©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
MH1, A Second-site Revertant of an Escherichia coli Mutant Lacking Na/H Antiporters (nhaAnhaB), Regains Na Resistance and a Capacity to Excrete Na in a t;ex2html_html_special_mark_amp;mgr;-independent Fashion (*)

(Received for publication, September 1, 1994; and in revised form, November 3, 1994)

Michal Harel-Bronstein Pavel Dibrov Yael Olami (§) Elhanan Pinner Shimon Schuldiner Etana Padan

From the Division of Microbial and Molecular Ecology, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The Escherichia coli mutant DeltanhaADeltanhaB (EP432), which lacks the two specific Na/H antiporter genes, is incapable of efficiently excreting Na. Accordingly at low K (6 mM) medium, its intracellular Na concentration is only slightly lower (1.5-2times) than the extracellular concentration (50 mM), explaining the high sensitivity to Na (geq30 mM) of the mutant. This Na sensitivity is shown to be a powerful selection for spontaneous second-site suppressor mutations that allow growth on high Na (leq0.6 M) with a rate similar to that of the wild type. One such mutation, MH1, maps at 25.7 min on the E. coli chromosome. It confers Na but not Li resistance upon DeltanhaADeltanhaB cells and exposes a Na-excreting capacity, maintaining a Na gradient of about 8-10 (at 50 mM extracellular Na), which is similar to that of the wild type. Although lower, Na excretion capacity is also observed in the DeltanhaADeltanhaB mutant when grown in medium containing higher K (70 mM). This capacity is accompanied with a shift in the sensitivity of the mutant to higher Na concentrations (geq300 mM). Whereas Na excretion by a wild type carrying Deltaunc is uncoupler sensitive, that of MH1Deltaunc is dependent on respiration in an uncoupler-insensitive fashion. It is concluded that under some conditions (high K in the medium or in MH1-like mutants), a primary pump driven by respiration is responsible for Na extrusion when the Na/H antiporters are not active.


INTRODUCTION

Na/H antiporters are essential for adaptation to Na during growth since Escherichia coli mutants lacking the Na/H antiporters are sensitive to Na (Padan et al., 1989; Pinner et al., 1993). In accordance, upon transformation of the mutant strains with multicopy nhaA or nhaB plasmid, the wild type capacity to grow in the presence of Na is restored.

The pattern of regulation of nhaA studied in a strain carrying nhaA`-`lacZ protein fusion reflects the importance of nhaA in salt adaptation (Karpel et al., 1991; Rahav-Manor et al., 1992). Its expression, which is positively regulated by nhaR, is induced by Na. The induction is increased with pH similar to the Na sensitivity of DeltanhaA (Padan et al., 1989).

In view of the Na sensitivity found in the antiporter mutants (DeltanhaA or DeltanhaADeltanhaB), it can be anticipated that unless other Na-expelling machinery exists and functions in the absence of the antiporters, Na will accumulate in the cells to growth inhibitory levels. Primary Na pumps were demonstrated in various bacteria (Dimroth, 1992a, 1992b; Tokuda and Unemoto, 1985; Heefner and Harold, 1982; Takase et al., 1993). Furthermore, based on studies with intact cells (Dibrov, 1991; Skulachev, 1984, 1988) and isolated membrane vesicles (Avetisyan et al., 1989, 1993), it has also been implied that primary Na pumps exist in E. coli. Therefore, the DeltanhaADeltanhaB mutant affords new approaches to the search for genes and proteins involved in Na-excreting machinery other than that of the specific Na/H antiporters. Given that there exist nonspecific antiporter systems or pumps with low affinity and/or V(max) to Na, amplification of the respective genes by their cloning on multicopy plasmid may confer some Na resistance upon transformation into DeltanhaADeltanhaB. Indeed, with this approach, chaA has been cloned (Ivey et al., 1993). On the basis of its homology to calsequestrin and its promotion of Ca/H exchange activity in membranes, ChaA appears to be a Ca/H antiporter with low affinity to Na.

The Na sensitivity of the DeltanhaADeltanhaB strain provides yet another approach to look for systems conferring Na resistance (a powerful selection (growth on Na) of second-site revertants resistant to Na). The mechanism of resistance of such mutants should bear upon new Na-excreting machinery and/or other mechanisms relevant to Na resistance and to the identification of their genes.

