Replacement of Alanine 58 by Asparagine Enables the Melibiose Carrier of KZebsieZZapneumoniae to Couple Sugar Transport to Na+*

The melibiose carrier of Klebsiella pneumoniae couples sugar transport to H+ and Li+, while that of Esch- erichia coli uses Na+ besides the other two cation species (Hama and Wilson, 1992). We have shown that the K. pneumoniae melibiose carrier is capable of recog- nizing Na+ when the amino-terminal 81 residues are replaced by the corresponding region of the E. coli melibiose carrier (Hama and Wilson, 1993). In this amino-terminal region there are 5 residues that are not conserved between the two carriers. In this study, we changed each of the 5 residues of the K . pneumoniae carrier to the one in the E. coli carrier. The substitutions are Ile-36 + Val, Val-43+Leu, Leu-54+Trp, Ala-58+Asn, and Cys-684Ala. With four of the five mutants, Ile-364Va1, Val-43+ Leu, Leu-54+Trp, and Cys-6€bAla, sugar accumula- tion was not affected by Na+. In striking contrast, melibiose and methyl-1-thio-B-D-galactopyranoside accumulation was greatly stimulated by Na+ with the Ala-58+Asn mutant. Furthermore, Na+ uptake cou- pled to downhill melibiose transport was observed with the Ala-SB+Asn mutant. These results indicate that the Ala-SBdAsn substitution enables the K . pneumoniue melibiose carrier to couple sugar transport

All living cells take up various solutes from the environment. One common mechanism for solute uptake is cationsubstrate cotransport, which can be found in cells ranging from bacteria to mammals. In general, the coupling cation for cotransport systems is correlated to the ion species that is primarily involved in the energy transduction at the membrane in which they are located. For example, many of the cotransporters in the mammalian plasma membrane, where Na+ is the primary cation for energy transduction, use this cation for cotransport. On the other hand, in bacterial membranes where H+ is the primary cation for energy transduction, H+-solute cotransporters are most common. There are interesting exceptions, such as the Escherichia coli melibiose carrier, which is a cytoplasmic membrane protein responsible for cotransport of a-and p-galactosides with monovalent cations. Although H+ is the primary ion that is involved in energy transduction at the cytoplasmic membrane of E. coli, this carrier uses Na+, Li+, or H ' . Furthermore, the preferable coupling cation is variable depending on sugar substrates (see * This work was supported by National Science Foundation Grant DCB-90-17255 and National Institute of Health Grant DK05736. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "ndvertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. Tsuchiya et al. (1985) for a review). This unique feature makes the carrier a useful system to study the mechanism of cationsugar coupling. The melibiose carrier of E. coli is encoded by the melB gene. The melibiose carrier consists of 469 amino acid residues (Yazyu et al., 1984) and is considered to have 12 transmembrane a-helices (Botfield et al., 1992). Several groups have been studying cation and sugar recognition sites of this carrier. Approximately 30 different mutants have been isolated, which shows changes in cation or sugar recognition (Yazyu et al., 1985;Kawakami et al., 1988;Botfield and Wilson, 1988). Pourcher et al. (1991Pourcher et al. ( , 1993 made site-specific mutants at aspartate 31,51,55, and 120, which are located in the putative transmembrane regions, and showed that these residues are in or near the Na+ binding site. Wilson and Wilson (1992) also showed that aspartate 51 and 120 are important for Na+stimulated melibiose accumulation.
Recently we undertook another approach to learn more about the Na+ recognition site in the melibiose carrier. We cloned and sequenced the melB gene of Klebsiella pneumoniae and showed that this carrier uses H+ and Li+ but not Na+ (Hama and Wilson, 1992). Although its primary structure was 78% identical to the E. coli melibiose carrier, we were not able to identify region(s) involved in Na+ recognition by comparing the amino acid sequences, because the differences were seen throughout the molecule. Therefore, we made chimeric carriers to narrow the choice of the amino acid residues on which we should focus. When the amino-terminal 81 residues of the K. pneumoniue carrier were replaced by the corresponding 77 residues of the E. coli carrier, the resulting chimeric carrier, E2K10, was able to couple sugar transport to Na+ (Hama and Wilson, 1993). There are only 5 amino acid residues that are not conserved between the two carriers in this amino-terminal region, indicating that one (or more) of the 5 residues must be directly involved in Na+ recognition.
