Altered cation coupling to melibiose transport in mutants of Escherichia coli.

The alpha-galactoside transport system of Escherichia coli utilizes either H+ of Na+ as a coupling cation for melibiose transport. Mutants were isolated which showed altered cation recognition for melibiose transport. The transport carrier of the mutants has lost the ability to accept H+ but can utilize either Na/ or Li+ for co-transport with melibiose. Such mutants might be predicted if the melibiose carrier were viewed as a descendant of an evolutionary transition between the H+-substrate co-transport systems of primitive cells and the Na+-substrate systems of more complex organisms. A second mutation was found in these mutants which involves the Na+ (Li+)-H+ exchange carrier. The maximal rate of Li+-H+ exchange in one of the mutants was 11 times higher than the parent and the Km for Li+ by this carrier was one-sixth that of the parent.

K' pump establishes a large Na' gradient and the co-transport systems use exclusively Na'. During evolution there must have been a transition between the "proton economy" of the primitive cell and the ''Na' economy" of the animal cell (1). It is reasonable to suppose that, during this transition period, a co-transport system existed that recognized both H' and Na' and that it later lost the capacity to utilize H'.
From an evolutionary point of view, it is therefore of interest to consider the unusual properties of the Escherichia coli melibiose membrane carrier which shows cation-sugar cotransport with either H' or Na' (2). If this carrier represents a descendant of a transport protein that appeared during the period of transition between H' and Na' membrane economies, one might predict that it would be possible to isolate by a simple mutation a carrier that recognizes only one of the two cations. This paper describes two mutants of E. coli in which the melibiose carrier (that normally utilizes either Hf or Na~') has lost the ability to recognize H' and developed an absolute * This research was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
8 To whom reprint request should be addressed. requirement for Na+ (or Li'). The properties of these two Na+(Li+)-requiring mutants are described.

EXPERIMENTAL PROCEDURES
Strains and Growth of Cells-The primary strain utilized in these studies was W3133-2, lac ZY deleted and possessing a temperatureresistant melibiose carrier (3). A plasmid from the Clarke-Carbon collection (4) carrying the me1 A (a-galactosidase) and me1 B (melibiose transport) genes was inserted into two transport mutants. The mating experiments were carried out as follows: W3133-2s (or W3133-2T) was grown in Tris medium (see below) containing melibiose and 10 mM LiCL Cells were diluted 100-fold and 0.1 ml spread on agar plates containing Tris medium plus 10 mM melibiose and 100 pg/ml of streptomycin. Onto this plate were streaked cells of JA200/pLC 25-33 previously grown on a nutrient agar plate. The donor fails to grow due to its sensitivity to streptomycin and amino acid requirements while mutants fail to grow in the absence of added Na' or Li'. The mating on the plate gave rise to colonies which were purified and tested for the presence of the plasmid. medium) consisting of 100 mM Tris-C1, pH 7.3, 1 m~ (NH4)2SO4. 1 For the following investigation, cells were grown in a medium (Tris mM MgHP04, plus 10 mM melibiose with or without 10 m~ LiCl and supplemented in some experiments with 1% Bacto-tryptone (Difco). Cells were grown at 37 "C and harvested at late exponential phase of growth.
Isolation of Mutants with Altered Cation Specificity-E. coli W3133-2 (3) were treated with a mutagen, N-methyl-N'-nitrosoguanidine (5). Treated cells were divided into 10 portions in order to isolate at least 10 independent mutants. Cells in 10 tubes were grown in nutrient medium, then inoculated into the Tris medium described above plus 10 m~ melibiose and 10 m~ LiCI. Potassium was not added to the medium to minimize the contamination of Na'. The final concentration of both K' and Na' in the medium due to impurities in the other salts were 2 to 3 p~. The growth rate and extent of growth was about half that observed in other minimal media. Since Li' inhibited H'-melibiose co-transport (2, 6), a mutant which can grow on melibiose as sole source of carbon in the presence of Li' should possess some alteration in the transport carrier. After 2 days of shaking at 37 "C in the Li+-containing medium, the culture medium became turbid. Cells were spread on 10 agar plates which contained various salts as described above (Tris medium plus 10 mM melibiose and 10 m~ LiCl) and 1.5% agar. Plates were incubated at 37 "C for 2 days, and the largest colonies on each plate were picked. Thus, we obtained 10 mutants which can grow on melibiose in the presence of Li'. Six of the mutants were found to require Li+ or Na' for growth when melibiose was added as a sole carbon source. The four other mutants grew either in the absence or in the presence of Li'. Two of such Li'(Na+)-dependent mutants (W3133-2S, W3133-2T) were studied in detail.
in the Tris medium supplemented with 1% Bacto-tryptone. The cells Preparation ofEverted Membrane Vesicles-The cells were grown were washed once with 10 m~ MOPS'-KOH, pH 7.2, containing 100 mM KCI, 5 mM MgS04, and 2 mM 2-mercaptoethanol, and resuspended in the same buffer containing 2 pg/ml of DNase to 5 volumes/ g of wet cells. The cells were disrupted by a passage through a French press cell (Ohtake Co., Tokyo) using a pressure of 20,000 p.s.i. The ' The abbreviations used are: MOPS, morpholinopropanesulfonic acid; TMG, methyl-b-D-thiogalactoside. resulting suspension was centrifuged at 11,000 X g for 10 min to remove unbroken cells and large cell debris. The supernatant solution was centrifuged at 105,000 X g for 1 h. The pellet was resuspended to give about 40 mg of protein/ml in the same buffer, and an equal volume of glycerol was added. As reported previously (7), glycerol is very useful to stabilize the vesicles. All steps were performed at approximately 4 "C. The vesicles were frozen in dry ice-acetone, and stored at -80 "C until use.
Measurements of H+ Movement and N u ' Movement-The pH change or pNa change of the extracellular medium resulting from transport of melibiose were measured as described previously (2) with slight modification. For pH measurements, cells were washed twice with 120 mM choline C1, and suspended in the same solution. For pNa measurement, experiments were carried out at various concentrations of NaCl ranging from 25 )AM to 10 mM. Anaerobic solutions of sugars were added to give a final concentration of 10 mM. pNa was measured with a Na'-selective electrode (Radiometer, Copenhagen). Initial velocity of the Na' entry was measured.