We show that DeltanhaADeltanhaB is Na sensitive and defective in Na excretion. This Na sensitivity has provided a strong selection for the isolation of a spontaneous second-site revertant, which bears a mutation at locus 25.7 min on the E. coli chromosome. The mutation confers Na but not Li resistance and exposes a Na excretion capacity, which is driven by respiration but not by Deltat;ex2html_html_special_mark_amp;mgr;.


MATERIALS AND METHODS

Bacterial Strains and Culture Conditions

The bacterial strains used in this study are all E. coli K12 derivatives described in Table 1. TA15Deltaunc was constructed by P1 transduction of Deltaunc702 using TA3952 as a donor and TA15 as a recipient strain. MH1Deltaunc was constructed in the same way as TA15Deltaunc using MH1 instead of TA15 as a recipient strain. The Tet-resistant extransductants grew in minimal medium on glucose but not on succinate. The UE5 was kindly provided by W. Boos (University of Konstanz). The Hfr strains used for mapping were CAG5051, CAG5052, CAG5053, CAG5054, CAG5055, CAG8209, and CAG8160.



In several experiments, cells were grown in modified L broth (pH 7.5) from which concentrations of K and/or Na were modified and indicated, respectively, in mM by a number (n, m) following the atomic symbol (LBKnNam). Thus, LBK70Na7 contains 70 mM KCl, and although Na is not added, it contains 7 mM Na (the endogenous contaminating Na as measured by atomic absorption); LBK6Na300 contains 6 mM endogenous contaminating K (as determined by atomic absorption) and 300 mM NaCl, etc. In other experiments, the L broth medium was further modified by the addition of 50 mM 1,3-bis(tris[hydroxymethyl]methylamino)-proprane (pH 7.5) and 50 mM glucose and the K or Na concentration indicated as above (LBBGKnNam).

Cells were also grown in two minimal media. Medium A (Davies and Mingioli, 1950) was used without sodium citrate but supplemented with thiamine (2.5 µg/ml) and the carbon source 10 mM melibiose or 10 mM glucose as indicated. When required, threonine (0.1 mg/ml) and/or thiamine (0.1 mg/ml) were added. The other minimal medium was GMM containing 30 mM K(2)HPO(4), 50 mM NaCl, 50 mM 1,3-bis(tris[hydroxymethyl]methylamino)-proprane (pH 7.5), 7.5 mM (NH(4))(2)SO(4), 1 mM MgSO(4), and 50 mM glucose. For plates, 1.6% agar was used. Concentrations of antibiotics were 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 12.5 µg/ml tetracycline. Resistance of cells to Na was tested by determining colony-forming units on unmodified L broth plates containing various concentrations of Na. Conjugation and P1 transduction were conducted as described (Miller, 1992).

Plasmids

Plasmid pGM36 is a pBR322 derivative bearing nhaA (Karpel et al., 1988). pEL24 is a pUC18 derivative that carries nhaB (Pinner et al., 1992). pSE330 is a pSE420 (Brosius, 1992) derivative bearing chaA (Ivey et al., 1993).

Everted Membrane Vesicles and Measurement of Na/HAntiporter Activity

Everted membrane vesicles were prepared (Goldberg et al., 1987) essentially as described by Rosen(1986). Na/H antiporter activity in everted membrane vesicles was estimated based on its ability to collapse a transmembrane pH gradient. Acridine orange fluorescence was monitored to estimate DeltapH, as previously described (Goldberg et al., 1987). The fluorescence was monitored in a Perkin-Elmer fluorimeter (Luminescence Spectrometer, LS-5). Exciting light was 490 nm, and emission light was measured at 530 nm.

Protein concentration in membrane vesicles was determined as described (Bradford, 1976).

