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J. Biol. Chem., Vol. 275, Issue 39, 30287-30292, September 29, 2000

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Bacillus subtilis YqkI Is a Novel Malic/Na⁺-Lactate Antiporter That Enhances Growth on Malate at Low Protonmotive Force^*

Yi Wei, Arthur A. Guffanti, Masahiro Ito^§, and Terry A. Krulwich^¶

From the  Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029 and ^§ Department of Life Science, Toyo University, Oura-gun, Gunma 374-0193, Japan

Received for publication, February 10, 2000, and in revised form, June 25, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacillus subtilis yheL encodes a Na⁺/H⁺ antiporter, whereas its paralogue, yqkI, encodes a novel antiporter that achieves a simultaneous Na⁺/H⁺ and malolactate antiport. B. subtilis yufR, a control in some experiments, encodes a Na⁺/malate symporter. YqkI complemented a malate transport mutant of Escherichia coli if Na⁺ and lactate were present. YheL conferred Na⁺ uptake capacity on everted membrane vesicles from an antiporter-deficient E. coli mutant that was consistent with a secondary Na⁺/H⁺ antiport, but YqkI-dependent Na⁺ uptake depended on intravesicular malate and extravesicular lactate. YqkI-dependent lactate uptake depended on intravesicular malate and extravesicular Na⁺. YqkI mediated an electroneutral exchange, which is proposed to be a malic²-2H⁺ (or fully protonated malate)/Na⁺-lactate¹ antiport. Because the composite YqkI-mediated exchanges could be driven by gradients of the malate-lactate pair, this transporter could play a role in growth of B. subtilis on malate at low protonmotive force. A mutant with a disruption of yqkI exhibited an abrupt arrest in the mid-logarithmic phase of growth on malate when low concentrations of protonophore were present. Thus growth of B. subtilis to high density on a putatively nonfermentative dicarboxylic acid substrate depends on a malolactate exchange at suboptimal protonmotive force.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The catalogue of diverse structural and mechanistic types of Na⁺ efflux systems in prokaryotes is still expanding rapidly, an eloquent testimony to the importance of maintaining low cytoplasmic concentrations of Na⁺ (1-3). Those Na⁺ efflux systems which are secondary active transport systems are dominated thus far by Na⁺/H⁺ antiporters (3, 4). They are energized by the electrochemical proton gradient, p¹, that is produced by distinct redox-, light-, or ATP-driven proton extrusion systems (5, 6). These antiporters sometimes catalyze efflux of Li⁺ and/or K⁺ in addition to Na⁺ and thus can have a role in Li⁺ resistance and K⁺ homeostasis, respectively, as well as in Na⁺ resistance (7). Na⁺/H⁺ antiporters can support net accumulation of H⁺ when the coupling ratio of H⁺/Na⁺ is greater than unity (8). Accordingly, they can also contribute importantly to pH homeostasis when cells are growing in alkaline media (7, 9, 10). However, reliance on these mechanisms makes these important stress responses vulnerable to conditions in which the p is low, i.e. at which the driving force for the secondary antiporters is reduced.

At the level of amino acid sequence and likely structural category, the secondary Na⁺/H⁺ antiporters reported to date are all distinct from one another except for orthologues, which are simply closely related gene products in different organisms (3). The diversity of the proteins that catalyze Na⁺/H⁺ antiport indicates that there is flexibility in the structures that can support this particular transport function and in some instances reflects a capacity for transporting additional substrates (3, 9). The current study focuses on two paralogous Bacillus subtilis genes whose deduced products both have strong sequence similarity to the Na⁺/H⁺ antiporter NhaC. NhaC was first described in alkaliphilic Bacillus pseudofirmus OF4 (formerly B. firmus OF4 (11)) (12, 13). In that organism, NhaC appears to be one of the relatively high affinity Na⁺/H⁺ antiporters with a role in pH homeostasis at high pH and low [Na⁺] but no evident role in resistance to growth inhibition by elevated Na⁺ (13). Expression of the two homologous B. subtilis genes, yheL and yqkI, was recently examined in tetA(L) deletion mutants of B. subtilis to ascertain whether their up-regulation might occur upon loss of the Na⁺/H⁺ antiporter function of multifunctional TetA(L) (14). Expression of one of these genes, yheL, was indeed up-regulated. In addition, B. subtilis mutants deleted in yheL had a phenotype consistent with YheL being an NhaC-type of Na⁺/H⁺ antiporter. The experiments reported here were subsequently undertaken to confirm that YheL indeed catalyzed this exchange and to probe the possibility that the product of the other paralogue, yqkI, whose expression was not up-regulated, might catalyze a different, novel exchange.

