Competition is the basis of the transport mechanism of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae

Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.


Introduction
Na + /H + exchangers are ubiquitous in nature as they ensure that organisms are capable of regulating their intracellular Na + concentration, pH and volume [1]. The best studied Na + /H + exchangers belong to the cation proton antiporter (CPA) superfamily, which includes both electrogenic and electroneutral members [2]. To this date, the crystal structures of four CPA Na + /H + exchangers have been solved, beginning with the prototype of the family, NhaA from E. coli (EcNhaA) [3] and continuing in recent years with the structures of NapA from T. PLOS  electrophysiological investigation of an NhaB Na + /H + exchanger. We found that the competition-based kinetic model [7] that is valid for CPA Na + /H + exchangers also describes the non-CPA KpNhaB transporter. Additionally, we found that, despite their high homology to EcN-haA, KpNhaA1 and KpNhaA2 have altered activity profiles. Overall, the profiles of the three investigated Na + /H + exchangers from K. pneumoniae are complementary, indicating that they contribute to the survival of the bacterium under different conditions of salinity and pH.

Results
Overexpression of KpNhaB, KpNhaA1 and KpNhaA2 in E. coli The genes encoding for KpNhaB, KpNhaA1 and KpNhaA2, containing a C-terminal His-tag and cloned in either the pET21d or the pTrcHis2 TOPO expression vectors, were used to transform E. coli strains BL21(DE3) or KNabc. Expression of the target proteins in E. coli membranes was verified by performing SDS-PAGE followed by Western blot, using anti-His IgG as primary antibody (Fig 2A). Clear bands could be observed for all three proteins on the Western blot. As NhaA was shown to be a dimer [33], we assigned the higher mass bands observed for KpNhaA1 and KpNhaA2 as the dimer forms of the protein. The existence of dimer bands even in the presence of the harsh detergent SDS has previously been reported for CPA Na + /H + exchangers [34]. No dimer band was observed for KpNhaB. In all three cases, the monomeric forms of the proteins migrated on the gel to masses lower than their calculated molecular weights (KpNhaB-57 kDa; KpNhaA1-43 kDa; KpNhaA2-41 kDa), which is typical for membrane proteins [35]. Following solubilization of the E. coli membranes and purification using immobilized metal affinity chromatography, proteins were subjected again to SDS-PAGE and visualized using Coomassie Blue staining of the gel (Fig 2B). Essentially only the same bands seen in Fig 2A were observed, indicating that proteins were purified to a high degree.

Survival assays of E. coli KNabc expressing the K. pneumoniae transporters
In order to check the functionality of the expressed transporters in E. coli 7and also to verify their role as Na + export systems, we assayed the capability of the K. pneumoniae exchangers of rescuing the survival of the Na + /H + exchanger-deficient E. coli KNabc strain under conditions of high salinity. Under the conditions tested (Table 1), we observed that E. coli KNabc could only grow in medium where Na + was replaced by K + (LBK), while high concentrations of either Na + or Li + prevented the growth of the vector-transformed strain. When either the control NhaA from H. pylori (HpNhaA) or one of the K. pneumoniae Na + /H + exchangers were expressed, survival of the cells was restored under high amounts of Li + and Na + at pH 7. However, at pH 8.3 only KpNhaA1 and HpNhaA could provide survival in presence of a high Li + concentration, while none of the exchangers could provide survival under a high Na + concentration.

Acridine orange dequenching assays
In order to ascertain the functionality of the overexpressed proteins in E. coli membranes, acridine orange dequenching assays were performed by using everted E. coli membrane vesicles that were acidified by addition of Tris-D-lactate. All three transporters were active (Fig 3A-3C), as shown by the dequenching of the acridine orange fluorescence observed when Na + was added to the outside of the vesicles. As EcNhaB, which has high homology to KpNhaB ( Fig  1B), was reported to show a pH-independent activity [36,37], we recorded the pH dependence of the dequenching for KpNhaB and found essentially no pH dependence in the pH range tested (Fig 3D).

