Novel insights into pivotal risk factors for rectal carriage of extended-spectrum-ß-lactamase-producing enterobacterales within the general population in Lower Saxony, Germany.

AIMS: To estimate the prevalence of extended-spectrum-ß-lactamase (ESBL)-producing enterobacterales (ESBL-E) carriage in the general population of Lower Saxony, Germany, and to identify risk factors for being colonized. METHODS AND RESULTS: Participants were recruited through local press and information events. Detection of ESBL-E by culture was conducted using ESBL-selective chromagar plates containing third-generation cephalosporins. Identification of pathogens was performed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)_technology on Vitek mass spectrometry. Antibiotic susceptibility testing was conducted by microdilution (Vitek II) and an ESBL confirmation assay was carried out using a combination disk test. Of 527 randomly collected stool samples from healthy volunteers, 5.5% were tested positive for ESBL-E. Post-stratification for age and gender yielded a similar population estimate (5.9%). People traveling abroad and taking antibiotics had the greatest rectal ESBL-E carriage. CONCLUSIONS: Potential risk factors (eg, working in healthcare facilities, recent inpatient stay) did not attribute to rectal ESBL-E carriage as other factors (eg, travelling, taking antibiotics). Rectal ESBL-E carriage within the general population seems to be high. SIGNIFICANCE AND IMPACT OF THE STUDY: The known risk factors for carriage with MDRO might not be fully applicable to ESBL-E and require further examination in order to develop effective strategies for the prevention of ESBL-E dissemination within the general population.

Individual interactions of the b subunits within the stator of the Escherichia coli ATP synthase.

FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-ß, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/ß cleft, with bI close to subunit α, whereas bII is proximal to subunit ß. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-ß, α-bI-bII-ß, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase.

Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase.

The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K(+) and H(+) were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K(+) concentrations and pK values for H(+) binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K(+) into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post-Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.

Construction and characterization of a gradually inducible expression vector for Halobacterium salinarum, based on the kdp promoter.

Gradually inducible expression vectors which are governed by variations of growth conditions are powerful tools for gene expression of conditionally lethal mutants. Furthermore, controlled expression allows monitoring of overproduction of proteins at various stages in their expressing hosts. For Halobacterium salinarum, which is often used as a paradigm for halophilic archaea, such an inducible expression system is not available to date. Here we show that the kdp promoter (Pkdp), which facilitates gene expression upon K(+) limitation, can be used to establish such a system for molecular applications. Pkdp features a rather high expression rate, with an approximately 50-fold increase that can be easily varied by K(+) concentrations in the growth medium. Besides the construction of an expression vector, our work describes the characterization of expression patterns and, thus, offers a gradually inducible expression system to the scientific community.

Synthesis and conformational analysis of efrapeptins.

The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo-efrapeptins from the fungus Geotrichum candidumare inhibitors of F(1)-ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in C(α)-dialkyl amino acids (Aib, Iva, Acc) and contain one ß-alanine and several pipecolic acid residues. The C-terminus bears an unusual heterocyclic cationic cap. The efrapeptins C-G and three analogues of efrapeptin C were synthesized using α-azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F(1)-ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT-IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 3(10)-helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 3(10)-helical conformation.

Archaeal transcriptional regulation of the prokaryotic KdpFABC complex mediating K(+) uptake in H. salinarum.

The genome of the halophilic archaeon Halobacterium salinarum encodes the high-affinity ATP-dependent K(+) uptake system Kdp. Previous studies have shown that the genes coding for the KdpFABC complex are arranged in a kdpFABCQ gene cluster together with an additional gene kdpQ. In bacteria, expression of the kdpFABC genes is generally regulated by the dedicated sensor kinase/response regulator pair KdpD/KdpE, which are absent in H. salinarum. Surprisingly, H. salinarum expresses the kdp genes in a manner which is strikingly similar to Escherichia coli. In this study, we show that the halobacterial kdpFABCQ genes constitute an operon and that kdpFABCQ expression is subject to a complex regulatory mechanism involving a negative transcriptional regulator and is further modulated via a so far unknown mechanism. We describe how the regulation of kdp gene expression is facilitated in H. salinarum in contrast to its bacterial counterparts. Whereas the Kdp system fulfils the same core function as an ATP-driven K(+) uptake system in both archaea and bacteria, the different mechanisms involved in the regulation of gene expression appear to have evolved separately, possibly reflecting a different physiological role of ATP-driven K(+) uptake in halophilic archaea.