The genomic context of yqkI provided an interesting clue to such an alternative exchange. The yqkI gene is in an apparent operon with yqkJ. The strongly predicted product of yqkJ in BLAST (15) analyses is a malolactate enzyme. The malolactate enzymes of lactic acid bacteria catalyze the formation of lactate and carbon dioxide from malate, with the consumption of a cytoplasmic proton (16, 17). The malolactate enzyme, together with a transporter (or two separate transporters) that exchanges the external precursor (malate) for the product of the malolactate enzyme (lactate), comprise malolactate fermentation (MLF) (18, 19). MLF plays an important role in determining the acidity of wine (20-22). The basis for energy conservation for the bacteria that carry out the MLF pathway depends in part on the consumption of the cytoplasmic proton, so that a net pH, acid outside, results (17). Depending on the details of substrate and product exchange, energy conservation may be further enhanced (18, 19, 23), e.g. as in Lactobacillus lactis in which the malate-lactate exchange catalyzed by an antiporter is accompanied by inward movement of net negative charge, producing (24). The deduced yqkI product does not resemble the malolactate exchange transporters or malate transporters that have been described in association with malolactate enzymes in fermentative bacteria (24-26).

The current study was designed to test the possibility that in fact YqkI is a novel transporter that combines Na⁺/H⁺ and malate/lactate antiports in a malate-proton/Na-lactate exchange. Comparisons were carried out on the YheL-, YqkI-, and YufR-mediated transport activities in cells of a malate-minus Escherichia coli strain and in membrane vesicles from a Na⁺/H⁺ antiporter-deficient E. coli strain. YufR is a distinct B. subtilis transport protein of unconfirmed function that was strongly predicted by both sequence and context to encode a Na⁺/malate symporter and hence was developed as a positive control for some of the experiments. The results support the expected catalytic properties for YheL and YufR and support the proposal that YqkI is a new structural type of malate/lactate antiporter that has novel ion coupling properties, i.e. coupling Na⁺ efflux and H⁺ uptake to the substrate-product antiport. Because the concentration gradients of the organic acid substrate and product could drive the coupled antiport, this combined exchange could be less vulnerable to reductions in the p such as those accompanying increased cell densities or encounters with protonophoric agents. Studies of a mutant in which yqkI was disrupted support this hypothesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Plasmids-- Three E. coli strains were used in this study: E. coli strain DH5a (Life Technologies, Inc.) was used for initial cloning work; E. coli EP432 (27), a Na⁺/H⁺-antiporter-deficient strain; and E. coli CBT312 (28), which is deficient in malic acid transport. The E. coli strains were grown at 37 °C in the media described in connection with specific experiments. Two strains of B. subtilis were employed, the wild type, BD99 strain obtained from A. Garro, and the VK3 strain constructed as part of this study. The cells were grown at 37 °C in Spizizen salts medium with either glucose or malate as the major carbon source, as described previously (14).

The plasmid constructs were made by cloning PCR products into shuttle vector pBK36 (obtained from K. Zen). The primers and sites used for cloning the genes are listed in Table I. The various plasmids are designated pBK36 (control plasmid), pYqkI, pYqkJ, pYqkIJ, pYufR, and pYheL.