Determination of Intracellular Na

The method of Borbolla and Rosen(1984) was modified to determine the distribution of Na across the cytoplasmic membrane in growing rather than resting cells. Cells were grown (0.2-0.5 A) in LBBGK70 and the indicated Na concentrations. Aliquots of 0.4 ml were incubated in each of 24 wells of cell culture dishes (well diameter, 16 mm) (Costar Corp., Cambridge, MA) under agitation at 37° for 1 h in the presence of 0.5 µCi carrier-free Na. This time period was sufficient to attain a steady state of the Na across the membrane (not shown). Cells were then filtered on filter membrane (Schleicher and Schuell, 0.45-µm diameter) and washed with 4 ml of prewarmed fresh growth medium; radioactivity was then measured with a -counter (GAMMAmatic, Kontron). Toluenized cells were used to subtract nonspecific absorption of the isotope. [Na] was calculated from values obtained in a parallel experiment for the amount of radioactivity in deenergized cells, i.e. incubated with 50 µM CCCP (^1)under anaerobic conditions (covered with 0.4 ml of light mineral oil (Sigma)) in which Na is equilibrated across the membrane. For calculation of intracellular Na, the value of intracellular volume was made by interpolation at the appropriate osmolarity using the calibration curve described in Castle et al.(1986). All experiments were carried out in quadruplicate. Proteins of intact cells were quantitated as described (Lowry et al., 1951).


RESULTS

K Improves the Growth Rate of EP432 (DeltanhaADeltanhaB) and Decreases Its Na Sensitivity

As previously shown (Pinner et al., 1993), the mutant EP432, DeltanhaADeltanhaB, grows in LBK70Na7 medium at a growth rate that is 50-60% slower than that of the wild type strain (TA15). In similar medium supplemented with buffer (pH 7.5) and glucose (LBBGK70Na7), the rate of both strains increases so that the difference in their growth rates is maintained (Fig. 1, A and B). However, the growth rate of the mutant (Fig. 1B) but not of the wild type (Fig. 1A) is reduced by 2-fold if no K is added to the rich medium, which already contains 6 and 7 mM endogenous K and Na, respectively (LBBGK6Na7). We therefore reexamined the Na sensitivity of EP432 in high K medium (LBBGK70) as opposed to low K medium (LBBGK6) (Fig. 1). Whereas the Na sensitivity of the wild type is identical in both media (Fig. 1A), the Na sensitivity of the mutant is shifted to higher Na concentrations in the presence of the high K (Fig. 1B). Osmolites (100 mM) like betaine, trehalose, and sucrose could not replace K in improving the growth rate of DeltanhaADeltanhaB even without added Na (not shown). Therefore, the effect of K cannot be ascribed to its osmotic activity.


Figure 1: High K improves the growth rate and decreases the Na sensitivity of the DeltanhaADeltanhaB mutant. Wild type (TA15) (A), DeltanhaADeltanhaB (EP432) (B), and MH1 (C) cells were grown in LBBGK6-based medium (circle) or in LBBGK70-based medium (bullet) in the presence of various concentrations of Na. Note that the lowest [Na] = 7 mM, the contaminating Na.



Isolation of Na-resistant Second-site Revertants of DeltanhaADeltanhaB

When transformed with multicopy plasmid pGM36 bearing nhaA, the Na sensitivity of DeltanhaADeltanhaB is totally relieved (Fig. 2A). We have therefore presumed that this Na-sensitive phenotype provides a powerful selection to search for Na resistance conferring systems in the DeltanhaADeltanhaB strain. Indeed, if the culture inocculated in LBK6Na300 with DeltanhaADeltanhaB cells is further incubated in the presence of the salt, growth of apparently spontaneous second-site revertants is observed after about 30 h of incubation (Fig. 2A).


Figure 2: MH1 mutation confers Na but not Li resistance. A, cells EP432/pGM36 (circle) or EP432 (bullet), as indicated, were grown in LBK70Na7 (pH 7.5) to the logarithmic phase and at zero time diluted (1:100) in the medium containing 0.3 M NaCl (LBK6Na300). B-D, cells (TA15, box; EP432, bullet; and MH1, ) were grown in LBK70Na7 (pH 7.5) as above and at zero time diluted (1:100) at the same pH in LBK70Na7 (B), LBK6Na300 (C), or (LBK6Li100) (D).



The salt-resistant phenotype (0.6 M NaCl) of these mutants is stable even after several transfers on medium devoid of added Na (LBK70Na7). As expected for a genotype derived from EP432, these cells are NhaANhaBThrLacKanCM. The frequency of spontaneous appearance of such mutants is about 1/10^7 cells. One mutant designated MH1 was isolated for further study.