Solution structure of the KdpFABC P-type ATPase from Escherichia coli by electron microscopic single particle analysis.

The K+-translocating KdpFABC complex from Escherichia coli functions as a high affinity potassium uptake system and belongs to the superfamily of P-type ATPases, although it exhibits some unique features. It comprises four subunits, and the sites of ATP hydrolysis and substrate transport are located on two different polypeptides. No structural data are so far available for elucidating the correspondingly unique mechanism of coupling ion transport and catalysis in this P-type ATPase. By use of electron microscopy and single particle analysis of negatively stained, solubilized KdpFABC complexes, we solved the structure of the complex at a resolution of 19A, which allowed us to model the arrangement of subunits within the holoenzyme and, thus, to identify the interfaces between subunits. The model showed that the K+-translocating KdpA subunit is in close contact with the transmembrane region of the ATP-hydrolyzing subunit KdpB. The cytosolic C-terminal domain of the KdpC subunit, which is assumed to play a role in cooperative ATP binding together with KdpB, is located in close vicinity to the nucleotide binding domain of KdpB. Overall, the arrangement of subunits agrees with biochemical data and the predictions on subunit interactions.

The stoichiometry of subunit c of Escherichia coli ATP synthase is independent of its rate of synthesis.

Immunoblot quantitation of Escherichia coli ATP synthase isolated from atp wildtype and mutant cells, the latter comprising a reduced expression of the atpE gene coding for subunit c due to a point mutation within its Shine-Dalgarno sequence, suggested a variable stoichiometry of subunit c [Schemidt et al. (1995) Arch. Biochem. Biophys. 323, 423-428]. To study the c ring of the mutant strain and its stoichiometry in more detail, F O isolated from wildtype and mutant were investigated by quantitation, reconstitution, and cross-linking. Direct quantitation by staining with SYPRO Ruby revealed a reduction of subunit c in the mutant by a factor of 2 compared to F O subunits a and b. Rates of passive H (+) translocation correlated with the amount of subunit c present. Lower rates for mutant F O could be increased by addition of subunit c, whereas translocation rates remained constant by coreconstitution with nonfunctional subunit cD61G arguing against the presence of smaller c rings that are filled up with coreconstituted subunit c. Intermolecular cross-linking by oxidation of bicysteine-substituted subunit c ( cA21C/ cM65C) revealed an equal pattern of oligomer formation in wildtype and mutant also favoring a comparable subunit c stoichiometry. Cross-linking of membrane vesicles containing cysteine-substituted subunits a ( aN214C) and c ( cM65C) characterized the mutant F O preparation as a heterogeneous population, which consists of assembled F O and free ab 2 subcomplexes each present to approximately 50%. Thus, these data clearly demonstrate that the stoichiometry of the subunit c rings remains constant even after reduction of the synthesis of subunit c.

The extremely halophilic archaeon Halobacterium salinarum R1 responds to potassium limitation by expression of the K+-transporting KdpFABC P-type ATPase and by a decrease in intracellular K+.