Complementation and Resistance Studies in E. coli-- The complementation of E. coli EP432 by the different plasmid constructs was tested as described previously (29) in LBK medium (30), supplemented with various concentrations of NaCl. In some experiments 50% LBK plus either 50 mM potassium malate or glucose was used. Complementation of E. coli CBT312 for growth on malate was tested in two ways. In one protocol, LT medium (26) containing L-malate and bromcresol blue was used to detect the consumption of L-malate by the appearance of a deep blue color. In other experiments, M9 minimal medium (32), with either L-malate or glucose as sole carbon source, was used for complementation studies. In some experiments, potassium was substituted for the sodium salts in M9 medium.

Transport Assays in E. coli Vesicles-- RSO membrane vesicles were prepared by the method of Kaback (33) by shocking spheroplasts in 50 mM Tris-HCl, pH 7.5, plus 2 mM MgSO₄. Everted membrane vesicles were prepared, as described by others (34), in either 50 mM Tris-HCl, pH 7.5, or 50 mM potassium phosphate, pH 7.5. For some reactions, the vesicles were preloaded by incubating for 18 h at 4 °C with the appropriate substrate. Transport of various radioactive substrates was assayed, by filtration, as described elsewhere (29). Protein was determined by the method of Lowry et al. (35), using lysozyme as the standard.

Construction and Characterization of B. subtilis VK3-- A mutant strain of B. subtilis was constructed in which yqkI was disrupted with a Sp^R cassette with its own promoter. PCR was performed on purified B. subtilis BD99 chromosomal DNA by using primers YqkIUP and YqkIDN that were contained within the yqkI gene. YqkIUP (5'-CAAACCAGGTATCGCAGGGAAAGA-3') corresponded to base pairs 265255-265278 of the data base entry (GenBank^TM accession number D84432). YqkIDN (5'-TACCGTCTCCACTTCACCAATATCC-3') corresponded to base pairs 266899-266867 of the same data base entry. The purified PCR product was first ligated into HincII-digested pGEM11zf(+) (Ap^R; Promega). The recombinant plasmid was selected by blue/white screening in E. coli DH5a. After isolation, the yqkI fragment was digested with PmlI, and a gene encoding Sp^R was ligated to this linear plasmid, resulting in a recombinant plasmid containing a fragment of yqkI disrupted by the Sp^R gene (yqkI::Sp^R). After isolation, the plasmid was introduced into B. subtilis BD99 by competent cell transformation. Transformed cells were resistant to Sp (150 µg/ml). Both Southern hybridization and PCR analyses confirmed that the gene disruption event had taken place in the candidates. The yqkI-disrupted strain was designated VK3.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Assays of YufR-dependent Na⁺/Malate Symport in RSO Membrane Vesicles of E. coli EP432 and CBT312-- Initial studies were directed toward determining whether YufR was indeed a Na⁺/malate symporter by determining whether the gene product catalyzed Na⁺-dependent uptake of malate and malate-dependent uptake of Na⁺. RSO membrane vesicles were prepared from E. coli strains EP432 and CBT312 expressing either the control plasmid pBK36 or pYufR. The vesicles were pretreated with 10 mM KCN, which was also present in the assay medium, to inhibit p-dependent fluxes of malate or Na⁺. As shown in Fig. 1, substantial uptake of radiolabeled malate occurred in YufR vesicles of E. coli EP432 in the presence of Na⁺ but not in the control vesicles or in the absence of the Na⁺ gradient. As shown in Fig. 2, uptake of Na⁺ by KCN-treated RSO vesicles of E. coli CBT312/pBK36 was modest and somewhat greater in the absence than in the presence of malate. By contrast, vesicles prepared from E. coli CBT312/pYufR exhibited substantial malate-dependent uptake of ²²Na⁺. Taken together, these results supported the expectation that YufR is a Na⁺/malate symporter and would be an appropriate positive control in complementation experiments with a malate-minus E. coli strain.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The effect of an inwardly directed Na+ gradient on uptake of L-malic acid in RSO membrane vesicles prepared from E. coli EP432 transformed with plasmid pBK36 or pYufR. RSO membrane vesicles were pretreated with 10 mM KCN for 10 min. Transport was initiated by diluting the vesicles 10-fold into a reaction mixture, at pH 7.5, containing 14 µM L-[1,4-(2,3)-14C]malic acid (0.8 µCi/ml) and 10 mM KCN, with or without 10 mM NaCl. Each reaction mixture contained 100 µg of vesicle protein. Samples were taken at various times and filtered. The radioactivity was counted by liquid scintillation spectrometry.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   The effect of an inwardly directed malic acid gradient on 22Na+ uptake by RSO membrane vesicles prepared from E. coli CBT312 transformed with plasmid pBK36 or pYufR. The vesicles were treated and assayed in the presence of KCN as described in the legend to Fig. 1. The vesicles were diluted into a reaction mixture, at pH 7.5, containing 500 µM 22NaCl (10 µCi/ml), with or without 20 µM L-malic acid, and assayed as in Fig. 1.

