INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The mrp operon was first identified in the genome of Bacillus subtilis as a homologue of a locus that had been found to be centrally important to cytoplasmic pH regulation in alkaliphilic Bacillus halodurans C-125 (3, 16). A point mutation in the first gene of the alkaliphile homologue resulted in loss of Na⁺/H⁺ antiporter activity (3). Such antiport is widely used by prokaryotes for alkali and Na⁺ resistance inasmuch as coupled Na⁺ exclusion and H⁺ accumulation can be accomplished via electrogenic exchange of cytoplasmic Na⁺ for a greater number of H⁺ (14, 20). The complete mrp operon of B. subtilis is predicted to encode seven hydrophobic gene products (9, 12), as is also posited for homologues from diverse organisms, including alkaliphilic Bacillus pseudofirmus OF4 (14, 15), Rhizobium meliloti (21), Staphylococcus aureus (6), and others annotated in genome databases. Apart from the apparent role of the alkaliphile mrp operons in Na⁺-dependent pH homeostasis, studies with mutants have suggested that the R. meliloti homologue, pha, may encode a K⁺/H⁺ antiporter that is required for symbiotic nitrogen fixation (21) and that B. subtilis mrp (called ntr and sha by other investigators [12, 13]) has multiple functions. First, the B. subtilis mrp locus has been shown to play a role in Na⁺ resistance and in both Na⁺- and K⁺-dependent cytoplasmic pH homeostasis (9, 12). This is consistent with one or more mrp genes encoding an Na⁺(K⁺)/H⁺ antiport activity. Recently, Kosono et al. (13) showed that a B. subtilis mrpA (shaA) mutant fails to sporulate normally and suggested that an early step in sporulation is sensitive to the elevated cytoplasmic Na⁺ concentration that results from mrp mutations. The second B. subtilis mrp activity, in which the mrpF gene has been implicated, encompasses cholate and Na⁺ efflux activities, which may be mechanistically coupled. Demonstration of cholate efflux activity has thus far been made only in a mutant with a disruption in mrpF that also lowered expression of mrpG (9), but in the current study, separate mutations in mrpF and mrpG have been examined.

Before the discovery of the mrp operon and its homologues, the well-studied examples of Na⁺/H⁺ antiporters all involved a single structural gene product (20). Data to date suggest that monovalent cation/H⁺ antiporter activity requires the first gene of the operon, mrpA, in B. subtilis, but that other genes of the operon are required for some combination of antiporter activity, expression, and assembly (9, 12). That is, MrpA is necessary but not sufficient for Na⁺/H⁺ activity. Similarly, Hiramatsu et al. (6) have suggested from studies in which the S. aureus homologue, designated mnh, was expressed in an Na⁺-sensitive Escherichia coli mutant, that all the genes of the operon may be required for the Na⁺ resistance conferred in that system. There are recent reports of secondary multidrug transporters with two heterologous protein components (10, 18) but the complexity of the mrp product interactions might be of a much higher order. In addition, the long-recognized sequence similarity of several mrp products to membrane-embedded subunits of energy-coupled NADH dehydrogenase complexes (3, 9) raises the possibility that there is a capacity for electron transport that could provide a primary energy coupling option for mrp functions.

In the current study, individual in-frame deletions were made in each of the B. subtilis mrp genes for which no such mutations had been made earlier, i.e., mrpB, -C, -D, -E, -F, and -G. For each of those strains, a version was also made in which an active copy of the disrupted gene was returned to the amyE locus of the chromosome under the control of an IPTG (isopropyl--D-thiogalactopyranoside)-inducible promoter. Each mrp gene was similarly introduced into the amyE locus of an mrp null mutant, VKN1, of B. subtilis, and into that of a polar mutant, VK6, that lacks mrpA and expresses mrpB to -G at greatly reduced levels. Resistance and transport studies have supported earlier indications that MrpF is the Na⁺-cholate efflux protein and further show that MrpF activity is independent of the expression of additional mrp genes. By contrast, MrpA function, which is shown to correlate with a protonophore-sensitive Na⁺ efflux activity, requires all six other mrp genes. In addition, evidence is presented for a complex regulatory relationship between loss of function of particular mrp genes and expression of the polycistronic mrp mRNA.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains, plasmids, and general growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. The bacteria were routinely grown at 30°C in malate-containing, potassium-replete TKM medium (9). For experiments in which mrp genes were introduced into the amyE locus under control of the p_spac promoter, 200 µM IPTG was included in the growth medium.