SSM-based electrophysiological measurements
Proteoliposomes containing the reconstituted K. pneumoniae Na + /H + exchangers were investigated via SSM-based electrophysiology. Thus, the proteoliposomes were subjected to Na + concentration jumps under conditions where the pH was the same inside and outside the proteoliposomes (symmetrical pH). In the case of all exchangers, transient currents of negative polarity were recorded (Fig 4A-4C). The negative polarity of these currents indicates the transport of positive charge out of the proteoliposomes (or net negative charge inside), and is in line with the expected electrogenicity of the Na + /H + exchange. Concentration jumps of Li + (data not shown) were also performed, yielding transient currents of the same polarity and shape as the ones recorded for Na + concentration jumps. pH dependence of the transport activity As previously shown [38], the amplitude of the recorded transient currents is a good measure of the steady-state transport activity of the exchanger, in the case where the recorded transient currents show no pre steady-state component. This was true for nearly all of the recordings done on the K. pneumoniae Na + /H + exchangers, with the exception of Na + concentration jumps on KpNhaB performed at pH 9 and pH 9.5, where the transient currents showed a pre steady-state component ( Fig 4A). As this component appears only at high pH, it is likely associated with the Na + binding or translocation event in the transporter's reaction cycle, as was previously seen in the EcNhaA G338S mutant [38] or in the NhaP exchangers from M. jannaschii [22] and P. abyssi [39]. For these recordings, the transport currents were numerically reconstructed [40] and the stationary component of the reconstructed currents was determined (S1 Fig).
The pH-dependent activity profile of KpNhaB determined by SSM-based electrophysiology is shown in Fig 4D. A high pH dependence of the transport activity was observed (Fig 4D), unlike the profile determined by acridine orange dequenching measurements ( Fig 3D). The exchanger was down-regulated in the acidic, with the activity increasing with pH up to a maximum that is reached at pH 8-8.5, after which activity decreased rapidly, reaching almost zero at pH 9.5 ( Fig 4D). By comparison, KpNhaA1 and KpNhaA2 (Fig 4E and 4F) showed the same down-regulation in the acidic, but the activity remained high even at higher pH. Na + and Li + dependence of the transport activity At pH 8.5, increasing the value of the Na + concentration jumps yielded a hyperbolic increase of the transporter-dependent currents, which was observed in all the investigated transporters ( Fig 5A-5F). The determined values for the Na + apparent affinities (K m values) at pH 8.5 are similar for all three transporters, in the range of 7-14 mM Na + , and are also close to the value of 11 mM previously determined for EcNhaA at the same pH (Table 2).
When pH was lowered, however, the peak currents recorded for KpNhaB at pH 7 showed a sigmoidal dependence (Fig 5D), indicating cooperativity in the substrate binding. In this case, the data could be fitted by the use of a Hill equation, yielding a Hill coefficient n = 2. The dependence of the currents on the Na + concentration remained hyperbolical for KpNhaA1 ( Fig 5E) and KpNhaA2 (Fig 5F). In all investigated exchangers, lowering pH had the effect of decreasing the affinity for Na + (Fig 5 and Table 2), indicating the presence of competition between Na + and H + .
We also performed Li + concentration jumps at pH 8.5, given that most Na + /H + exchangers are also capable of transporting Li + . KpNhaB displayed a twofold lower affinity for Li + than for Na + (Fig 5G). For KpNhaA1 ( Fig 5H) and KpNhaA2 (Fig 5I), the affinities for Li + were higher than the affinity for Na + , which is in line with the behavior previously seen in EcNhaA ( Table 2). Competition-based transport mechanism in NhaB from K. pneumoniae PLOS ONE | https://doi.org/10.1371/journal.pone.0182293 July 27, 2017 Determination of kinetic parameters Fig 6 shows the kinetic model describing the transport mechanism of Na + /H + exchange, which is in accordance to the alternating access model proposed by Jardetzky [42]. In brief, the transporter can bind either of the substrates H + and Na + to the same binding site in either its inward-or outward-open conformations. This causes a conformational transition that exposes the bound substrate ion to the opposite side of the membrane, where it is released. One of the advantages of this simple model is that, in the absence of cooperative binding, the activity of each transporter can be described by a set of only 3 kinetic parameters (Fig 6), corresponding to the affinity of the transporter for H + (pK), the affinity for Na + (K Na D ) and the ratio between the rates of H + and Na + transport (k 2 /k 1 ). We previously used the competition-based kinetic model in order to characterize several CPA exchangers, including transporters from the NhaA and NhaP families [7]. The pHdependent activity profile of KpNhaB ( Fig 4D) as well as the existence of competition ( Fig 5D) indicated that the model might also be appropriate for the characterization of this non-CPA exchanger. In order to account for the cooperativity observed for KpNhaB at pH 7 we included two additional kinetic parameters, which are the Hill coefficients n for the Na + binding reaction and m for the H + binding reaction, respectively (Fig 6).
A kinetic analysis of each of the investigated exchangers using the competition-based model was performed. Table 2 shows the obtained kinetic parameters for the investigated exchangers, and the fits of the model to the experimental data are shown in Fig 7. The m and n Hill coefficients were fixed as 1 for the NhaA exchangers, where no cooperativity was apparent.
As it can be seen (Table 2), the three transporters differ in their pK values, with KpNhaB being the most acid-shifted and KpNhaA1 the most alkaline-shifted. The determined K Na D values are similar, in the range of 1.6-3.6 mM Na + .
Regarding k 2 /k 1 , a major aspect of obtaining a precise value is measuring the activity of the exchanger in a range where down-regulation at both acidic and alkaline pH can be observed. While this was the case for KpNhaB and KpNhaA1, where we obtained values for k 2 /k 1 of 23  Table 2. Exp. = experimentally determined data points, fit = modeled curve following the fit of the kinetic model to the experimental data. https://doi.org/10.1371/journal.pone.0182293.g007 Competition-based transport mechanism in NhaB from K. pneumoniae PLOS ONE | https://doi.org/10.1371/journal.pone.0182293 July 27, 2017 and 13, respectively, in the case of KpNhaA2 this value was less defined, most likely due to the weak down-regulation in the alkaline. Thus, although our fit gave a value of~2000 to k 2 /k 1 for KpNhaA2, we assigned it a k 2 /k 1 value of 100, as values above 100 for this parameter yielded essentially identical pK and K Na D values.