Complementation and Resistance Conferred by YheL, YqkI, YqkIJ, and YufR-- Although not shown, only YqkIJ and YufR gave a positive result for complementation of the malate transport-minus phenotype of E. coli CBT312 in LT medium containing malate; YufR was further tested, on both solid and liquid media for substrate specificity, and was found not to support growth on citrate, fumarate, or succinate. As assessed qualitatively from the blue color that developed, YufR was slightly more efficacious than YqkIJ in promoting growth on malate, and neither YqkI nor YheL differed from control plasmid. As shown in Table II, although all the transformants grew well on glucose-containing M9 medium, only YqkIJ and YufR supported growth of E. coli CBT312 on malate. Growth was dependent on the presence of Na⁺ in the medium. The results were consistent with YqkI being a malate uptake system that depended on Na⁺ and probably also on adequate lactate, given the requirement for YqkJ.

As shown in Fig. 3A, the Na⁺ sensitivity of E. coli EP432 in LBK medium was exacerbated by YufR. In this very undefined rich medium there is probably enough contaminating malate for this symporter, which mediates Na⁺-coupled solute uptake, to take in an inhibitory level of Na⁺. The transformants containing these genes were further examined versus the control transformant in malate- and glucose-containing media. Although all three transformants were equally sensitive to growth inhibition by Na⁺ in glucose-containing medium (Fig. 3C), YqkI and YqkIJ conferred significant resistance to Na⁺ when E. coli EP432 was grown on malate (Fig. 3B). The results of the growth studies were thus consistent with YufR being a Na⁺/solute symporter and YqkI being a transporter that mediates Na⁺ efflux, which is somehow coupled to a capacity for malate uptake. Transformation of E. coli EP432 with a yheL-containing plasmid did not result in substantial complementation of Na⁺ sensitivity, as expected for a relatively high affinity system of the NhaC type (13).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   The effect of pBK36, pYufR, pYqkI, and pYqkIJ on the growth of E. coli EP432 on different carbon sources as a function of NaCl concentration. E. coli EP432 transformed with pBK36 (), pYufR (), pYqkI (), or pYqkIJ () was grown on LBK medium (A), 50% LBK plus 50 mM K-malate (B), or 50% LBK plus 50 mM glucose (C) in the presence of the indicated NaCl concentrations. The pH of all the growth media was adjusted to 7.5. The A600 was recorded after 15 h of growth at 37 °C.