Northern analyses. Cells were grown, RNA was prepared, and the Northern procedures were carried out according to methods described by others (2). Three different probes were employed: (i) a probe to a gene upstream of the mrp operon that encodes maeN (yufR) was prepared using primers yufR1 and yufR2; (ii) a probe to part of mrpA was prepared using primers BsmrpA1 and mrpA2T7; and (iii) a probe to part of mrpG was prepared using primers BsmrpG1 and mrpG2T7. For these preparations as well as other PCRs, either Pfx (Life Tech) or Vent (New England Biolabs) DNA polymerase was used. The PCR products were first gel purified and then eluted using a gel extraction kit (Qiagen). The probes were then used as DNA templates for random priming ³²P labeling using a random-primed DNA labeling kit (Roche Diagnostics). The locations of the probes are indicated in Fig. 1.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of the yufR (maeN)-mrp region of the B. subtilis chromosome that indicates the probes used for Northern analyses and the primers used in construction of new mrp deletion strains.

Construction of mutant strains. For each type of mutant, the phenotype of the strain used in the studies was the same as that of several other strains from the construction protocol. Each mutant that was used in the subsequent studies was initially confirmed to have the expected PCR profile and was then directly shown to contain the expected sequence. Sequencing was conducted at the Biotechnology Center at Utah State University (Logan, Utah) with an ABI-100 model 377 sequencer. All in-frame mutations were constructed by gene splicing via overlap extension, as described by others (7). For the deletions of mrpB, mrpC, and mrpE, the approach was precisely as used in earlier construction of the VK1A (mrpA deletion, previously called VK1) strain (9). For deletions in mrpD, mrpF, and mrpG, the earlier approach alone did not yield the desired mutations; therefore, gene splicing via overlap extension was used as the first step, followed by steps that resulted in the introduction of a spectinomycin resistance (Sp^r) cassette gene, spc (19), into the deleted area.

Determination of MICs of Na⁺ and inhibition profile for cholate. The MIC of Na⁺ was determined in TKM medium at pH 7.0 or 8.3 exactly as described elsewhere (9). Sensitivity to cholate was also assessed as previously described (9).

Transport assays. Measurements of cholate efflux were conducted on whole cells that were preloaded with cholate and then assayed using a filtration assay as described in connection with earlier studies of the mrp operon (9). Measurements of ²²Na⁺ efflux were conducted in both whole cells and right-side-out membrane vesicles. The whole-cell ²²Na⁺ efflux assays conducted in connection with MrpF function were carried out precisely as described earlier (9). For the assays in membrane vesicles, right-side-out vesicles were prepared by a modification of the method of Kaback (11). Protoplasts were prepared from logarithmic-phase cells by incubating at 37°C in 100 mM potassium phosphate (pH 7.5)-20% sucrose-300 µg of lysozyme per ml until the cells had rounded up. The protoplasts were shocked in 50 mM potassium phosphate (pH 7.5) plus 1 mM MgSO₄ by diluting 100-fold and passing through a syringe with a 19G1/2 needle. Vesicles were passively loaded with 5 mM ²²NaCl (10 µCi/ml) for 18 h at 4°C. For assays of Na⁺ efflux energized by the electron transport chain, vesicles (100 µg of protein/ml) were incubated at 10°C. No further additions were made, or 10 mM potassium ascorbate-0.1 mM phenazine methosulfate (PMS) was added. The protonophore carbonyl cyanide p-chlorophenylhydrazone (CCCP) was added to some reactions, as indicated. Samples were taken at various times and filtered, and radioactivity was measured by liquid scintillation counting.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Northern analyses of the mutant strains. Northern analyses were conducted on each of the newly constructed strains, the VK1A strain constructed earlier (9), and the wild type. Three different probes were used. One was to the upstream maeN (yufR) gene, which has been shown to encode an Na⁺/malate symporter (Y. Wei, unpublished data). This gene is transcribed in the same direction as the mrp genes, and the possibility that it is cotranscribed or coregulated with them was of interest. The other two probes were to the mrp genes at the ends of the known operon, mrpA and mrpG. These probes were used to determine whether the new in-frame deletion mutations were in fact nonpolar, and whether all of the new mutants exhibited increased mrp RNA abundance. The mrpA deletion in strain VK1A had earlier been shown to be nonpolar and to result in much higher levels of mrp RNA than the wild type (9). As shown in Fig. 2, all the new inframe deletion mutations (mrp BCDEF) are nonpolar inasmuch as the cells express mrpG at wild-type levels of RNA abundance or greater. In Fig. 2, an arrow indicates the location of the wild-type mrp band. This band could only be visualized more distinctly upon longer exposures, which made it too difficult to discern any detail in most of the other lanes. The sizes of the mrp bands in the mutants were as expected from the size of the deletion combined, in three of them, with the introduction of a cassette. As had been observed for mrp RNA in VK1A (9) (which is shown again in Fig. 2 together with the new mutants), the new mutants with deletions in mrpB, mrpE, mrpD, and mrpG all exhibited a significant increase in mrp RNA, as did the retested mrpA mutant. Two of the new mutants with deletions in mrpC or mrpF did not exhibit an elevation of mrp RNA comparable to that seen in the other strains, although there was still an increase relative to the wild-type strain. In the RNA preparations from some of the mutants, there were also bands that reacted with mrp gene probes that were smaller than the major, expected band and may represent degradation products.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Northern analyses of mutants with individual deletions in each of the seven B. subtilis mrp genes. RNA was prepared from wild-type B. subtilis and probed with the DNA probes corresponding to parts of the first and last mrp genes and to part of the upstream maeN (yufR) gene. The locations of the probes are shown in Fig. 1, and the procedures are described in Materials and Methods. The strain is indicated above each lane, e.g., Wt, wild type; A, VK1A; etc. The probe used for the particular blot is indicated below the panel.