In this study we changed each one of the 5 residues of the K. pneumoniue carrier to the one found in the E. coli carrier.
With four of the five mutants, Ile-3hVa1, Val-43+Leu, Leu-54+Trp, and Cys-GhAla, Na+ had no effect on sugar transport. In striking contrast, the Ala-58-Asn mutation had a remarkable effect. This mutant had the ability to couple sugar transport to Na+. These results show that Asn-58 (Asn-54 in the E. coli carrier) is involved in Na+ recognition.   to 213 Oligonucleotides with the suffix "C" and " A represent coding and anticoding sequences, respectively.

E. coli TG1 ( A b -p r o ] supE thi hsdD5/F'traD36proA+B+
host strains for transport assay and sugar fermentation assay, re-locP locZAMI5) (Amersham International) was used as a host strain for cloning and plasmid isolation.
pSUBS25 (Hama and Wilson, 1992), which has the BglII-SmaI 2.5-kilobase fragment containing the K. pneumoniae melB gene at the BamHI-HincII sites of pSU2718 (Martinez et al., 1988), was used as a source of the wild type gene.  Ala-58 + Asn 0.1 0.6 3.8 Cvs-68 + Ala 0.3 0.3 0.9 "DWl(pcn) cells expressing each carrier were incubated with 0.2 * Cations were added to give a final concentration of 10 mM.

Site-directed
Mutagenesis-Site-directed mutagenesis was carried out by the overlap extension method (Ho et al., 1989). Oligonucleotides used as primers are listed in Table I. DNA amplification was carried out with Pfu DNA polymerase as follows: a cycle of 5 min at 95 "C, 1 min at 40 "C, and 30 s at 75 "C; 29 cycles of 1 min at 95 'C, 1 min at 40 "C, and 30 s at 75 "C; 5 min at 75 'C. One hundred pmoles of each primer was added to a total of 100 pl of reaction mixture. pSUBS25 (3 ng) was used as a template for the first sets of amplification. The combinations of primers for the first amplification were: KpmelAC-KpI36VA and KpI36VC-Kpl51A for the Ile-3hVal mutation; KpmelAC-KpV43LA and KpV43LC-Kp151A for the Val-43+ Leu mutation; KpmelAC-KpL54WA and KpL54WC-Kp151A for the Leu-5kTrp mutation; KplC-KpA58NA and KpA58NC-Kp151A for the Ala-5hAsn mutation; KplC-KpC68AA and KpC68AC-Kpl51A for the Cys-6hAla mutation. After the first amplification, the reaction mixtures were diluted 100-fold, and 5 p1 of each was used as a template for the second amplification. The second amplification was mM ["C]lactose for 60 min.
FIG. 3. The uptake of Na+ and H+ driven by downhill melibiose entry into cells. Na+ and H' uptake measurements were carried out as described under "Experimental Procedures." After a base line value was recorded for 2-3 min, an anaerobic 1 M melibiose solution was added at the point indicated by arrows to give a final concentration of 10 mM. Wild type, DWl(pcn)/pSUBS25; A58N, DWl(pcn)/pSUA58N. carried out with primers KpmelAC-Kpl51A for Ile-3hVa1, Val-4h Leu, and L e u -5 k T r p mutations and KplC-Kpl51A for Ala-58+ Asn and Cys-6&Ala mutations. To clone DNA fragments containing Ile-3hVa1, Val-43+Leu, or L e u -5 k T r p mutation, the amplified products were digested with S m I and BamHI of which recognition sites were in the KpmelAC primer and in the melB gene (positions 156 and 236): respectively. DNA fragments containing each mutation were used to replace the corresponding region of pSUBS25. With the Ile-3hVal and the Val-43-Leu mutants, the SO-base pair BamHI-BamHI fragment (156-236), which was lost during this cloning procedure, was inserted later. This was not necessary for the Leu"+ Trp mutant because the first BamHI site had been disrupted to create the mutation. The resulting plasmids, pSUI36V, pSUV43L, and pSUL54W, were sequenced. DNA fragments containing the Ala-5& Asn or the Cys-68+Ala mutation were cloned into the SmaI site of pUC18 and sequenced. The BarnHI-BarnHI SO-base pair fragment (156-236) containing each mutation was subsequently cut out and used to replace the corresponding segment of pSUBS25. The resulting plasmids were named pSUA5SN and pSUC68A.