Growth Phenotype of MH1

The growth phenotype of MH1 at pH 7.5 as a function of various Na concentrations is shown in Fig. 1and Fig. 2. In LBK70Na7, wild type and MH1 grow with identical rates while the growth rate of EP432 is somewhat slower (Fig. 2B). In LBK6Na300, EP432 is inhibited (Pinner et al., 1993 and Fig. 2A), but MH1 grows like the wild type (Fig. 2C). At further higher Na concentrations both in LBK70 (not shown) or LBBGK70 media, the growth of MH1 is only slightly inhibited as compared with the wild type above 0.6 M NaCl (Fig. 1, A and C). Neither pH (7.5-8.5) nor carbon sources (glycerol, glucose, or succinate tested in minimal medium) affected the salt tolerance of MH1.

In marked contrast to the significant Na resistance conferred by the MH1 mutation, this mutant remains as sensitive to Li as EP432 (Fig. 2D). Thus, MH1 is a mutation that specifically affects Na resistance.

Mapping of MH1

To map the MH1 mutation by conjugation, we used MH1 (Cam^R Kan^R) as recipient cells, while the donors were E. coli Hfr strains that have their start transfer at different points on the genetic linkage map and carry a Tn10 insertion in close proximity to their origin of transfer. After crossing for 80 min, Tet^R Cam^R Kan^R exconjugants were selected and screened for the capacity to grow on salt (0.4 M NaCl at pH 7.5 inhibits both DeltanhaA (NM81) and DeltanhaADeltanhaB (EP432) but not MH1). Of the seven different crosses, in only one (with the donor CAG5054-KL96) 120 out of the 330 exconjugants lost their Na resistance (Na^R). This frequency suggests that the mutation is located on the side of the Tn10 (27.7 min), remote from the entry site of the donor chromosome (49 min).

Since all of these exconjugants were Cam^R (in the recipient chromosome, 25.6 min) (Pinner et al., 1992), it is suggested that the mutation is localized between 25.6 and 27.7 min. It should be noted that 10 out of 11 other MH1-like mutations, isolated in the same way and shown to have similar growth phenotype, were found by conjugation to map in the same region.

To define the position of MH1 more closely, we used P1 transduction (Table 2). When the donor strain was the exconjugant (obtained in one of the crosses described above) Kan Cam Tet Na^R (DeltanhaA::kanDeltanhaB::cattrp::Tn10 MH1) and the recipient strain NM81 (DeltanhaA1::kannhaBMH), Cam^R or Tet^R transductants were selected, and each scored for Na resistance. Out of 253 Cam^R transductants, 199 were Na resistant, and out of 410 Tet^R colonies, 54 were Na resistant. None of the transductants were both Cam^R and Tet^R. The cotransduction to each marker suggests map location between 25.8 and 26.5 min.



When tre::Tn10 was the donor strain and the recipient was MH1, Tet^R transductants were selected, and each scored for Cam^R and Na resistance. Out of 266 Tet transductants, 193 became Cam^S as expected from the distance between nhaB::cat (25.6 min) and tre::Tn10 marker (26 min). Since not all Tet^R transductants are Na^S, it is apparent that the MH1 mutation does not reside in tre. Furthermore, out of the 266 Tet^R transductants, 173 became Na^S (Table 2), suggesting that MH1 is located in close proximity to tre (within 0.3 min).

To determine on which side of tre MH1 is located, an extransductant of the latter transduction, DeltanhaA1::kanDeltanhaB1::catMH1tre::Tn10 (Kan^R Cam^R Na^R Tet^R), was used as a donor strain and NM81 as an acceptor. All tetcat transductants were also Na^R as expected from the location of MH1 between tre and nhaB. Hence, MH1 maps at 25.7 min on the E. coli chromosome.