MrpF-dependent cholate resistance, cholate efflux, and Na⁺ efflux. Cholate resistance was examined in our initial studies of the B. subtilis mrp operon because the BLAST analysis (1) of the deduced mrpF product revealed sequence similarities to Na⁺-coupled bile acid transporters. The difference in cholate resistance found between the wild type and mrpF (polar) mutant, VK15, was significant and reproducible but not large enough to be captured as a difference in MIC (9). As shown in Fig. 3 for each of the single mrp gene mutants and their complemented versions, a large increase in sensitivity to cholate was found only in the new mrpF (nonpolar) mutant VK1F, and only in that mutant was the restoration of an active gene in the amyE locus accompanied by significantly greater resistance to cholate. To correlate the resistance with cholate transport capacity, the efflux of cholate from preloaded cells of the wild type was compared to efflux from the mrpF and mrpG mutants, VK1F and VK1G, respectively, and the versions of each in which the affected gene was expressed from the amyE locus. As shown in Fig. 4A, efflux of cholate was not defective in the mrpG mutant strain but was significantly reduced in the mrpF mutant strain VK1F. Expression in trans of the mrpF gene in VK1F increased the cholate efflux activity of the mutant almost to the level in VK1G (Fig. 4A) or the wild-type strain (Fig. 4B). Earlier studies indicated that expression of mrpF in the mrp null strain of B. subtilis did not restore cholate resistance to that strain (9). However, the results here led us to reexamine the possibility that MrpF can function independently of any other mrp genes in experiments in which transport of cholate itself was assayed. Efflux and resistance might not be observed in parallel, for example, if cholate reentry was pronounced relative to the capacity and rate of efflux. As shown in Fig. 4B, expression of mrpF under control of an IPTG-inducible promoter in the amyE locus of mrp null strain VKN1 resulted in a dramatic increase in cholate efflux. Expression of mrpF in strain VKN1 also resulted in an increase in Na⁺ efflux (Fig. 5), although, as noted below, this increase too was not sufficient to be reflected in an increase in Na⁺ resistance.

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Sensitivity of wild type (wt) and mrp mutant strains of B. subtilis to growth inhibition by cholate. Cells were grown in TKM medium (pH 7.0) in the presence (hatched bars) or absence (open bars) of 0.08% (wt/vol) cholate. After 6 h of incubation with shaking at 30°C, the A600 was determined. The results represent the mean of at least eight determinations, and standard deviations are shown as error bars.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Cholate efflux from whole cells of wild-type and selected mrp mutant strains of B. subtilis. The cells were starved and loaded with 20 µM [14C]cholate. Efflux was initiated by diluting 100-fold into buffer containing 10 mM glucose. Samples were taken at various times, filtered, and washed. The radioactivity was determined by liquid scintillation counting. (A) mrpF mutant VKIF and mrpG mutant VK1G, both without (solid symbols) and with (open symbols) the deleted gene expressed from an IPTG-inducible promoter in the amyE locus. (B) Wild-type strain, the mrp null mutant VKN1, and VKN1 expressing mrpF under control of an IPTG-inducible promoter in the amyE locus.