Sequencing was carried out with double strand plasmid DNA by the chain termination method (Sanger et a!., 1977) using T7 DNA polymerase (Sequenase, United States Biochemical).

DWl(pcn).
In all cases the melB genes were expressed constitutively in Sugar Transport Assay-Cells were grown in LB medium containing 10 pg/ml tetracycline and 30 pg/ml chloramphenicol. Transport assay was carried out as described previously (Hama and Wilson, 1993).
Measurement of Cation Mouenent-Cells were grown in LB medium containing 10 pg/ml tetracycline and 30 pg/ml chloramphenicol. Proton and Na+ uptake coupled to sugar transport was measured as described previously (Hama and Wilson, 1993). Melibiose was added to give a final concentration of 10 mM.

RESULTS
Five amino acid residues of the K. pneumoniue melibiose carrier, Ile-36, Val-43, Leu-54, Ala-58, and Cys-68, were changed to Val, Leu, Trp, Asn, and Ala, respectively, by sitedirected mutagenesis. At first, we intended to overproduce the mutated carriers using the pKK223-3 (Pharmacia KLB Biotechnology Inc.) expression vector. However, the growth of the cells overexpressing these carriers was very slow and deletions of a part of the melB gene occurred frequently. We therefore used pSUBS25, which has been successfully used for the physiological study of the K. pneumoniae melibiose carrier to clone the mutated genes. A part of the melB gene containing each mutation was used to replace the corresponding region of the wild type melB gene of pSUBS25 as described under "Experimental Procedures." The plasmids pSUI36V, based on Hama and Wilson (1992).
* The nucleotide numbering for the melB gene of K. pneurnoniae is pSUV43L, pSUL54W, pSUA58N, and pSUC68A express melibiose carriers with Ile-36+Val, Val-43+Leu, Leu-5LkTrp, Ala-5&Asn, and Cys-6hAla mutations, respectively. During the construction of these plasmids, we usedpcn strains as hosts. This mutation has been shown to lower the copy number of multicopy plasmids (Lopilato et al., 1986). pcn strains carrying each of the plasmids grew much faster than the corresponding strain without the pcn mutation (data not shown). When transformed with each of the plasmids, ~010nies of DWZ(pcn), which has the a-galactosidase but lacks its own melibiose carrier, turned red on melibiose-MacConkey agar plates indicating that all of the mutant carriers had melibiose transport activity (data not shown).