Halobacterium species balance high external osmolality by the accumulation of almost equimolar amounts of KCl. Thus, steady K(+) supply is a vital prerequisite for life of these extreme halophiles. So far, K(+) is reported to enter the halobacterial cell only passively by use of potential-driven uniporters. However, the genome of both the extreme halophilic archaeon Halobacterium sp. NRC-1 and H. salinarum R1 comprises one single gene cluster containing the genes kdpFABC coding for homologs of the bacterial ATP-driven K(+) uptake system KdpFABC, together with an additional ORF so far annotated as cat3 in Halobacterium sp. NRC-1 and as UspA protein in H. salinarum R1 (the ORF is only referred to as cat3 in the following). Deletion of the kdpFABCcat3 genes led to a reduced ability to grow under limiting K(+) concentrations, whereas real-time RT-PCR measurements revealed cat3-dependent high expression rates of the Kdp system in case of external K(+) depletion. Synthesis of the KdpFABC complex enables H. salinarum R1 to grow under extreme potassium-limiting conditions of >20 microM K(+). These results provide the first experimental evidence of an ATP-driven K(+) uptake system in Halobacterium. Moreover, H. salinarum R1 was shown to further adapt to K(+) limitation by a significant decrease of the intracellular K(+) level, which suggests a rather complex mechanism of K(+) homeostasis, in which the adaptation of cellular K(+) concentrations and the concomitant transcriptional regulation of genes coding for a high-affinity ATP-driven K(+) uptake system ensure the essential potassium supply under limiting conditions.

Synthesis, and structural and biological studies of efrapeptin C analogues.

A series of analogues of efrapeptin C (1), with variations in the central tripeptide epitope (positions 6-8), were prepared by a combination of solid- and solution-phase peptide syntheses. The conformations of the modified compounds 2-6 were investigated by circular-dichroism (CD) spectroscopy to differentiate between 3(10)- and alpha-helical secondary structures. The inhibitory activities of the new compounds towards F(1)-ATPase from E. coli were determined. The modified congeners 3-5 were less active by one order of magnitude compared to 1 (K(i) 10 microM), and 6 was completely inactive. Our experiments demonstrate that the flexible, central tripeptide epitope, comprising positions 6-8 in 1, is crucial for molecular recognition, even slight sequence modifications being hardly tolerated.

An ATP-driven potassium pump promotes long-term survival of Halobacterium salinarum within salt crystals.

Many extremely halophilic archaea belonging to the Halobacteriales have remarkable longevity. They are even known to persist for millions of years within fluid inclusions of salt crystals. However, the key systems responsible for this remarkable ability and the underlying physiological mechanisms have not yet been deciphered. This study revealed that the ATP-dependent K(+) uptake system KdpFABC of Halobacterium salinarum is essential for survival under desiccation and salt crystal inclusion and, thus, can be identified as at least one of these systems in this organism. The presence of the kdp genes promoted survival of H. salinarum entombed in halite, compared with cells in which these genes were deleted. Expression of the kdp operon was found to be induced already under desiccating conditions without halite entombment. The morphology of cells included in halite resembled that of cells grown under potassium limitation. Therefore, a steady potassium supply, even under unfavourable energetic conditions, plays a key role in long-term survival and desiccation tolerance.

The KdpFABC complex from Escherichia coli: a chimeric K+ transporter merging ion pumps with ion channels.

The KdpFABC complex represents a multi-subunit ATP-driven potassium pump, which is only found in bacteria and archaea. Based on the properties of the ATP-hydrolyzing subunit (KdpB) the transporter has been classified as a type IA P-type ATPase. However, structural and functional properties of the remaining subunits clearly show homologies to members of the potassium channel as well as the ABC transporter family, thus rendering the KdpFABC complex to represent an inimitable chimera of ion pumps and ion channels. Accordingly, this striking juxtaposition entails special features of KdpFABC with respect to typical members of each of the transporter families, involving not only the concepts but also the structures of ion channels and ion pumps. For example, the sites of ATP hydrolysis and substrate transport are spatially separated on two different polypeptides, which, in turn, leads to a unique coupling mechanism. During catalysis, the KdpFABC complex cycles between two main conformational states, each of which comprises different structural properties together with different binding affinities for both ATP and the transport substrate. These structural configurations have recently been directly visualized in the working enzyme. Translocation of potassium is mediated by the KdpA subunit, which comprises structural as well as functional homologies to potassium channels of the MPM-type. The KdpC subunit participates in the binding of ATP, thus acting as a catalytic chaperone, which increases the ATP binding affinity of the KdpB subunit via a mechanism typical of nucleotide binding in ABC transporters.