Discussion
KpNhaB, KpNhaA1 and KpNhaA2 are high turnover, electrogenic Na + export systems EcNhaA and EcNhaB have previously been shown to play a vital role in the survival of E. coli under conditions of high salinity [19]. Indeed, E. coli strains deficient in EcNhaA and EcNhaB such as the EP432 strain [43] or the KNabc strain that we employed in this work [44] do not survive under conditions of high Na + or Li + concentration. As our results (Table 1) show, expressing either of the electrogenic K. pneumoniae Na + /H + exchangers in E. coli KNabc restored the salt resistance of this strain at neutral pH. Conversely, at pH 8/0.6 M Na + , none of the expressed Na + /H + exchangers, including the control HpNhaA, were able to restore resistance. This is in line with the higher susceptibility of E. coli KNabc to salt stress compared to other Na + /H + exchanger-deficient strains such as EP432 [45,46], and most likely results from the fact that, besides EcNhaA and EcNhaB, E. coli KNabc is missing also the unspecific ChaA transporter.
Thus, considering our results as well as the role of the NhaA and NhaB exchangers in other organisms [19,45], we can conclude that the KpNhaB, KpNhaA1 and KpNhaA2 exchangers contribute to salt resistance in K. pneumoniae. This conclusion is additionally substantiated by the pH-dependent activity profiles of the exchangers (Fig 4D-4F), which are in line with the expected profile for an exchanger that has as a main role Na + export [for an in-depth discussion, see Refs. 7 and 22].
An analysis of the transient currents recorded using SSM-based electrophysiology following Na + (or Li + ) concentration jumps clearly shows that all investigated exchangers are electrogenic, carrying net positive charge out of the proteoliposomes (or net negative charge inside) when Na + is applied from the outside. This conclusion is substantiated by the negative polarity of the recorded transients, the decay time constants that are Na + concentration dependent ( Fig  5A-5C) and by the increase of the decay time constants of the transient currents with the increase of the lipid to protein ratio (LPR, S2 Fig), as previously shown for EcNhaA by Zuber et al. [41].
In addition, based on the maximum amplitude of the transient currents recorded for all K. pneumoniae Na + /H + exchangers (Table 2), we can conclude that all of them are high turnover systems, as the recorded amplitudes are comparable with those previously recorded for EcN-haA (Table 2), which has a turnover of more than 1000 ions/second [47].
KpNhaB can be described by the same competition-based mechanism as the NhaA exchangers Our previous research activity has led to a simple competition-based transport mechanism (Fig 6) that we have shown to be valid for Na + /H + exchangers of the CPA superfamily [7]. So far, the transporters that were shown to follow this mechanism were the NhaA exchangers from E. coli [38], H. pylori [22] and S. typhimurium [48], the NhaP exchangers from M. jannaschii [22] and P. abyssi [39] and the NapA exchanger from T. thermophilus [49]. The mechanism explains the highly pH-dependent activity of Na + /H + exchangers as a consequence of its transport mechanism, not necessitating additional pH-sensitive regions (so-called "pH sensors") other than the substrate binding site.
As we expected, the characterization of KpNhaA1 and KpNhaA2 was fully in line with the competition-based mechanism. Thus, competition is readily apparent in both exchangers as shown by the reduction of the Na + affinity when pH decreases (Fig 5). Interestingly, we found that KpNhaB can equally be described by the competition mechanism. Thus, competition can be observed in KpNhaB by the same decrease in Na + affinity with the lowering of pH as observed for the NhaA exchangers (Fig 5D). An intrinsic property of this mechanism (Fig 6) is that transport is down-regulated at extreme pH: in the acidic, down-regulation is explained by the H + out-competing the Na + ions, while in the alkaline, it is explained by H + depletion that reduces the overall availability of this substrate and hence, turnover. Indeed, the pH profile of KpNhaB (Fig 4D) clearly shows both alkaline and acidic down-regulation while for KpNhaA1 ( Fig 4E) and KpNhaA2 (Fig 4F) only acidic down-regulation is observed, most probably because alkaline down-regulation is shifted out of the experimentally accessible pH-range.