Effect of Cation on Sugar Transport-Each plasmid containing either one of the mutated genes or the normal meZB gene was placed in DWl(pcn), which lacks both the melibiose carrier and the a-galactosidase, and used for physiological study. When exposed to radioactive melibiose in the absence of NaCl or LiCl, DWl(pcn)/pSUBS25, which expressed the wild type carrier, accumulated this sugar to a concentration 90 times higher than that in the external medium (Fig. 1). The accumulation was not affected by adding NaCl and was slightly lowered by LiC1. This result is consistent with what we obtained with DWl/pSUBS25 (Hama and Wilson, 1992). With DWl(pcn)/pSUI36V, pSUV43L, or pSUC68A, melibiose accumulation was about 70% of that with the wild type carrier. The effect of NaCl and LiCl on melibiose accumulation with the three mutants was the same as the wild type carrier. Melibiose accumulation in the cells carrying pSUL54W was slightly lower than in the cells with the wild type carrier in the absence of cations (about 60% of the wild type), and NaCl had no effect. LiCl stimulated the accumulation of this sugar 1.4-fold ( Fig. 1 and Table 11), suggesting the possibility that the Leu-5hTrp mutant had acquired the ability to couple melibiose transport to Li+. In striking contrast, melibiose accumulation in DWl(pcn)/pSUA58N was only 22-fold (or 23% of normal) in the absence of cations and greatly stimulated by adding NaCl or LiCl. The Na' and Li' stimulation were 3.2-and 2.2-fold, respectively. This result strongly suggests that the Ala-5kAsn mutation enables the carrier to couple melibiose transport to Na' or Li'. TMG transport is coupled to H' and Li' in the K. pneum o n k melibiose carrier (Hama and Wilson, 1992). TMG accumulation in DWl(pcn)/pSUBS25 was 9.3-fold without added cations (Fig. 2). Li' stimulated the accumulation 6-fold while Na' had no effect with the wild type carrier. With Ile-36-Va1, Val-43+Leu, and Cys-6hAla mutants, TMG accumulation in the absence or presence of cations was about the same as with the wild type carrier. In the cells expressing the carrier with L e u -5 h T r p or Ala-5&Asn mutations the accumulation was very poor in the absence of cations (1.8and 1.0-fold, respectively), while it was comparable with the others in the presence of Li', indicating the possibility of impaired H'-TMG coupling. A striking resuIt is that Na' stimulated TMG accumulation in DWl(pcn)/pSUA58N 18fold, while there was no effect with the Leu-5hT1-p mutant. This result is consistent with the idea that the Ala-5hAsn mutant can use Na+ for sugar transport. Lactose is a relatively poor substrate for the K. pneumoniae melibiose carrier. The cells with the K. pneurnoniae carrier accumulated this sugar only in the presence of Li' (Hama and Wilson, 1992). The accumulation of lactose was very low when DWl(pcn) was used as a host strain, and Li' stimulation was barely seen (Table 111). With Ile-3hVa1, Val-43+Leu, Leu-54+Trp, and Cys-6&Ala mutants, lactose accumulation was as low as in the case with the wild type carrier.  (Botfield et al., 1992). Amino acid numbering is different from the one for E. coli, because the K. pneumonioe carrier has 4 additional residues at the amino terminus. The 5 residues mutated in this study are indicated by filled circles. The aspartic acid residues in the putative transmembrane regions  are marked with @. These Asp residues correspond to positions 31,51,55, and 120 in the E. coli carrier.
However, in the presence of Li+, cells expressing the Ala-5& Asn mutant carrier accumulated lactose 3 times as much as the cells with the wild type carrier. It was not clear if Na+ had a stimulatory effect.
Cation Movement Induced by Downhill Melibiose Transport-When a high concentration of substrate of a cotransporter is added to cell suspensions, rapid downhill entry of the substrate occurs through the carrier and is associated with entry of cations into the cells. The decrease of the extracellular concentration of the cation caused by such uptake can be measured by an ion-selective electrode. H+ and Na' uptake coupled to melibiose transport has been demonstrated with the E. coli melibiose carrier (Tsuchiya and Wilson, 1978) and chimeric carriers derived from the E. coli and the K. pneumoniue melibiose carriers (Hama and Wilson, 1993). We tested melibiose-induced Na' uptake by DWl(pcn)/ pSUA58N. As we reported previously, no Na' uptake was observed when 10 mM melibiose was added to cells expressing the K. pneumoniae melibiose carrier (Fig. 3). On the other hand, rapid Na+ uptake was observed with DWl(pcn)/ pSUA58N. This result unambiguously indicates that melibiose transport is coupled to Na+ in the Ala-5hAsn mutant.