The KdpC subunit of the Escherichia coli K+-transporting KdpB P-type ATPase acts as a catalytic chaperone.

In Bacteria and Archaea, high-affinity potassium uptake is mediated by the ATP-driven KdpFABC complex. On the basis of the biochemical properties of the ATP-hydrolyzing subunit KdpB, the transport complex is classified as type IA P-type ATPase. However, the KdpA subunit, which promotes K(+) transport, clearly resembles a potassium channel, such that the KdpFABC complex represents a chimera of ion pumps and ion channels. In the present study, we demonstrate that the blending of these two groups of transporters in KdpFABC also entails a nucleotide-binding mechanism in which the KdpC subunit acts as a catalytic chaperone. This mechanism is found neither in P-type ATPases nor in ion channels, although parallels are found in ABC transporters. In the latter, the ATP nucleotide is coordinated by the LSGGQ signature motif via double hydrogen bonds at a conserved glutamine residue, which is also present in KdpC. High-affinity nucleotide binding to the KdpFABC complex was dependent on the presence of this conserved glutamine residue in KdpC. In addition, both ATP binding to KdpC and ATP hydrolysis activity of KdpFABC were sensitive to the accessibility, presence or absence of the hydroxyl groups at the ribose moiety of the nucleotide. Furthermore, the KdpC subunit was shown to interact with the nucleotide-binding loop of KdpB in an ATP-dependent manner around the ATP-binding pocket, thereby increasing the ATP-binding affinity by the formation of a transient KdpB/KdpC/ATP ternary complex.

Osmotic Stress.

Escherichia coli and Salmonella encounter osmotic pressure variations in natural environments that include host tissues, food, soil, and water. Osmotic stress causes water to flow into or out of cells, changing their structure, physics, and chemistry in ways that perturb cell functions. E. coli and Salmonella limit osmotically induced water fluxes by accumulating and releasing electrolytes and small organic solutes, some denoted compatible solutes because they accumulate to high levels without disturbing cell functions. Osmotic upshifts inhibit membrane-based energy transduction and macromolecule synthesis while activating existing osmoregulatory systems and specifically inducing osmoregulatory genes. The osmoregulatory response depends on the availability of osmoprotectants (exogenous organic compounds that can be taken up to become compatible solutes). Without osmoprotectants, K+ accumulates with counterion glutamate, and compatible solute trehalose is synthesized. Available osmoprotectants are taken up via transporters ProP, ProU, BetT, and BetU. The resulting compatible solute accumulation attenuates the K+ glutamate response and more effectively restores cell hydration and growth. Osmotic downshifts abruptly increase turgor pressure and strain the cytoplasmic membrane. Mechanosensitive channels like MscS and MscL open to allow nonspecific solute efflux and forestall cell lysis. Research frontiers include (i) the osmoadaptive remodeling of cell structure, (ii) the mechanisms by which osmotic stress alters gene expression, (iii) the mechanisms by which transporters and channels detect and respond to osmotic pressure changes, (iv) the coordination of osmoregulatory programs and selection of available osmoprotectants, and (v) the roles played by osmoregulatory mechanisms as E. coli and Salmonella survive or thrive in their natural environments.

K+-translocating KdpFABC P-type ATPase from Escherichia coli acts as a functional and structural dimer.