View larger version (10K): [in this window] [in a new window]   FIG. 5.   22Na+ efflux from wild-type (wt) B. subtilis, the mrp null strain VKN1, and VKN1 expressing mrpF in trans. The cells were washed, energy depleted, and loaded with 5 mM 22NaCl as described previously (9). Efflux was initiated by diluting the cell suspension 100-fold into buffer containing 5 mM NaCl with no further additions () or in the presence of 10 mM glucose (). Samples were taken at various times, filtered, and washed. The radioactivity was determined by liquid scintillation counting.

Na⁺ and alkali- sensitivity of the mrp mutants. The MIC of Na⁺ was determined in the wild type (BD99), in the mrp null strain (VKN1), in each of the single mrp gene deletion strains, and in each of the deletion strains with the gene restored in the amyE locus. The determinations were made at pH 7.0 and at pH 8.3 as described in Materials and Methods. The two pHs were examined because of the role of the Na⁺/H⁺ antiport in both Na⁺ and alkali resistance and because Na⁺ cytotoxicity is elevated at alkaline pH (14, 20). A record was kept of the A₆₀₀ after 15 h of growth in the absence of added Na⁺ at each pH (i.e., the same time at which growth was recorded for Na⁺-containing cultures) as an indicator of cell yield of each mutant at the two pHs. As shown in Table 2, all of the mrp mutants were from 6 to >10 times more sensitive to Na⁺ than the wild-type strain at pH 7.0, and there were no major differences in the absorbances reached by the different mutants and the wild type in the absence of added Na⁺. Restoration of the mutated gene to each of the single deletion mutants resulted in complementation in trans that resulted in essentially wild-type MICs for each of the mutants. At pH 8.3, the patterns were more complex. First, the mrpA and mrpD mutants, VK1A and VK1D, respectively, exhibited poor growth at the elevated pH in the absence of added Na⁺. This deficit was especially pronounced with VK1D, in which reintroduction of the gene in the amyE locus did not completely restore wild-type levels of growth. Second, all of the mutants exhibited at least 10-fold-greater sensitivity to Na⁺ than the wild type at pH 8.3, but the sensitivity of the mrpA, mrpB, mrpD, and mrpE mutants (VK1A, VK1B, VK1D, and VK1E, respectively) was reproducibly greater than that of the other three strains, with VK1D being the most Na⁺ sensitive of all the mutants. The most sensitive strains were also those that were not completely complemented up to wild-type levels upon restoration of the affected gene in trans.

Effects of expression of individual mrp genes in trans on the Na⁺ resistance of VK6 and VKN1 mutant strains. Unlike cholate resistance, which depended upon the status of a single mrp gene, wild-type levels of Na⁺ resistance clearly depended upon the status of multiple mrp genes. Previous work had shown that expression of mrpA from an IPTG-inducible promoter in the amyE locus of mutant strain VK6 led to a significant increase in the Na⁺ resistance as well as the ²²Na⁺ efflux activity of that strain. VK6 has a disrupted mrpA and reduced expression of mrpB through -G (9). No such increase in either resistance or efflux was observed upon expression of mrpA in the mrp null strain VKN1. We sought to assess whether MrpA is the likeliest candidate for the Na⁺-translocating protein in Mrp-dependent Na⁺/H⁺ antiport, albeit dependent in some manner on all the other mrp gene products as well. Each mrp gene was individually expressed in VK6 (VK6/mrpA through VK6/mrpG) and VKN1, and the MIC of Na⁺ was determined pH 7.0 and 8.3. As expected from earlier assays (9), expression of mrpA and mrpF increased the resistance of strain VK6 to Na⁺. Only mrpB expression among all the remaining mrp genes caused a significant increase in resistance and that was restricted to pH 7.0 (Table 3). In the mrp null strain VKN1, no single mrp gene caused a significant, reproducible increase in Na⁺ resistance (data not shown).