There Is No Specific Na/HAntiporter Activity in MH1 Mutant

EP432 is devoid of the specific Na/H antiporters of E. coli (Pinner et al., 1993), and therefore membrane vesicles isolated from this strain show only the nonspecific K/H antiporter activity (Pinner et al., 1993), and the ionophore monensin collapses the DeltapH created by these membranes (Fig. 3). It is thus conceivable that the MH1 mutation affects the K/H antiporter or other transporters so that Na excretion is resumed by an alternative Na/H exchanging system. However, whether grown in the presence or absence of added Na (0.3 M) in LBK6 or LBBGK70 media, there is no measurable Na/H antiporter activity in membrane vesicles isolated from the MH1 cells (Fig. 3), and the activity of the K/H antiporter in these vesicles is identical to that of EP432 (not shown). Upon transformation with multicopy plasmid bearing nhaA (pGM36, Fig. 3) or chaA (pES330, not shown), MH1 transformants, like EP432 transformants, show Na/H exchange activity.


Figure 3: Membrane vesicles isolated from MH1 do not exhibit Na/H antiporter activity. Everted membrane vesicles were isolated from cells grown in LBK70Na7; EP432, MH1, or MH1 transformed with pGM36, a multicopy plasmid bearing nhaA (Karpel et al., 1988). DeltapH was monitored with acridine orange in a medium containing 140 mM potassium chloride, 10 mM tricine (pH 8), 5 mM MgCl(2), acridine orange (0.5 µM), and membrane vesicles (50 µg of protein). At the onset of the experiment, Tris-D-lactate (5 mM) was added, and the fluorescence quenching was recorded. NaCl (10 mM) was then added, and the new steady state of fluorescence obtained (dequenching) was monitored. When indicated, monensin (1 µM) was added.



The Capacity of the Wild Type, MH1, and DeltanhaADeltanhaB Strains to Maintain a NaGradient at Various Concentrations of Extracellular Na

Since both EP432 and MH1 lack the Na/H antiporters, yet MH1 is Na resistant, it was crucial to determine in these strains the intracellular Na concentration, under various growth conditions, as compared with the wild type (Fig. 4). For this purpose, cells were first grown to the logarithmic phase of growth (A = 0.25) in LBBGK6Na50 (Fig. 4, A and 4B), a medium in which DeltanhaADeltanhaB still grows albeit 3-4 times slower than both the wild type and MH1 (Fig. 1). Then, Na was added (zero time), and measurement of intracellular Na commenced after a steady state level of the label had been established (at 1 h). The results show that within the following 2 h of the experiment, through which logarithmic growth continues, TA15 maintains a low concentration of intracellular Na (5 mM) and thus a Na gradient of 10 (Fig. 4A). On the other hand, EP432 grows 3-4 times slower, its intracellular Na concentration is 6 times higher (28 mM), and thus its gradient is less than 2 (Fig. 4B). Most interestingly, MH1 maintains a low intracellular Na of about 8 mM, only slightly higher than that of the wild type and therefore a substantial Na gradient of 6 (Fig. 4B). Very similar data were obtained when the experiment was conducted in minimal medium (GMM) (not shown).


Figure 4: MH1 but not EP432 maintains a low concentration of intracellular Na at low K medium. TA15 (box), EP432 (circle), or MH1 (up triangle) cells were grown to the logarithmic phase (A = 0.25) in LBBGK6Na50, Na (0.5 µCi, carrier free) was added at zero time, and measurement of intracellular Na commenced at 1 h, after a steady state level of the label had been established. The calculated intracellular Na quantity and concentrations are shown. Opensymbols, no additions; closedsymbols, anaerobic conditions in the presence of 50 µM CCCP.



Since the presence of 70 mM K in the rich medium increased the growth rate of DeltanhaADeltanhaB with no effect on either the wild type or MH1 strain (Fig. 1), we also measured the Na gradient maintained by the different strains in the high K medium (LBBGK70) at various [Na] (Fig. 5). It is apparent that, up to 400 mM Na, TA15 maintains a relatively constant gradient of 15-20-fold, which results in a low [Na]. MH1, up to 350 mM [Na], also maintains a gradient of 10-11-fold, which is only slightly lower than that of the wild type. Beyond this concentration, [Na] of MH1 increases much more drastically than that of the wild type. At 400 mM [Na], the [Na] of these strains are 20 and 80 mM, respectively. It was impossible to measure accurately [Na] at still higher extracellular Na concentrations.