Our finding for KpNhaB is especially relevant taking into account that previous reports regarding the pH dependence of NhaB exchangers gave mixed conclusions [8,36] and that NhaB belongs to a different transporter family than NhaA. In addition, NhaB has a distinct structure as far as conclusions from sequence analysis are predictive in this respect. We will treat these considerations in turn.
The first investigated NhaB exchanger was NhaB from E. coli, which was identified more than 25 years ago [50]. One of the earliest reported properties of EcNhaB was that its transport activity seemed independent of pH in the range 6.5 to 8.5, as seen in acridine orange dequenching assays [36,37]. On the other hand, the activity of the NhaB exchanger from V. alginolyticus (VaNhaB) was shown to be highly pH dependent using the same assay [51]. The acridine orange dequenching assays performed on KpNhaB (Fig 3D) also showed no pH dependence of the transport activity in the same pH range. However, our electrophysiological results clearly show that the activity of KpNhaB is highly pH dependent (Fig 4D). Therefore, the apparent "pH insensitivity" observed in the acridine orange dequenching assay is most probably due to the limited dynamic range of the dequenching assays, as previously reported for H. pylori NhaA [52]. Here it is interesting to note that KpNhaB, just as H. pylori NhaA [52], has an acid-shifted activity profile with respect to EcNhaA. Using only the acridine orange dequenching assay, KpNhaB might, therefore, wrongly be qualified as "pH insensitive", as HpNhaA was previously thought to be [52]. Thus, it is important that, when such considerations are made, a more sensitive assay than acridine orange dequenching is used.
According to the TC system [18], NhaB exchangers are classified into the Ion Transporter (IT) superfamily, while NhaA and NhaP exchangers belong to the Cation Proton Antiporter (CPA) superfamily. Furthermore, a topological study of VaNhaB showed that NhaB exchangers possess 9 transmembrane helices (TMs), unlike the 12 or 13 TMs present in NhaA or NhaP exchangers [27]. Also, NhaB exchangers, while electrogenic as NhaA exchangers, do not seem to possess obvious conserved motifs as were shown to be required for substrate transport in NhaA such as the two adjacent Asp residues present in TM V of NhaA (Fig 1A). However, a definite answer to the question how NhaA and NhaB transporters compare in terms of functional groups requires a high resolution structure which is presently not available.
Notwithstanding the structural dissimilarities between NhaA and NhaB type exchangers, we could establish that KpNhaB follows the same general mechanism as shown for the CPA exchangers. In particular, competition between H + and Na + obviously is a key element of the Na + /H + exchange mechanism in these transporters and may be a general concept for all Na + / H + exchangers.
The major difference between the behavior of KpNhaB and the NhaA exchangers was the observed cooperativity of Na + binding in KpNhaB (Fig 5D), which was not found for the NhaA exchangers. This is a clear indication that more than one Na + ion is involved in the transport mechanism and indeed this agrees with the different stoichiometries of Na + /H + exchange: while NhaA transports 1 Na + ion for 2 H + [20], NhaB exchanges 2 Na + ions for 3 H + [21].