Energy-dependent efflux of ²²Na⁺ from right-side-out membrane vesicles of B. subtilis wild-type and selected mrp mutant strains. Prior studies of mrp-dependent Na⁺ fluxes had been conducted on respiring whole cells, in which significant cyanide- and protonophore-dependent inhibition was observed, especially for efflux from cells of mutant VK1A, in which mrpA expression was induced in trans (9). Reproducible data showing that an imposed diffusion potential could also energize such efflux had not been obtained in the whole-cell system. In order to further explore an MrpA-dependent capacity for secondary Na⁺/H⁺ antiport, right-side-out membrane vesicles of the wild-type and selected mutant strains were assayed for MrpA- and energy-dependent ²²Na⁺ efflux. Ascorbate-PMS was found to support significant enhancement of the rate of ²²Na⁺ efflux from vesicles prepared from the wild type and from VK1A/mrpA but only slight enhancement of efflux from the control preparations from the null VKN1 or VK1A strains (Fig. 6). The protonophore CCCP abolished the ascorbate-PMS-dependent efflux. The four preparations were then examined at a range of imposed valinomycin-mediated K⁺ diffusion potentials, with points taken during the initial efflux period under each condition. The percent ²²Na⁺ remaining at 5 s is plotted in Fig. 7 for each preparation as a function of the magnitude of the imposed diffusion potential. The difference among the preparations correlated well, especially at the lowest diffusion potential established, 60 mV, with the ascorbate-PMS-dependent pattern. That is, Na⁺ efflux from the wild type and VK1A/mrpA was significantly greater than that from VKN1 or VK1A. At higher imposed potentials, efflux via some other transporter may be a more dominant contributor to the observed pattern.

View larger version (11K): [in this window] [in a new window]   FIG. 6.   Ascorbate-PMS-dependent 22Na+ efflux from right-side-out membrane vesicles of wild-type B. subtilis (BD99), mrp null mutant VKN1, mrpA mutant VK1A, and VK1A to which mrpA is restored in the amyE locus. The vesicles were passively loaded overnight at 4°C with 5 mM 22NaCl in 100 mM potassium phosphate (pH 7.5) plus 5 mM MgSO4. For assays of Na+ efflux, vesicles (100 µg of protein/ml) were incubated at 10°C. No further additions were made (), or 10 mM potassium ascorbate plus 0.1 mM PMS was added in the absence () or presence () of 10 µM CCCP. Samples were taken at various times and rapidly filtered. The radioactivity on the filters was measured by liquid scintillation counting.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   Efflux of 22Na+ from right-side-out membrane vesicles of wild-type B. subtilis (BD99) and strains VKN1, VK1A, and VK1A/mrpA as a function of the magnitude of an imposed potassium diffusion potential. Membrane vesicles were loaded with 22Na+ as described in the legend to Fig. 6 in buffer containing 100 mM potassium phosphate and 10 µM valinomycin. These vesicles were diluted to different extents into 50 mM Tris-HCl (pH 7.5), in order to generate diffusion potentials of different magnitudes (indicated on the bottom of the figure). Samples were taken at 5 s after dilution and rapidly filtered. The radioactivity was determined by scintillation counting.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The mrp operon and its homologues are widely distributed among diverse prokaryotes and function in multiple processes involving ion-coupled transport reactions. Thus far, only one of these operons, the mrp operon of B. subtilis, has been shown to catalyze different transport reactions that relate to different gene products within the operon. The major finding of the current study is that MrpF can function in cholate and Na⁺ efflux independently of any other mrp gene product, whereas MrpA-dependent Na⁺/H⁺ antiport activity and Na⁺ resistance are highly dependent upon other mrp gene products, probably requiring all six of them. MrpA is a strong candidate for a major, if not sole, structural gene for Mrp-encoded Na⁺/H⁺ antiport, since the antiport activity of mrpA mutant VK1A, which has elevated levels of all the remaining mrp genes, has low Na⁺ resistance and efflux activities. In addition, mrpA overexpression in mutant VK6 (polar) elevates the Na⁺ resistance of that strain more than overexpression of any of the other mrp genes.

We tentatively hypothesize that the Na⁺ efflux catalyzed by MrpF is coupled to solute efflux (e.g., endogenous cholate-like substrate and/or exogenous cholate-like compounds) rather than being a true Na⁺/H⁺ antiport mode of this independent transporter. However, the assays conducted to date of MrpF-dependent cholate and Na⁺ efflux have not shown that there actually is coupling between the two substrates. In the whole cells in which the assays have thus far been conducted, there are complications of high contaminating Na⁺ levels, multiple antiporters apart from mrp, and the potential presence of an endogenous substrate that could substitute for preloaded cholate. Attempts to assess coupling of cholate efflux by MrpF in vesicles from the current B. subtilis strains were not undertaken because of their substantial Na⁺/H⁺ antiport activities as well as difficulties that we have had in making good everted vesicle preparations from these strains. Future studies will attempt to clarify the possible Na⁺-cholate coupling in everted vesicles from appropriate Escherichia coli strains if, as is now anticipated, expression of MrpF alone yields an active transporter. Moreover, attempts will be made to identify a transport activity for additional mrp gene products, especially MrpB and MrpC.