Figure 5: The capacity of the wild type, DeltanhaADeltanhaB, and MH1 to maintain a Na gradient at various extracellular Na concentrations. TA15 (), EP432 (bullet), or MH1 (circle) cells were grown in LBBGK70 (pH 7.5) medium. NaCl was added to the indicated concentrations 1 h before measurement together with Na. Internal Na was determined as described under ``Materials and Methods.'' Dashedline, [Na] = [Na].



In contrast to MH1 and TA15, intracellular concentration of DeltanhaADeltanhaB mutant increases linearly with a higher slope; at 50 mM [Na], the gradient was 3-5-fold (Fig. 5) and at 300 mM [Na], whereas [Na] of MH1 or TA15 is 10-20 mM, that of EP432 is above 100 mM (Fig. 5).

MH1 and EP432 Possess an Active Na-excreting Machinery

To study whether MH1 and EP432 have an active Na-excreting machinery, we tested the effect of inhibition of energy metabolism on the capacity of these strains to maintain a low intracellular concentration. Both in the wild type and the mutant strains, simultaneous applications of the uncoupler CCCP and anaerobic conditions led to total equilibration of Na (Fig. 4). Hence, MH1 like EP432 actively maintains its Na gradient, and even the small gradient seen in EP432 in low K-rich medium is energy dependent.

NaExcretion in MH1 Is Independent of Deltat;ex2html_html_special_mark_amp;mgr;but Dependent on Respiration

It is difficult to identify the driving force for active transport in a strain possessing the H/ATPase since the main forms of cell energy (oxidation energy, Deltat;ex2html_html_special_mark_amp;mgr;, and ATP) can be interconverted by this enzyme via Deltat;ex2html_html_special_mark_amp;mgr;.

To compare the pattern of energization of Na transport in MH1 with that of the wild type (TA15), we have deleted most of the H/ATPase genes of these strains (Deltaunc derivatives). Both MH1 Deltaunc and TA15Deltaunc (Fig. 6) maintained a similar Na gradient of about 5-6. Similar to previous results obtained by Borbolla and Rosen(1984) in another wild type-derived strain, addition of CCCP to TA15Deltaunc collapsed the Na gradient (Fig. 6A). Note that in the high K medium dissipation of the gradient by CCCP was faster than in the low K medium, suggesting that K serves as a counter-ion to the movement of Na across the membrane. This result agrees with the notion that Delta via the Na/H antiporters maintains the Na gradient in E. coli cells. Furthermore, this result shows that under these conditions, any Deltat;ex2html_html_special_mark_amp;mgr;-independent mechanism of Na excretion, if present, has a very weak activity.


Figure 6: Respiration but not Deltat;ex2html_html_special_mark_amp;mgr; drives Na excretion in MH1Deltaunc cells. A, TA15Deltaunc cells were grown in medium LBBGK6Na50 (box) or LBBK70Na50 (up triangle) and manipulated as described in Fig. 4with minor modifications needed for rapid inter-conversion between anaerobic and aerobic conditions. At zero time, 15-ml aliquots of mid-logarithmic phase cells were placed into 125-ml Erlenmeyer flasks, and Na was added; after 1 h of aerobic growth (with shaking) in the presence of the isotope, CCCP (final concentration of 50 µM) was added () (fullsymbols). B, MH1Deltaunc cells grown in LBBK6Na50 and manipulated as above. box, no addition , 50 µM CCCP added at 60 min (). For anaerobic cells (circle), immediately after addition of CCCP, cell suspension was transferred into a 50 ml-test tube and covered with 1 ml of the light mineral oil. 1 h later, the cell suspension was exposed to oxygen (indicated by an arrow) by rapid transfer into the original flask, and aerobic incubation continued with shaking (bullet).



In marked contrast to TA15Deltaunc, addition of CCCP to MH1Deltaunc had no effect on the Na gradient (Fig. 6B). This result is consistent with the lack of the Na/H antiporters in the latter strain and reveals that MH1 possesses a Na excretion machinery, which is independent of Deltat;ex2html_html_special_mark_amp;mgr;.