Kinetic analysis of the investigated exchangers
The pH and Na + dependence of all three investigated Na + /H + exchangers from Klebsiella pneumoniae could be described by a simple kinetic model requiring only 3 (for NhaA type transporters) and 5 (for the NhaB type transporter) kinetic parameters (Fig 6). The fits of the model to the experimental data obtained for the three K. pneumoniae Na + /H + exchangers are shown in Fig 7. The good quality of the fits and the low number of kinetic parameters represent strong support for the competition-based mechanism in all three transporters. The obtained pK and K Na D values for the K. pneumoniae Na + /H + exchangers ( Table 2) are in line with previously determined kinetic parameters for Na + /H + antiporters of other organisms. Thus, the Na + affinity is virtually the same as that determined for the NhaA exchangers of E. coli, H. pylori and S. typhimurium [52]. The lowest pK value was determined for KpNhaB (8.0) and is the same as for H. pylori NhaA [52], while the highest was determined for KpNhaA1 (9.2) and is the same as for S. typhimurium NhaA [52].

The role of K. pneumoniae Na + /H + exchangers in bacterial survival
The pH-dependent activity profiles of the three investigated exchangers obtained under symmetrical pH conditions are compared in Fig 8A, and can provide, along with the determined kinetic parameters, the basis of understanding the role of these exchangers in K. pneumoniae. However, it has to be kept in mind that a full comparison would require knowledge of the expression rate of the three exchangers, which we did not have access to. pneumoniae Na + /H + exchangers calculated using the determined kinetic parameters in Table 2 under physiological stress conditions and normalized to the transport rate determined at pH out = 5.5. Parameters used are listed in the panel. Dashed lines in B denote pH 7 and 8.3, where the survival assays presented in Table 1 were performed. In A, out and in denote the exterior and interior of the proteoliposomes, respectively, while in B, out and in denote the periplasmic and cytoplasmic space, respectively. https://doi.org/10.1371/journal.pone.0182293.g008 Competition-based transport mechanism in NhaB from K. pneumoniae In E. coli, EcNhaB is the main Na + export system at acidic and neutral pH [19], while EcN-haA is highly up-regulated under conditions of salt stress [53] and is more efficient at removing Na + at alkaline pH compared to EcNhaB [19]. Based on the kinetic parameters determined for the K. pneumoniae exchangers we modeled ( Fig 8B) the behavior of these transporters under physiological stress conditions that K. pneumoniae can encounter, such as the ones that we used in our survival assays presented in Table 1. Unlike conditions used in the SSM experimental setup (Fig 8A), the physiological activity of the exchangers occurs under a negativeinside membrane potential and potentially at a different periplasmic pH and Na + concentration compared to the cytoplasmic pH and Na + concentration (Fig 8B).
It can be observed in Fig 8B that all three transporters are capable of exporting Na + against a high concentration gradient at acidic pH. At high values of the salinity, KpNhaB is hard pressed to export Na + once the periplasmic pH reaches neutral; under those conditions the two NhaA exchangers can perform this role, with KpNhaA1 being capable of functioning better at higher periplasmic pH. Overall, the three exchangers have complementary activity profiles (Fig 8B).
One more factor to consider is the behavior of the transporters when high amounts of Li + are present. We have shown that KpNhaB has a comparatively lower affinity for Li + (Table 2), whereas both KpNhaA1 and KpNhaA2 have much higher Li + affinities (Table 2). Thus, it stands to reason that when the cell is faced with Li + stress, the two NhaA exchangers are more adapted to ensure survival.
The modeled activity profiles can also be excellently correlated with the experimental results obtained in our survival assays (Table 1). Thus, at pH 7, in the presence of 600 mM Na + , all three exchangers are capable of ensuring survival of E. col KNabc, with the KpNhaA1-expressing cells surviving better. When pH is increased to 8.3, none of the expressed antiporters can restore survival of the strain in presence of 600 mM Na + , which fits very well the modeled activity in Fig 8B. An exception at pH 8.3 is represented by the capability of KpNhaA1 cells of surviving in 100 mM Li + , which can be explained by both the fact that this protein has the highest affinity for Li + out of the exchangers investigated in this work and has also the most alkalineshifted pK.
A better understanding of the way in which K. pneumoniae adapts to various stress conditions is essential, considering the danger posed by K. pneumoniae infections and the fact that many strains are resistant to currently used antibiotics. In all, the existence of three Na + /H + exchangers with complementary transport profiles in K. pneumoniae indicates that this bacterium is well prepared to survive salt stress at various pH values. Designing specific, high affinity inhibitors against these Na + /H + exchange systems is a possible avenue to consider in the task of finding a treatment against carbanepem-resistant K. pneumoniae infections.