The basis for the MrpB-dependent increase in the Na⁺ resistance of strain VK6 at pH 7.0 is of interest. The Block+ program for motif analysis (4) does not provide useful clues vis à vis a specific MrpB transport activity, but indicates that MrpC has some similarities to Na⁺-coupled organic acid transporters. If there were one or more additional contributors to overall Mrp-dependent Na⁺ efflux, that would explain why mrpA and mrpF mutants have slightly but reproducibly higher Na⁺ resistance than the mrp null strain VKN1 at pH 7.0. No attempt can be made to interpret the differences in MIC at pH 8.3, since the levels of Na⁺ that are toxic to the mrp mutants at that pH are already in the range of contaminating Na⁺, and thus assessments of differences are unlikely to be accurate. The striking growth deficit of mrpA and mrpD mutants in the absence of added Na⁺ at pH 8.3 might in fact reflect a much greater Na⁺ sensitivity, effected by contaminating levels, in these strains. The evident importance of mrpD at high pH is intriguing, especially since its overexpression does not increase the Na⁺- resistance of polar mrpA strain VK6, and thus it is unlikely to be an antiport protein itself.

The studies of MrpA-dependent Na⁺ efflux in the right-side-out vesicles of VK1A/mrpA support the earlier indication from whole-cell assays (9) that the Na⁺/H⁺ antiporter can function as a secondary, proton motive force-dependent antiporter. However, although not shown, we were unable to demonstrate efflux at pH 8.0 and 8.3 in these vesicles even though Mrp-dependent Na⁺ efflux in malate-utilizing cells is clearly an important function at this pH. The possibility of a primary coupling mode for Mrp-dependent Na⁺/H⁺ antiport, using redox energy, is underscored by the importance and efficacy of the Mrp system in cells at elevated pH; by the complex dependence of the antiport on multiple mrp gene products; by the importance of MrpD at elevated pH; and by the strong sequence similarity between several mrp gene productsespecially MrpA, MrpB, MrpE, and MrpDto hydrophobic subunits of energy-coupled NADH dehydrogenase and to regions of other redox proteins (as analyzed via BLAST [1] and Block+ [4]). To explore possible primary energization, we are undertaking studies of the Bacillus mrp operons expressed in various E. coli strains. If a redox-dependent activity and complex formation are supported, it will be important to identify the electron donors and acceptors which would facilitate any subsequent efforts to study a purified Mrp complex.

Another final set of observations of interest in the current study emerge from the Northern analyses. Evidently, deletion of any of the mrp genes results in a significant increase in mrp expression, with the effect being somewhat smaller in mrpC and mrpF mutants. A simple interpretation would be that a rise in cytoplasmic Na⁺ leads to the overexpression. Even if correct, there are many features left to elucidate, including the basis for the difference between the effect in mrpC and mrpF mutant strains as well as the elements and mechanism of the putative regulatory effect. Another finding that will merit further exploration is that expression of maeN, which encodes an Na⁺-malate symporter, is partially coordinated with expression of mrp except that its basal expression is much greater and lower rather than greater levels of maeN RNA are observed in the mrpA mutant VK1A. The greatly reduced levels of maeN RNA in the VK1A strain may reflect disruption of some feature in the regulatory interaction between maeN and mrp that is lost during construction of the mrpA deletion. The regulatory coupling of maeN and mrp suggests the importance under at least some growth conditions of coordinating a full Na⁺ cycle. Such a cycle would encompass Na⁺/malate symport and Na⁺ reextrusion in exchange for H⁺ and also coupled to (MrpF-dependent) efflux of external chaotropes (e.g., cholate) and perhaps additional metabolic by-products. Under alkaline pH conditions, such an Na⁺-coupled cycle could be particularly important for achieving both substrate uptake and cytoplasmic pH regulation, and its efficacy would be enhanced if a primary, redox-coupled energization option existed for one or more of the efflux activities.