Total collapse of the Na gradient was obtained in MH1 by applying anaerobiosis in the presence of CCCP (Fig. 6B). Similar but somewhat slower decay of the gradient occurred under anaerobic conditions (not shown). The Na gradient was reestablished upon reaeration of the anaerobic experimental systems (Fig. 6B). We conclude that respiration drives Na excretion in MH1.


DISCUSSION

In this work, we show that MH1, a spontaneous point mutation in the mutant DeltanhaADeltanhaB (EP432) which lacks the two specific Na/H antiporters, renders Na resistance upon the cells and exposes a Na extrusion capacity that is independent of Deltat;ex2html_html_special_mark_amp;mgr; but driven by respiration.

The MH1 mutation was selected, without mutagenesis, by growth of the Na-sensitive EP432 cells on Na (0.3 M, pH 7.5) under conditions in which it is highly sensitive to Na (medium of low K (6 mM)). Similar selection previously allowed us to clone chaA, a putative Ca/H antiporter gene, from a DNA library prepared from and transformed into EP432 (Ivey et al., 1993). A recent study of a DeltachaA mutation in the genetic background of the wild type or the Na/H antiporter mutants (DeltanhaA, DeltanhaB, DeltanhaADeltanhaB) has shown that chaA may have a role in Na circulation at alkaline pH (pH 8.5) but not at neutral pH (Ohyama et al., 1994). Although both MH1 and chaA (Ivey et al., 1993) confer Na resistance but not Li resistance they are different loci and confer different phenotypes. MH1 maps at 25.7 min, whereas chaA maps at 27 min (Ivey et al., 1993) on the E. coli chromosome. In contrast to chaA, which apparently in multicopy increases the Na/H and the Ca/H antiporter activity of isolated membrane vesicles of both EP432 as well as MH1 transformants, we could not find any Na/H antiporter activity in MH1, and its Ca/H antiporter activity was identical to that of the wild type. MH1 was isolated at neutral pH and, in contrast to chaA, exerts its effect both at neutral and alkaline pH.

The spontaneous frequency of mutants, similar to MH1 in growth phenotype, is high (1:10^7cell). Interestingly, out of the 11 independently selected mutations analyzed, 10 were localized to the MH1 region. The ease in selection of the MH1-like mutations reflects the very strong selection pressure imposed on EP432 mutant by Na and the efficient capacity of MH1 to confer Na resistance.

Determination of Na distribution across the cytoplasmic membrane of the wild type cells (TA15) under growth conditions in low K medium (LBBGK6Na50, pH 7.5) shows a Na gradient of about 10 (Fig. 4A). Similar results were previously obtained with endogenously respiring resting cells of another wild type strain resuspended in buffer containing 80 mM Na and 5 mM K using NMR technology to quantitate intracellular Na ( Fig. 4in Castle et al., 1986; Pan and Macnab, 1990).

In marked contrast to the wild type cells, EP432 cells have very low capacity to maintain a Na gradient (Fig. 4B and 5). This result is consistent with the suggestion (West and Mitchell, 1974; Padan and Schuldiner, 1992, 1994) that the Na/H antiporters are the main Na extrusion machinery of wild type E. coli cells. As a result of the lack of the antiporters, the [Na] of EP432 is around 30 mM (at 50 mM [Na] in the low K medium), whereas that of the wild type is around 5 mM (compare Fig. 4, A and B). We therefore suggest that as opposed to the wild type that grows up to 0.8 M NaCl in this medium (Padan et al., 1989), this increase in intracellular Na is the cause of the dramatic growth inhibition of EP432 by extracellular Na (>30 mM) (Pinner et al., 1993).

Addition of K (to 70 mM), which does not affect the wild type growth (Fig. 1A), sensitivity to Na (Fig. 1A), or Na gradient (compare Fig. 4A with Fig. 5), increases the growth rate of EP432 even without added Na, and this effect seems to account for the shift in the Na sensitivity of EP432 toward higher concentrations observed in the high K medium (Fig. 1B). Most interestingly, concomitantly the addition of K improves the capacity of EP432 to maintain a Na gradient, and at 50 mM [Na], a gradient of 3-5 is maintained (Fig. 5). However, the capacity of EP432 to maintain a Na gradient is much lower than that of the wild type at increasing [Na]. At [Na] of 300 mM, [Na] of EP432 is 125 mM, as opposed to 15 mM of TA15. Therefore, it appears that by allowing certain capacity of Na excretion, the sensitivity of EP432 is shifted toward higher [Na] in the high K medium. Although as yet the mechanism of the effect of K is unknown, it may be related to a protective effect of the ion against the inhibitory effect of Na on many enzymatic reactions (Walderhaug et al., 1987).