YqkI- and YheLB-dependent ²²Na⁺ and [¹⁴C]Lactate Uptake by Everted Membrane Vesicles of E. coli EP432-- Uptake of ²²Na⁺ by everted vesicles made from control, pYheL, and pYqkI transformants of E. coli EP432 was examined as a function of intravesicular malate, extravesicular lactate, and an electron donor. The everted YheL vesicles took up ²²Na⁺ when an electron donor was added, either NADH alone or NADH and lactate, and exhibited no dependence on intravesicular malate or on the specific presence of lactate as long as NADH was added. By contrast, the everted YqkI vesicles exhibited significant ²²Na⁺ uptake, but only in the presence of both extravesicular lactate and intravesicular malate (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   The effect of an energy source and intravesicular malic acid on the uptake of 22Na+ by everted membrane vesicles prepared from E. coli EP432 transformed with pYqkI or pYheL. Everted membrane vesicles, in buffer at pH 7.5, were either pre-loaded (closed symbols) or not pre-loaded (open symbols) with 200 µM L-malic acid. They were assayed for 22Na+ uptake in the absence of an energy source (,), in the presence of 2.5 mM Tris-NADH (,), or in the presence of NADH plus 200 µM L-lactic acid (, ). The control preparation with pBK36 exhibited no uptake under any condition.

Because the apparent necessity of extravesicular lactate for ²²Na⁺ uptake by YqkI vesicles made it impossible to evaluate transport in the absence of an energy source, inhibitors were employed to probe the energetics of the exchanges catalyzed by YheL and YqkI. In the experiments shown in Fig. 5A, everted YqkI vesicles of E. coli EP432 were loaded with L-malate and energized with NADH. The uptake of ²²Na⁺ and the effect of inhibitors was assayed upon addition of both the radiolabeled Na⁺ and extravesicular lactate. The uptake of ²²Na⁺ was markedly inhibited by both CCCP and nigericin but not by valinomycin. The effect of nigericin is consistent with an energizing role for the pH component of p. The lack of inhibition by valinomycin suggests the absence of such a role for in the YqkI-mediated antiport. That is, the exchange is likely to involve movement of one or more protons with the malate and, overall, to be an electroneutral antiport. In the experiments shown in Fig. 5B, everted YheL vesicles of E. coli EP432 were assayed for ²²Na⁺ uptake upon being energized by NADH without any intravesicular malate or extravesicular lactate. In contrast to the inhibition pattern observed with the YqkI-mediated exchange, the YheL-mediated Na⁺/H⁺ antiport was markedly inhibited by both valinomycin and CCCP; nigericin inhibited YheL-mediated Na⁺/H⁺ antiport only modestly. The results are consistent with the YheL-mediated antiport being electrogenic, as anticipated for a Na⁺/H⁺ antiport that plays a role in alkali resistance (8).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   The effect of ionophores on the uptake of 22Na+ by everted membrane vesicles prepared from E. coli EP432 transformed with pYqkI or pYheL. Everted membrane vesicles prepared in 50 mM potassium phosphate, pH 7.5, were assayed for 22Na+ uptake by energizing with 2.5 mM K-NADH. The YqkI vesicles (A) were preloaded with 200 µM L-malic acid and were assayed in the presence of extravesicular L-lactic acid (200 µM). The YheL vesicles (B) were not preloaded with malic acid and were assayed for 22Na+ uptake in the absence of extravesicular lactic acid. The transport assays were conducted in the presence of: no further additions (); 0.1 µM nigericin (); 1 µM CCCP (); or 1 µM valinomycin ().