RESULTS
The Melibiose Carrier-An attempt was made to isolate a series of mutants with abnormal cation requirements for the melibiose transport system. Previous studies indicated that lithium-resistant mutants showed alterations in the cation recognition site of the membrane carrier (6). In this study, mutagenized cells were grown in liquid media containing melibiose as the sole carbon source in the presence of 10 mM Li' . The growth of parental strain was strongly inhibited but after 2 days mutant cells appeared in the culture. These survivors were plated on agar plates containing 10 mM Li' with melibiose as the sole carbon source. The largest clone on each plate was picked for further study. Ten independent mutants were tested for growth on melibiose in the presence and absence of Li'. Six of the mutants showed a Li' requirement for growth, four showed no effect of Li' . Fig. 1 compares the effect of Li' on the parental cell (W3133-2) and one of the mutant cells (W3133-2s). Lithium at a concentration of 10 mM completely inhibited the growth of the parental cell. The mutant on the other hand failed to grow unless Li+ was added to the growth medium. Sodium could replace Li' in the stimulation of growth of this mutant (data not shown). Five other mutants showed similar growth properties. Sodium at a concentration of 10 m~ caused a 40% inhibition of growth of the parental cell. Neither Na+ or Li' had an effect on the growth of the mutant cells when glucose, galactose, or amino acids were the sole source of carbon. The effect of Li' concentration on growth of the parent and the mutants was next investigated. The concentration of Li' which caused 50% inhibition of growth of the parental cell was approximately 0.02 mM (Fig. 2). The stimulation of growth in the mutants (W3133-2s and W3133-2T) required a very much higher concentration of Li+ (approximately 0.4 mM for half-maximal effect). The concentration of Na+ which gave half-maximal effect on the growth of the mutants was approximately 0.6 to 0.7 m~. Thus, Li' was slightly more effective than Na' in the stimulation of the growth of the mutants on melibiose.