H+ uptake coupled to downhill melibiose transport was observed with both DWl(pcn)/pSUBS25 and DWl(pcn)/ pSUA58N (Fig. 3), indicating that melibiose transport is coupled with H' in both the wild type and the mutant carrier. The initial rate was somewhat lower with the Ala-5kAsn mutant. Because the accumulation of melibiose and TMG was also lower in the cells with the Ala-5bAsn mutant than with the wild type carrier in the absence of cation (Figs. 1  and 2), it appears that H+-coupled pathway is less efficient in the mutant.

DISCUSSION
The most important observations with the mutant Ala-5& Asn were: 1) melibiose and TMG accumulation was stimulated by Na', and 2) Na' uptake was coupled to downhill melibiose transport. It is concluded that this mutant couples sugar transport to Na' . This study provides strong evidence for the idea that the region surrounding the position 58 of the K. pneumoniae melibiose carrier (54 in the E. coli carrier) forms a critical part of the Na' recognition site.
The Leu-5bTrp mutant seems to have Li'-coupled melibiose transport activity. With the E2K10 chimeric carrier, which contains all of the five substitutions made in this study, Na' and Li+ stimulated melibiose accumulation to the same extent. Because Li+, but not Na' , was effective on melibiose accumulation with the Leu-5bTrp mutant and Na' was more effective than Li' with the Ala-5hAsn mutant, the effect of the cations on melibiose transport with the E2K10 carrier appears to be caused by the combination of these two substitutions.
The locations of the 5 residues targeted in this study are shown in Fig. 4. The Ala-58 is very close to the Asp-59 and the Asp-55 residues, which have been shown to be important for Na+ recognition in the E. coli carrier (Pourcher et al., 1991;Wilson and Wilson, 1992;Pourcher et al., 1993). In addition, the Ala-58 may also be close to the Asp-35 and the Asp-124, which are also implicated in Na' recognition by studies in E. coli. Thus, the 4 Asp residues and the Asn-58 may form a Na+ binding "pocket" in the mutant melibiose carrier. The way the Asn residue interacts with Na+ could be either direct or indirect. The replacement of Ala-58 by Asn might enable the carrier to form an additional hydrogen bond required to bind Na+. Alternatively, Asn-58 (Asn-54 in E. coli) might be necessary for the correct conformation of one (or more) of the Asp side chains, which may form hydrogen bonds with Na' . Further mutagenesis study at the position 58 (54 in E. coli) would provide valuable information to answer these questions.
It is of interest to note that Ala-5hAsn mutant has higher lactose transport activity than the wild type K . pneumoniae carrier (Table 111). In other words, the mutation caused improved recognition for both Na' and lactose. Botfield and Wilson (1988) identified 22 single mutations that altered both Li' and TMG recognition. These observations suggest that the cation and the sugar recognition sites may overlap.
The fact that H+-sugar coupling is slightly lowered in the Ala-5hAsn mutant (Figs. 1-3) suggests that the mutant has acquired functional Na+ recognition site at the expense of a slight distortion of the H+ recognition site. Thus, Na+ and H' recognition sites in the melibiose carrier appear to overlap. According to a systematic analysis of primary structure of transporters carried out by Marger and Saier (1993), Na'solute cotransporters make distinct groups from H+-solute cotransporters and facilitators. We believe the melibiose carriers could be considered as intermediate type, because they have the capacity to bind either H' or Na' . Another example is the alanine carrier of thermophilic bacteria, which is one of the few transporters with sequence similarity to the melibiose carrier and also uses both H+ and Na+ (Kamata et al., 1992).
We speculate that spontaneous Na+-coupled mutants of the K. pneumoniue melibiose carrier would arise under certain selective pressure such as alkaline pH, high external Na+ concentration, or high temperature, because Na+-coupled cotransporters are found in bacteria living in these environments (Dimroth, 1987;de Vrij et. al., 1989). It would be an interesting system in which a transporter undergoes diversification in response to the environment.