The experiments with the mutant E. coli CBT312 directly supported a role for YqkI in malate transport, and the vesicle experiments above demonstrated YqkI-mediated Na⁺ flux. Direct evidence for YqkI-mediated transport of the lactate via the putative malate/Na-lactate exchange was then sought by assaying [¹⁴C]lactic acid uptake by everted YqkI vesicles of E. coli EP432 as a function of the presence of NADH, intravesicular malate, and extravesicular Na⁺. In the absence of added Na⁺ (Fig. 6A), modest stimulation of uptake, over a lactate-only reaction mixture, was caused by the inclusion of NADH and intravesicular malate. By contrast, vesicles assayed in the "complete" reaction mixture, with intravesicular malate, extravesicular NADH, Na⁺, and [¹⁴C]lactate, exhibited impressive uptake of the labeled transport substrate (Fig. 6B). The rate of antiport was much higher in the complete mixture than in mixtures from which either NADH or intravesicular malate was omitted.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   The effect of extravesicular Na+ and intravesicular malic acid on the uptake of L-[14C]lactic acid by everted membrane vesicles prepared from E. coli EP432 transformed with pYqkI. All reaction mixtures were at pH 7.5 and contained 100 µM L-[(U)14C]lactic acid (1 µCi/ml) as the transport substrate for the assay. In the experiments shown in A, no Na+ was added to the reaction mixtures, whereas in the experiments in B, 1 mM NaCl was added to all the reaction mixtures. Vesicles were either preloaded with 200 µM L-malic acid (closed symbols) or not (open symbols). Either no further additions were made (, ) or 2.5 mM Tris-NADH was added as an energy source (, ).

The absence of valinomycin inhibition of energized YqkI-mediated uptake of ²²Na⁺ by everted vesicles suggested that the antiport might be electroneutral, e.g. malic²-2H⁺/Na⁺-lactate¹. If so, energized uptake of [¹⁴C]lactate by everted YqkI vesicles, in the presence of intravesicular malate and extravesicular Na⁺, would be inhibited by CCCP and nigericin, which would abolish the pH. Valinomycin, which abolishes , would not inhibit. Stimulation by valinomycin might even be observed with this substrate if its transport was entirely dependent on YqkI, because the pH would be elevated upon abolition of the by valinomycin. As shown in Fig. 7A, both CCCP and nigericin indeed inhibited lactate uptake by YqkI vesicles in a complete reaction mix and valinomycin caused a reproducible stimulation of lactate uptake. That stimulation supported the conclusion that the antiport is electroneutral and can be energized by pH, but not by . A more direct probe of this conclusion was warranted, because the antiport catalyzed by some other malolactate antiporters has been proposed to involve net inward movement of negative charge, e.g. Hmalate¹ or malic²-H⁺/lactic or lactate¹-H⁺ antiport in L. lactis (24). Perhaps the YqkI-mediated antiport is also an electrogenic Hmalate¹ or malic²-H⁺/Na⁺-lactate¹ antiport instead of an electroneutral malic²-2H⁺/Na⁺-lactate¹ antiport. The stimulatory effect of valinomycin in energized vesicles might only reflect a dominance of the role of the pH under the particular assay conditions. To clarify this, YqkI-mediated uptake of [¹⁴C]lactic acid was assayed under de-energized conditions in cyanide-treated everted vesicles in which uptake was driven only by the gradients of the solutes. If the antiport were electrogenic, the developed during antiport should be a major constraint on the rate of lactic acid uptake and valinomycin should stimulate uptake significantly. If the antiport were electroneutral, valinomycin would be anticipated to have no effect. As shown in Fig. 7B, valinomycin had no effect.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   The effect of ionophores on [14C]lactic acid uptake by everted membrane vesicles prepared from E. coli EP432 transformed with pYqkI. Vesicles prepared in potassium phosphate buffer were preloaded with 200 µM L-malic acid and assayed, at pH 7.5 and in the presence of 1 mM NaCl, for [14C]lactic acid uptake as described in the legend to Fig. 6. In the experiments shown in A, the vesicles were energized by the addition of 2.5 mM K-NADH with: no further additions (); 1 µM CCCP (); 1 µM valinomycin (); or 0.1 µM nigericin (). In the assays shown in B, vesicles were pretreated and assayed with 10 mM KCN. The transport of [14C]lactic acid by these de-energized vesicles, which contained intravesicular malate and had extravesicular Na+, was assayed either with no further additions () or in the presence of 1 µM valinomycin ().