Measurement of H' and Na' Movements on the Melibiose
Carrier-Proton movement in response to addition of melibiose was measured in washed cells suspended in an unbuffered solution. The pH of the external medium was continuously monitored on a chart recorder. In these experiments, the uptake of protons with melibiose on the carrier of the wild type cell resulted in an alkalinization of the medium (an upward deflection on the chart, Fig. 3A). This alkalinization process was followed by an acidification of the medium (downward deflection on the chart) due to the metabolism of glucose and galactose resulting from hydrolysis of the disaccharide. In contrast, the two mutants showed no initial alkalinization of the medium on melibiose addition but showed only continuous acidification due to metabolism of the hexoses. These experiments were carried out in the presence of 120 m~ choline chloride to avoid the presence of Na' or Li'. The experiment was repeated in the presence of 10 m~ LiCl (Fig. 3B). Lithium  : W3133-2 ( a ) , W3133-2s ( b ) , W3133-2T (c).
completely prevented the alkalinization of the medium in the parental cell (W3133-2), owing to its known inhibition of the membrane carrier (2, 6). Lithium had little effect on the acidification reaction arising from metabolism. In the case of the two mutants, the addition of melibiose in the presence of Li' resulted in a rapid acidification of the medium during the fist 15 s compared with the Li+-free experiment in A. This was followed by a metabolic acidification reaction. It was assumed that in the presence of Li' this cation entered the cell with melibiose on the carrier resulting in a membrane potential (inside positive) which provided a driving force for the exit of protons. A similar melibiose-induced proton exit was previously observed in the presence of sodium ions with wild type cells (2).
Another substrate of the melibiose carrier, TMG, was tested for its effect on proton movement in the parent and two mutant cells. Previous experiments have shown that TMG is transported by the melibiose carrier with Na' or Li+ but not protons (2,3,9). The addition of TMG to the two mutants (in the absence of Na' or Li') results in no proton movement (Fig. 4A). In the presence of 10 mM Lit or Na' the addition of TMG to the parental cell caused a marked acidification of the medium as a result of the exit of protons from the cell (Fig.  4B). This effect is believed to be due to a positive internal membrane potential due to the entry of Li' (or Na+) with TMG. It should be noted that TMG is not metabolized by these cells and thus does not result in the prolonged acidification seen with melibiose. The two mutants showed much less acidification on the addition of TMG in the presence of Li'. This is consistent with the view that TMG transport in these two mutants cannot couple to Li' effectively. Na'-TMG co-transport appears to be normal. Addition of 10 mM Na' stimulates TMG accumulation by W3133-2s 6-fold. Cation specificity of methyl-Lu-galactoside transport in the two mutants was also tested. No proton movement was observed when a-methylgalactoside was added in the absence of Na+ or Li'. On the other hand, proton efflux (acidification of the medium) was observed when the sugar was added in the presence of either Na' or Li+. When sugar was added to cells in the presence of Na+, this cation entered the cell as indicated by the fall in external Na' concentration (measured with a Na+-electrode).
It has been previously shown that in the presence of Na' melibiose enters the cells on the carrier with Na' (2, 9). The uptake of Na+ by the parent and mutant cells was measured with a sodium electrode. The addition of melibiose resulted in a prompt fall in the concentration of Na' in the external medium. The kinetic parameters for Na' stimulation of melibiose transport in the parent and mutant cells were similar. The parent showed a K, for Na' of 0. ng ions/min/mg of protein, while the mutant (W3133-2s) gave a K,,, of 0.4 mM and a V,,, of 19 (Fig. 5).
Additional evidence was sought to demonstrate that the mutation in the lithium-resistant mutant W3133-2s was indeed in the melibiose transport system. A plasmid carrying the melA melB genes (Clarke-Carbon collection) (4) was inserted into the mutant. In Fig. 6A the mutant shows a complete loss of proton entry on melibiose addition (H+melibiose co-transport), while the same cell carrying the plasmid (pLC 25-33) showed a restoration of proton co-transport.  Quenching a 9-aminoacridine fluorescence was assayed with membrane vesicles prepared by the French press. Addition of lactate produced quenching of fluorescence, following which varying concentrations of LiCl were added. The addition of LiCl caused the rapid increase in fluorescence as shown in Fig. 7. The initial rate of the fluorescence increase per s was calculated. Membrane vesicles used were: W3133-2 (O), W3133-25 (A), and W3133-2S/pLC 25-33 (m).
interest to assay the Na+(Li')/H' antiporter which is known to possess the capacity to extrude both Na' and Li' (8,11). The measurement of the Na+(Li')/H' antiporter activity was carried out with the 9-aminoacridine fluorescence technique. The internal pH of the everted membrane vesicles was monitored by changes in fluorescence of 9-aminoacridine. The addition of lactate activated the respiratory chain and acidification of the vesicle lumen which resulted in accumulation of 9-aminoacridine within the vesicle and quenching of its fluorescence (Fig. 7 ) . When a new steady state was reached LiCl (or NaC1) was added to a give a final concentration of 10 I " . The entry of Li' (or Na') on the carrier resulted in a loss of H' which decreased the ApH and resulted in an increase in fluorescence of 9-aminoacridine. The rate and extent of the increase in fluorescence is a good measure of the activity of the Na'(Li')/H+ antiporter (8,12). The response to the addition of Li' (and Na+) was greater than normal in the mutant W3133-2T and was very much more active than normal in the mutant W3133-2s. Lithium caused larger change of fluorescence than Na'.