The membrane-embedded K (+)-translocating KdpFABC complex from Escherichia coli belongs to the superfamily of P-type ATPases, which share common structural features as well as a well-studied catalytic mechanism. However, little is known about the oligomeric state of this class of enzymes. For many P-type ATPases, such as the Na (+)/K (+)-ATPase, Ca (2+)-ATPase, or H (+)-ATPase, an oligomeric state has been shown or is at least discussed but has not yet been characterized in detail. In the KdpFABC complex, kinetic analyses already indicated the presence of two cooperative ATP-binding sides within the functional enzyme and, thus, also point in the direction of a functional oligomer. However, the nature of this oligomeric state has not yet been fully elucidated. In the present work, a close vicinity of two KdpB subunits within the functional KdpFABC complex could be demonstrated by chemical cross-linking of native cysteine residues using copper 1,10-phenanthroline. The cysteines responsible for cross-link formation were identified by mutagenesis. Cross-linked and non-cross-linked KdpFABC complexes eluted with the same apparent molecular weight during gel filtration, which corresponded to the molecular weight of a homodimer, thereby clearly indicating that the KdpFABC complex was purified as a dimer. Isolated KdpFABC complexes were analyzed by transmission electron microscopy and exhibited an approximately 1:1 distribution of mono- and dimeric particles. Finally, reconstituted functional KdpFABC complexes were site-directedly labeled with flourescent dyes, and intermolecular single-molecule FRET analysis was carried out, from which a dissociation constant for a monomer/dimer equilibrium between 30 and 50 nM could be derived.

The K+-translocating KdpFABC complex from Escherichia coli: a P-type ATPase with unique features.

The prokaryotic KdpFABC complex from the enterobacterium Escherichia coli represents a unique type of P-type ATPase composed of four different subunits, in which a catalytically active P-type ATPase has evolutionary recruited a potassium channel module in order to facilitate ATP-driven potassium transport into the bacterial cell against steep concentration gradients. This unusual composition entails special features with respect to other P-type ATPases, for example the spatial separation of the sites of ATP hydrolysis and substrate transport on two different polypeptides within this multisubunit enzyme complex, which, in turn, leads to an interesting coupling mechanism. As all other P-type ATPases, also the KdpFABC complex cycles between the so-called E1 and E2 states during catalysis, each of which comprises different structural properties together with different binding affinities for both ATP and the transport substrate. Distinct configurations of this transport cycle have recently been visualized in the working enzyme. All typical features of P-type ATPases are attributed to the KdpB subunit, which also comprises strong structural homologies to other P-type ATPase family members. However, the translocation of the transport substrate, potassium, is mediated by the KdpA subunit, which comprises structural as well as functional homologies to MPM-type potassium channels like KcsA from Streptomyces lividans. Subunit KdpC has long been thought to exhibit an FXYD protein-like function in the regulation of KdpFABC activity. However, our latest results are in favor of the notion that KdpC might act as a catalytical chaperone, which cooperatively interacts with the nucleotide to be hydrolyzed and, thus, increases the rather untypical weak nucleotide binding affinity of the KdpB nucleotide binding domain.

Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex from Escherichia coli (Review).

P-type ATPases are ubiquitously abundant primary ion pumps, which are capable of transporting cations across the cell membrane at the expense of ATP. Since these ions comprise a large variety of vital biochemical functions, nature has developed rather sophisticated transport machineries in all kingdoms of life. Due to the importance of these enzymes, representatives of both eu- and prokaryotic as well as archaeal P-type ATPases have been studied intensively, resulting in detailed structural and functional information on their mode of action. During catalysis, P-type ATPases cycle between the so-called E1 and E2 states, each of which comprising different structural properties together with different binding affinities for both ATP and the transport substrate. Crucial for catalysis is the reversible phosphorylation of a conserved aspartate, which is the main trigger for the conformational changes within the protein. In contrast to the well-studied and closely related eukaryotic P-type ATPases, much less is known about their homologues in bacteria. Whereas in Eukarya there is predominantly only one subunit, which builds up the transport system, in bacteria there are multiple polypeptides involved in the formation of the active enzyme. Such a rather unusual prokaryotic P-type ATPase is the KdpFABC complex of the enterobacterium Escherichia coli, which serves as a highly specific K(+) transporter. A unique feature of this member of P-type ATPases is that catalytic activity and substrate transport are located on two different polypeptides. This review compares generic features of P-type ATPases with the rather unique KdpFABC complex and gives a comprehensive overview of common principles of catalysis as well as of special aspects connected to distinct enzyme functions.