The YheL vesicles of E. coli EP432 were, finally, assayed for [¹⁴C]lactate uptake, energized by NADH, that was dependent on extravesicular Na⁺ and/or intravesicular malate. No such uptake was observed, indicating that YheL does not have a malolactate mode in addition to its malate- and lactate-independent Na⁺/H⁺ antiport activity.

Growth Characteristics of B. subtilis VK3-- Growth studies of the mutant strain with a disrupted yqkI were conducted in comparison to the wild type to test the hypothesis that the pathway might be important for growth on Na-malate, especially when highly aerobic conditions are compromised by high cell densities or when the p is directly reduced by inclusion of sublethal protonophore concentrations in the medium. As shown in Fig. 8 for growth up to 10 h, B. subtilis wild type and VK3 grew similarly on glucose or malate under aerobic conditions in the absence of protonophore. In general, the growth of the mutant on malate slowed down slightly, relative to the wild type, after the first few hours of growth. In the presence of 1 or 2 µM CCCP, both strains exhibited a growth lag on Na-malate. In several independent experiments, the lag was slightly less pronounced in strain VK3, and the initial rate of growth on malate in the presence of CCCP was slightly faster in the mutant than in the wild type strain. However, once growth on malate in the presence of CCCP commenced in the wild type, it proceeded logarithmically, reaching the A₆₀₀ values of 1.4-1.5 shown for the wild type in the absence of CCCP by 14-18 h. By contrast, growth of VK3 on malate in the presence of CCCP was arrested when the A₆₀₀ of the cultures was only 0.15-0.25. At 2 µM CCCP, the growth turbidity declined thereafter for several hours at least, whereas at 1 µM CCCP the turbidity was steady. Although not shown, VK3 did exhibit a very slow renewed growth on malate after hours of growth arrest in the presence of CCCP. This may indicate the presence of another system that is slowly induced and can partially compensate for the role of YqkI. There was no difference in the CCCP effect on growth of the two strains on glucose.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Growth of B. subtilis wild type (BD99) and yqkI-disrupted mutant strain VK3 on glucose and on malate in the absence and presence of low CCCP concentrations. The two strains were grown on glucose (open circles) or malate (closed circles, triangles)-containing media at 37 °C as described under "Experimental Procedures." CCCP was added to some cultures at either 1 µM (open triangles) or 2 µM final concentration (closed triangles). The A600 was monitored.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The major findings of the current study are that B. subtilis possesses an antiporter, YqkI, that couples a malate-lactate exchange to proton uptake and Na⁺ efflux and that this transporter plays a role in supporting growth to high density on malate at reduced protonmotive force. We suggest that yqkI be designated mleN to reflect its commonality of function with other malolactate exchangers, and using the "N" to denote Na⁺. The coupling of Na⁺ efflux to such a precursor-product exchange has not been previously reported. The transport data strongly indicate that the MleN-mediated antiport is electroneutral, which further distinguishes it from the malolactate exchanges reported for both L. lactis (23, 24) and Leuconostoc oenos (25). The simplest MleN-mediated exchange that is consistent with all the data, and the near neutral pH used in the experiments, would be malic²-2H⁺/Na⁺-lactate¹ exchange. MLF pathways have been engineered into Saccharomyces cerevisiae strains, e.g. by introducing malate transport genes from Schizosaccharomyces pombe and the malolactate enzyme from L. lactis (36, 37), with a view toward improving the organic acid conversion during fermentation of grape juice. Perhaps MleN would be useful for fermentations in preparations with sodium ion concentrations that are above the optimum for the yeast.