Na'
added to the assay mixture, at the points indicated by arrows to give a final concentration of 10 mM. The chart speed of the recorder was increased at the point indicated by dotted lines to facilitate the measurement of the initial rate of change of fluorescence. A downward deflection indicates a decrease in fluorescence intensity, indicating the establishment of a ApH, acidic interior. An upward deflection represents a dissipation of the ApH. The effect of Li' concentration on proton movement was next investigated. Fig. 8 and Table I show that the mutant W3133-2s has a higher affinity for lithium and also increases in the maximum velocity of the reaction. The affinity of the antiporter for Na+ in the mutant (W3133-2s) was higher and the maximum velocity was also higher than the parent (W3133-2) ( Table I).
A second method for the assay of Li'/H' antiporter was investigated. In this technique, the extrusion of protons resulting from the addition of Li' to cell suspension was monitored with a pH electrode (11). Fig. 9 shows that when Li' was added to the mutant W3133-2S, there was a more rapid acidification of the external medium (0.77 ng ions H'/s X mg of protein) than in the comparable experiment with the parental cell (0.33 ng ion H'/s X mg of protein). These data are in general agreement with the findings obtained with the 9aminoacridine.
It was important to demonstrate that the abnormality of the Li'(Na+)/H+ antiporter activity was completely unrelated to the melibiose operon. To establish this fact, a mutant containing a normal melibiose operon from the plasmid was compared with the mutant strain W3133-2s. The insertion of a normal melibiose operon into the mutant did not modify the abnormality in the antiporter activity ( Fig. 8 and Table I). Thus, the mutant possesses two separate mutations, one in the melibiose transport system and a second in the Na'(Li+)/ H+ antiporter.

DISCUSSION
Previous studies of the melibiose transport system of E. coli have indicated that the carrier utilizes different cations for different substrates (2,6). Melibiose is transported with either H+ or Na+ but strongly inhibited by Li' ; TMG is transported with Na' or Li' but not Hf; methyl-a-galactoside is transported with any one of the three cations. Support for these conclusions has come from direct measurement of proton uptake with a pH electrode and Na' uptake with a Na+sensitive electrode. Furthermore, artificially imposed ion gradients drive accumulation of the appropriate sugar (3,9).
Although the uptake of Li+ (with TMG and methyl-agalactoside) has not been verified by direct chemical determination, it has been strongly inferred by two types of experiments. When an artificial Li' gradient was imposed across the membrane of energy-depleted cells marked TMG accumulation resulted (3). Furthermore, the addition of TMG or methyl-a-galactoside to cells in the presence of Li' causes an exit of H' from the cell (2,9,and Fig. 4). This proton exit was presumably due to the membrane potential (inside positive) resulting from entry of Li' into the cell. Such proton exit in the presence of Li+ was observed when melibiose was added to the two mutants and is taken as evidence for Li+-melibiose co-transport.
The properties of the two Li+(Na')-dependent mutants strongly suggest that when transporting melibiose the carrier has lost the ability to accept protons but can utilize either Na' or Li' for the co-transport process. The addition of melibiose to the mutants gave no H' uptake (Fig. 3A, Fig. 6A). However, in the presence of Na', this cation was taken up in the presence of melibiose presumably by sugar-Na' co-transport (Fig. 5). In the presence of Na' or Li+, proton extrusion was observed when melibiose was added, indirect evidence for uptake of Na' or Li' . Thus, these two mutants utilize Na' or Li+, but not H' , for melibiose transport as a coupling cation. This cation specificity is analogous to the amino acid transport by ascites tumor cells (13).
The second mutation in these two mutants involves the cation exchange carrier. In mutant W3133-2S, the maximal rate of Li'-H' exchange was 11 times higher than the parent and the K,,, for Li' reduced from the normal value of 5.5 mM to 0.9 mM (Table I). The second mutant W3133-2T showed similar although less marked changes. Apparently, the large amount of Li' entering the cell with melibiose on the mutant carrier becomes toxic unless it can be extruded at a rapid rate. The Na+(Li+)/H+ antiporter has been suggested to be responsible for the regulation of intracellular pH under alkaline external conditions (14,15). If this is the case, the mutants with elevated Na'(Li')/H' antiporter activity may show some different properties of growth under alkaline conditions. It has been suggested (1) that during the evolution of membrane bioenergetic processes, there was a change from the proton economy of the most primitive microorganisms to the Na' economy of the plasma membrane of animal cells. The ability to pump Na' with a Na+/H' antiport and thus develop Na+ gradients undoubtedly appeared very early in evolution, since sodium extrusion is essential for volume regulation (1). Subsequently, certain microorganisms (especially marine organisms) developed Na'-substrate co-transport systems (16). It has been proposed that the melibiose carrier of E. coli represents an example of the type of "transition" between the carriers utilizing H' and those using Na' (1, 2 ) . From this evolutionary standpoint, it is interesting to find a class of mutants that no longer accepts H' but utilize Na' (or Li'). Preliminary attempts to isolate the opposite extreme, a mutant utilizing H' but not Na+, have been reported (17). In this mutant, Na+ has far less effect on the carrier than in the parental cell. Thus, it appears that the mutants predicted based on the evolutionary hypothesis can be isolated.