Genetic constructs and bacterial strains
The genes encoding the K. pneumoniae exchangers KpNhaA1 (Strain MGH 78578, Uniprot accession number: A6T4F6), KpNhaA2 (Strain MGH 78578, Uniprot accession number: A6TJ58) and KpNhaB (Strain 342, Uniprot accession number: B5XQ77) were synthesized by Genscript (Piscataway, NJ, USA) in the pET-21d(+) vector (Merck Millipore, Billerica, MA, USA). The genes were optimized for E. coli expression and contained an additional C-terminal 6-His tag. These constructs were used for protein production in the BL21(DE3) E. coli strain. For expression in the Na + /H + exchanger deficient strain KNabc [44], the genes were cloned into the pTrcHis2 TOPO expression vector (Life technologies, Darmstadt, Germany) using the NcoI and EcoRI restriction sites. HpNhaA cloned in the pTrcHis2 TOPO expression vector obtained as previously described [22] was used as a control.

Survival assays
Bacterial survival in presence of high concentrations of Na + or Li + was assessed essentially as previously described [23]. Briefly, E. coli KNabc cells, deficient in the expression of the Na + /H + antiporters NhaA, NhaB and ChaA [44] were transformed with the KpNhaA1, KpNhaA2, KpNhaB, HpNhaA constructs or with the empty pTrcHis2 TOPO expression vector.
Cells were grown to OD 600 of 0.6-0.7 in modified Luria-Bertani medium in which NaCl was replaced by KCl (LBK). The medium was buffered with 50 mM MOPS and contained 50 μg/ml kanamycin, 36 μg/ml chloramphenicol and 100 μg/ml ampicillin.
2 μl samples of serial 10-fold dilutions of the cultures were spotted onto LBK agar (1.5%) plates containing 0.6 M NaCl or 0.1 M LiCl at either pH 7 or 8.3 and incubated for 48 h at 37 C. LBK agar plates with no addition of NaCl or LiCl were used as a control.