The yqkJ product that is the only other putative product of an operon, which includes mleN, is strongly predicted to be a malolactate enzyme that converts malate directly to lactate, as opposed to related malic enzymes that convert malate to pyruvate (38). The efficacy of YqkJ in providing sufficient cytoplasmic lactate to support strong malate uptake in E. coli CBT312 supports this tentative conclusion. Accordingly, we propose the designation mleA for yqkJ. Heretofore, the MLF pathway has been studied in anaerobes (17-19, 25, 26). Similarly, the other precursor-product pathways that can generate a p have also been found in fermentative bacteria (39-42). The finding of an apparent and novel MLF in B. subtilis is thus intriguing. This MLF pathway, encompassing the MleN antiport and MleA activity, would result in net conversion of malate to lactate and CO₂, with concomitant extrusion of Na⁺ and acidification of the cytoplasm (uptake of one net proton inasmuch as two protons enter with malate and one cytoplasmic proton is consumed in the malate-to-lactate conversion). Because the precursor and product gradients could drive the transport reaction even at low p, mimicking the protocol with cyanide-treated vesicles (Fig. 7), we anticipated that cells of B. subtilis could use this pathway as a malate uptake and Na⁺/H⁺ antiport mode that is relatively resistant to low oxygen or low p conditions. Increased use of the MLF pathway might, for example, underlie some of the energetics of malate-supported growth of protonophore-resistant mutants of B. subtilis in the presence of CCCP (43). The growth studies on B. subtilis VK3 support this hypothesis and specifically suggest that malate uptake via MleN (YqkI) is important past the initial hours of growth when the p has been reduced, e.g. as done here with protonophores. Similar decreases in p might occur in various natural environments via suboptimal reductions in oxygenation or encounters with chaotropic agents, bacteriocins, etc.

The transport reactions catalyzed by two other cation-coupled transporters have been established in this study. YheL is produced by the yheL gene, which is part of an operon with one other gene, yheK, which is strongly predicted to be a regulatory gene. The experiments in the current study support earlier observations in yheL deletion strains of B. subtilis that suggested that YheL is a secondary, electrogenic Na⁺/H⁺ antiporter that makes modest contributions to pH homeostasis in the alkaline range of pH but is not a contributor to Na⁺ resistance (14). YheL, despite its sequence similarity with MleN, did not exhibit a mode of Na⁺/H⁺ antiport that also includes a malolactate exchange. We propose that the yheKL operon be designated as nhaXnhaC. Several other genomes in the data bases, e.g. of Staphylococcus aureus and Borrelia burgdorferi, contain multiple genes with significant sequence similarity to the nhaC genes of Bacillus species. Perhaps in these organisms too, the paralogous transporters have different activities with some commonality.

Finally, the current studies support the prediction that the yufR gene product of B. subtilis is a Na⁺/malate symporter. The apparent specificity for malate as the organic acid substrate is also true for the closely related MaeP transporter of Streptococcus bovis, which is part of a family of transporters that includes citrate transporters CitP and CitS as well as the MleP from L. lactis (24, 26, 44). We propose that yufR be designated maeN to indicate the demonstrated Na⁺ coupling of this B. subtilis malate symporter. maeN is directly upstream of the mrp operon, which is importantly involved in Na⁺ resistance in B. subtilis (31). Perhaps yufR and mrp expression are coordinated under some conditions.

	FOOTNOTES

^* This work was supported by Research Grants GM52837 and GM28454 from the National Institutes of General Medical Sciences (to T. A. K.) and by The Inoue Enryo Memorial Foundation for Promoting Science (to M. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Box 1020, Dept. of Biochemistry & Molecular Biology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-7280; Fax: 212-241-996-7214; E-mail: terry.krulwich@mssm.edu.

Published, JBC Papers in Press, July 19, 2000, DOI 10.1074/jbc.M001112200

	ABBREVIATIONS

The abbreviations used are: p, transmembrane electrochemical proton gradient, conventionally positive and acid out for a bacterial cell or right-side-out vesicle; pH, transmembrane pH gradient; , transmembrane electrical potential; CCCP, carbonyl cyanide-m-chlorophenylhydrazone; MLF, malolactate fermentation; RSO, right-side-out; PCR, polymerase chain reaction; LBK, Luria Broth with KCl instead of NaCl.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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