was used to probe signal transduction in vivo. E. coli LMG194 [F- ΔlacX74 galE galK thi rpsL ΔphoA (PvuII) Δara714leu::Tn10]  was used for expression of the kdp-usp derivatives from the araBAD promoter.
To replace the Usp domain in E. coli KdpD with the E. coli Usp protein sequences, the corresponding usp genes were PCR amplified using genomic DNA of E. coli MG1655  as a template. The uspA, uspD, uspE, uspF, and uspG genes were amplified with primers complementary at least 21 bp to the 5' or the 3' ends of the corresponding genes with overhangs for a 5' NsiI site and a 3' SpeI site, respectively. uspC was amplified similarly, but with a 5' terminal SacI site. The amplified DNA fragments were cut with NsiI and SpeI, or SacI and SpeI, respectively, and ligated into equally treated vector pPV5-3, resulting in plasmids pPV5-3/UspA, pPV5-3/UspC, pPV5-3/UspD, pPV5-3/UspE, pPV5-3/UspF, and pPV5-3/UspG. To replace the Usp domain of E. coli KdpD with the Usp domain of the KdpD proteins of Agrobacterium tumefaciens, Salmonella enterica serotype Typhimurium, Streptomyces coelicolor, and Pseudomonas aeruginosa, respectively, the corresponding gene fragments were amplified by PCR using primers which were complementary to the corresponding gene locus with genomic DNA from the abovementioned bacteria as template. The corresponding regions of the kdpD gene were amplified with primers complementary at least 21 bp to the 5' or the 3' ends of the corresponding kdpD gene locus with overhangs for a 5' SacI site and a 3' SpeI site, respectively. The amplified DNA fragments were cut with SacI and SpeI, respectively, and ligated into equally treated vector pPV5-3, resulting in plasmids pPV5-3/Agrocoli-KdpD, pPV5-3/Salmocoli-KdpD, pPV5-3/Streptocoli-KdpD, and pPV5-3/Pseudocoli-KdpD. All hybrid genes were verified by sequencing each PCR-generated DNA segment through the ligation junctions in double-stranded plasmid DNA. The kdpD derivatives kdpD-uspA, kdpD-uspD, kdpD-uspE, kdpD-uspG, kdpD-uspF, agrocoli-kdpD, salmocoli-kdpD, and pseudocoli-kdpD were cloned into plasmid pBAD-18  using XmaI and HindIII; kdpD-uspC and pseudocoli-kdpD were cloned into plasmid pBD (kdpD in pBAD-18)  using XhoI and SpeI resulting in plasmids pBD/UspA, pBD/UspC, pBD/UspD, pBD/UspE, pBD/UspF, pBD/UspG, pBD/Agrocoli-KdpD, pBD/Salmocoli-KdpD, pBD/Streptocoli-KdpD, and pBD/Pseudocoli-KdpD, respectively. The correct insertion of the respective kdpD derivatives was checked by restriction analysis of the corresponding plasmids.
Cell fractionation and preparation of inverted membrane vesicles
E. coli strain TKR2000 transformed with plasmids pPV5-3 or its derivatives carrying different kdpD-usp derivatives was grown aerobically at 37°C in KML complex medium (1% tryptone, 0.5% yeast extract, and 1% KCl) supplemented with ampicillin (100 μg/ml). Cells were harvested at an absorbance at 600 nm of ~1.0, washed with buffer (50 mM Tris/HCl pH 7.5, 10 mM MgCl2) and disrupted by passage through a Cell disruptor (Constant Cell Disruption Systems, Northants, UK) at 1.35 kbar and 4°C in disruption buffer [50 mM Tris/HCl pH 7.5, 10% (v/v) glycerol, 10 mM MgCl2, 1 mM dithiotreitol, 0.5 mM phenylmethylsulfonylfluoride, and 0.03 mg/ml (w/v) DNAse]. After removal of intact cells and cell debris by centrifugation (9.000 × g, 10 min), membrane vesicles were collected by centrifugation at 160.000 × g for 60 min. Membrane vesicles were washed with low ionic strength buffer (10 mM Tris/HCl, pH 7.5, 3 mM EDTA), centrifuged again and resuspended in 50 mM Tris/HCl, pH 7.5 containing 10% (v/v) glycerol. Vesicles were frozen in liquid nitrogen and stored at -80°C until use.
Phosphorylation and Dephosphorylation Assays
Inverted membrane vesicles (2 mg protein/ml) containing about 10% KdpD were incubated at room temperature in phosphorylation buffer [50 mM Tris/HCl, pH 7.5, 10% glycerol (v/v), 0.5 M NaCl, 10 mM MgCl2 and 2 mM DTT]. Phosphorylation was initiated by addition of 20 μM [γ-32P]ATP (2.38 Ci/mmol). At different times, aliquots were removed and the reaction was stopped by mixing with SDS-sample buffer . After incubation for 4.5 min, an equimolar amount of purified KdpE was added to the KdpD-containing samples and the incubation was continued. Further aliquots were removed at different times and mixed with SDS-sample buffer . For dephosphorylation assays, 10His-KdpE~32P was obtained as described [16, 37]. Dephosphorylation was initiated by addition of inverted membrane vesicles (1 mg/ml) containing KdpD or KdpD chimeras, 20 mM MgCl2 in presence and absence of 20 μM ATP-γ-S. At different times, aliquots were removed, and the reaction was stopped by addition of SDS-sample buffer. All samples were immediately subjected to SDS-polyacrylamide gel electrophoresis
PAGE, an [γ-32P]ATP standard was loaded on the gels. Gels were dried, and protein phosphorylation was detected by exposure of the gels to a Storage Phosphor Screen. Phosphorylated proteins were quantified by image analysis using the Phosphorimager Storm (GE Healthcare).
Determination of kdpFABC expression in vivo
In vivo signal transduction was probed using E. coli strain HAK006 transformed with the plasmids as previously described. Cells were grown in minimal media containing different concentrations of K+  or in minimal medium containing 5 mM K+ with or without 0.4 M sodium chloride, and harvested in the mid-exponential growth phase by centrifugation. β-galactosidase activity was determined as described  and is given in Miller Units.
Proteins were assayed using a modified Lowry method , using bovine serum albumin as a standard. Immunodetection of KdpD was performed with polyclonal antibodies against KdpD as previously described .
Amino acid sequences were compared using the VectorNTI alignment tool AlignX (Invitrogen, Karlsruhe, Germany). Structure predictions were performed by ESyPred3D modeling  on the expasy server http://www.expasy.ch.
We thank Ivana Ristovski, Simone Holpert, and Sonja Kroll for technical assistance. This work was financially supported by the Deutsche Forschungsgemeinschaft (Exc114/1) and the BMBF (SysMO, project KOSMOBAC).
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