Remarkably, MH1, even though it lacks both nhaA and nhaB Na/H antiporters, maintains a Na gradient of 5-8 (directed inward) in the presence of 50 mM [Na], as does the wild type ( Fig. 4and Fig. 5). Furthermore, up to 350 mM [Na], the gradient of MH1 is only slightly lower than that of the wild type and, only beyond it, decreases (Fig. 5). At 400 mM [Na], [Na] of MH1 and the wild type are 90 and 15 mM, respectively. Most interestingly, at 400 mM [Na], when [Na] concentration of MH1 reaches 90 mM, a difference in growth rate between MH1 and wild type becomes apparent (Fig. 1C). These results imply that the mutation MH1 exposes a Na export machinery, which at least up to 350 mM [Na] is similar in its capacity to that of the wild type.

Deltaunc strains cannot interconvert phosphate bond energy with the electrochemical proton gradient, allowing conditions to be established in which the sources of energy available for transport are both phosphate bond energy and an electrochemical gradient (during metabolism of glucose), only phosphate bond energy (glucose metabolism in the presence of uncouplers or respiratory inhibitors), or only an electrochemical gradient of protons or redox (during respiration of substrates of the electron transport chain). As previously shown by Borbolla and Rosen(1984), the driving force for Na extrusion in wild type E. coli (in our case TA15) is Deltat;ex2html_html_special_mark_amp;mgr;. Thus, TA15Deltaunc maintains a Na gradient like TA15, and the uncoupler CCCP collapses the gradient in TA15Deltaunc. In contrast to TA15Deltaunc, MH1Deltaunc maintains the Na gradient even in the presence of uncoupler. It could be argued that MH1Deltaunc is insensitive to uncouplers. This is highly unlikely since, as described above, the isogenic strain TA15Deltaunc is uncoupler sensitive. Furthermore, the collapse of the Na gradient in MH1 by anaerobiosis is accelerated in the presence of uncouplers. Hence, Na extrusion in MH1Deltaunc is not driven by Deltat;ex2html_html_special_mark_amp;mgr;. Since only anaerobiosis in the presence or absence of uncouplers collapsed the gradient, we suggest that the Na extrusion in MH1 can be directly coupled to electron transport.

This report demonstrates the presence of a respiration-dependent, Deltat;ex2html_html_special_mark_amp;mgr;-independent, Na-extrusion mechanism in E. coli. This mechanism is evident in MH1, a mutant devoid of both antiporters and carrying a mutation. We suggest that a similar, although weaker device is responsible for the Na extrusion detected in EP432, a mutant devoid of both antiporters but still capable of generating a small Na gradient in the presence of high K. The two above mutants allow us to describe a mechanism that in the wild type, if present, is masked by the activity of two antiporters: NhaA and NhaB.

MH1 can be a mutation in a Na pump itself, increasing its activity and/or expression. On the other hand, it is also possible that MH1 affects another system that is needed for expression or that limits the activity of the pump. In this respect, as described above, EP432 shows a limited Na extrusion capacity. Hence, it is possible that this low activity can be increased by induction, under conditions unfavorable to the H cycle (in the presence of protonophores or at alkaline pH) as suggested before (Avetisyan et al., 1989, 1992, 1993; Skulachev, 1984, 1988; Dibrov, 1991) or by MH1-like mutations as shown here.


FOOTNOTES

*
This work was supported by a grant from the Ministry of Science and Technology of Israel and the Geselschaft Fuer Biotechnologische Forschung-GBF (to S. S.) and by the Ministry of Science and Technology (to P. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
An Alberman Fellow.

(^1)
The abbreviations used are: CCCP, carbonyl cyanide m-chlorophenylhydrazone; [Na], intracellular concentration; [Na], extracellular concentration.


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