Acridine orange dequenching in everted membrane vesicles
Everted vesicles from E. coli KNabc transformed with the recombinant plasmids for KpNhaA1, KpNhaA2 and KpNhaB were prepared as described previously [54]. Na + /H + antiport activity was assessed based on the measurement of Na + -induced changes in the ΔpH as measured by acridine orange, a fluorescent probe of ΔpH. Everted vesicles were resuspended in buffer containing 10 mM Tris (titrated to the pH 7 using HCl), 250 mM sucrose and 140 mM choline chloride. Total protein concentration was measured using the Bradford assay [55].
Fluorescence was measured using a Hitachi F4500 Fluorimeter (Hitachi High-Technologies Corporation, Tokyo, Japan) at excitation and emission wavelengths of 495 nm and 530 nm, respectively. Dequenching assays were performed in buffer containing 10 mM MES (titrated to the indicated pH using Tris), 145 mM choline chloride, 5 mM MgCl 2 , 2 μM acridine orange. 100 μg (total protein) of everted vesicles were added to 1 ml external buffer. Acidification of the vesicles was induced using 2.5 mM Tris-D-lactate (at the corresponding pH). After reaching steady-state fluorescence, dequenching was induced by adding 10 or 50 mM NaCl. Finally, the pH gradient was dissipated by addition of 8 mM NH 4 Cl.
Dequenching was calculated as Dequenching % ¼ F deq À F min F fin À F min Á 100 where F deq is the steadystate level of fluorescence achieved after dequenching, F min is the steady-state level of fluorescence after Tris-D-lactate addition, and before Na + addition, and F fin is the steady-state level of fluorescence after dissipation of the pH gradient.
Overexpression, purification and reconstitution C-terminally His-tagged proteins were produced in E. coli BL21(DE3) cells and purified using immobilized Ni 2+ affinity chromatography, as previously described [56]. Reconstitution of purified protein into proteoliposomes was performed using E. coli polar lipids extract (Avanti Polar Lipids, Alabaster, AL, USA) at a calculated LPR of 10 or 50, as previously described [38]. As previously shown [57], an LPR of 10 corresponts to a protein density of~1000 protein particles/μm 2 .

SSM-based electrophysiology
SSM measurements were performed as described previously [38]. Briefly, 30 μl of proteoliposomes at a lipid concentration of 3.3 mg/ml were adsorbed to an octadecanethiol / phospholipid hybrid bilayer on a gold surface (sensor). Unless otherwise stated, most measurements were performed using LPR 10 proteoliposomes. Proteoliposomes were allowed to adsorb to the sensor for 1 h. Electrogenic transport was initiated by a rapid change of substrate ion concentration in a single solution exchange protocol: non-activating solution (0.5 s)-activating solution (0.5 s)-non-activating solution (0.5 s). Currents were amplified using a current amplifier set to a gain of 10 8 −10 9 V/A and a rise time of 10 ms.
In most cases, the amplitude of the recorded transient currents following Na + concentration jumps was used in order to quantify steady-state Na + /H + exchange activity. An exception was made for currents recorded for KpNhaB at pH 9.0 and pH 9.5, which showed a substantial pre steady-state component in addition to the steady-state component. In these cases, the currents were reconstructed according to the procedure described by Mager et al. [38] that allowed the measurement of the stationary component of the reconstructed current.

Kinetic analysis
The steady-state solution for the kinetic model was calculated as described previously by Mager et al. [38]. This calculation yields turnover or activity of the transporter at a given Na + concentration and pH. For KpNhaB, the model was modified by the addition of the Hill coefficients m and n that denote cooperative H + and Na + binding, respectively. Simultaneous fits of the model to the experimentally determined pH and Na + dependences yielded values for the kinetic parameters pK, K Na D , k 2 /k 1 and, in the case of KpNhaB, also for m and n. For the NhaA exchangers, where no cooperativity was apparent, m and n were fixed to 1.
Supporting information S1 Fig. Measured vs reconstructed currents for KpNhaB. Current traces recorded for KpNhaB at pH 9.0 and 9.5 were reconstructed in order to determine the stationary component of the reconstructed current as described by Tadini-Buoninsegni and Fendler [40]. Presented is a trace recorded for a 100 mM Na + concentration jump at pH 9.5, where there is a significant pre steady-state component. (TIF)

S2 Fig. Transient currents recorded for different values of the lipid to protein ratio (LPR).
Current traces were recorded following 100 mM Na + concentration jumps at pH 8.5 for KpNhaA1 (A), KpNhaA2 (B) and KpNhaB (C). For a better comparison of decay time constants at different LPR values, currents were normalized to their maximum amplitude. (TIF)