The conserved dipole in transmembrane helix 5 of KdpB in the Escherichia coli KdpFABC P-type ATPase is crucial for coupling and the electrogenic K+-translocation step.

The KdpFABC complex of Escherichia coli, a high-affinity K+-uptake system, belongs to the group of P-type ATPases and is responsible for ATP-driven K+ uptake in the case of K+ limitation. Sequence alignments identified two conserved charged residues, D583 and K586, which are located at the center of transmembrane helix 5 (TM 5) of the catalytic KdpB subunit, and which are supposed to establish a dipole involved in energy coupling. Cells in which the two charges were eliminated or inverted by mutagenesis displayed a clearly slower growth rate with respect to wild-type cells under K+-limiting conditions. Purified KdpFABC complexes from several K586 mutants and a D583K:K586D double mutant showed a reduced K+-stimulated ATPase activity together with an increased resistance to orthovanadate. Upon reconstitution into liposomes, only the conservative K586R mutant was able to facilitate K+ transport, whereas the elimination of the positive charge at position 586 as well as inverting the charges at positions 583 and 586 (D583K:K586D) led to an uncoupling of ATP hydrolysis and K+ transport. Electrophysiological measurements with KdpFABC-containing proteoliposomes adsorbed to planar lipid bilayers revealed that in case of the D583K:K586D double mutant the characteristic K+-independent electrogenic step within the reaction cycle is lacking, thereby clearly arguing for an exact positioning of the dipole for coupling within the functional enzyme complex. In addition, these findings strongly suggest that the dipole residues in KdpB are not directly responsible for the characteristic electrogenic reaction step of KdpFABC, which most likely occurs within the K+-translocating KdpA subunit.

ATP binding properties of the soluble part of the KdpC subunit from the Escherichia coli K(+)-transporting KdpFABC P-type ATPase.

P-Type ATPases catalyze the transport of cations across the cell envelope via site-specific hydrolysis of ATP. Modulation of enzyme activity by additional small subunits and/or a second regulatory nucleotide binding site is still a subject of discussion. In the K(+)-transporting KdpFABC complex of Escherichia coli, KdpB resembles the catalytic P-type ATPase subunit, but ATP binding also occurs in the essential but noncatalytic subunit, KdpC. For further characterization, the soluble portion of KdpC (KdpC(sol), residues Asn39-Glu190) was synthesized separately and purified to homogeneity via affinity and size exclusion chromatography. Protein integrity was confirmed by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy, which revealed an alpha-helical content of 44% together with an 8% beta-sheet conformation consistent with the values deduced from the primary sequence. The overall protein structure was not affected by the addition of ATP to a concentration of up to 2 mM. In contrast, labeling of KdpC(sol) with the photoreactive ATP analogue 8-azido-ATP resulted in the specific incorporation of one molecule of 8-azido-ATP per peptide. No labeling could be observed upon denaturation of the protein with 0.2% sodium dodecyl sulfate, which suggests the presence of a structured nucleotide binding site. Labeling could be inhibited by preincubation with either ATP, ADP, AMP, GTP, or CTP, thus demonstrating a low specificity for nucleotides. Following 8-azido-ATP labeling and tryptic digestion of KdpC(sol), mass spectrometry showed that ATP binding occurred within the Val144-Lys161 peptide located within the C-terminal part of KdpC, thereby further demonstrating a defined nucleotide binding site. On the basis of these findings, a cooperative model in which the soluble part of KdpC activates catalysis of KdpB is suggested.