Resistance of the Melibiose Carrier to Inhibition by the Phosphotransferase System Due to Substitutions of Amino Acid Residues in the Carrier of Salmonella typhimurium"

The melibiose carrier of Salmonella typhimurium is under the control of the phosphoeno1pyruvate:carbohydrate phosphotransferase system (PTS). We isolated mutants of the melibiose carrier that showed resistance to inhibition via the PTS. Growth of the mutants on melibiose was not inhibited by 2-deoxyglucose, a nonmetabolizable substrate of the PTS, although growth of the parent strain was inhibited. Transport activity of the melibiose carrier in the mutants was fairly resistant to inhibition by 2-deoxyglucose, although the activity in the parent was sensitive to inhibition. We cloned the mutated melB gene that encodes the melibiose carrier, determined the nucleotide sequences, and identified replaced nucleotides. The mutations resulted in substitutions of Asp-438 with Tyr, Arg-441 with Ser, or Ile-446 with Asn. All of these residues are in the COOH-terminal region of the carrier. The secondary structure of this region is predicted to be an ahelix, and the mutated residues were on the same side of the helix. This region showed sequence similarity to a region of the MalK protein, in which substitution of amino acid residues also resulted in PTS-resistant mutants. Thus the COOH-terminal portion of the melibiose carrier is important for the interaction of dephosphorylated IIIG'", which is an entity causing reversible inactivation of the carrier.

The melibiose carrier of Salmonella typhimurium is under the control of the phosphoeno1pyruvate:carbohydrate phosphotransferase system (PTS). We isolated mutants of the melibiose carrier that showed resistance to inhibition via the PTS. Growth of the mutants on melibiose was not inhibited by 2-deoxyglucose, a nonmetabolizable substrate of the PTS, although growth of the parent strain was inhibited. Transport activity of the melibiose carrier in the mutants was fairly resistant to inhibition by 2-deoxyglucose, although the activity in the parent was sensitive to inhibition. We cloned the mutated melB gene that encodes the melibiose carrier, determined the nucleotide sequences, and identified replaced nucleotides. The mutations resulted in substitutions of Asp-438 with Tyr, Arg-441 with Ser, or Ile-446 with Asn. All of these residues are in the COOH-terminal region of the carrier. The secondary structure of this region is predicted to be an ahelix, and the mutated residues were on the same side of the helix. This region showed sequence similarity to a region of the MalK protein, in which substitution of amino acid residues also resulted in PTS-resistant mutants. Thus the COOH-terminal portion of the melibiose carrier is important for the interaction of dephosphorylated IIIG'", which is an entity causing reversible inactivation of the carrier.
When cells of Escherichia coli or Salmonella typhimurium are grown in the presence of glucose and melibiose, the growth curve exhibits two successive growth cycles separated by a lag period (1,2). This type of growth behavior has been termed diauxie. Glucose is utilized in the first growth cycle and melibiose in the second. In other words, the utilization of melibiose is inhibited by glucose. The two mechanisms responsible for this phenomenon are catabolite repression and inducer exclusion (2, 3). Catabolite repression is a control mechanism for gene expression, and inducer exclusion is a * This research was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (to T. T.) and by a grant from the Netherlands Organization for Scientific Research (NWO) under the auspices of the Netherlands Foundation for Chemical Research (SON) (to P. P.). 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.
control mechanism for transport. The PTS' is involved in both mechanisms (4, 5). We have been investigating membrane transport and are interested in the role and the mechanism of inducer exclusion. We have shown previously that, in E. coli, inhibition of melibiose utilization still occurs even if catabolite repression is released by adding cyclic AMP to the growth medium (6).
Transport of substrate not only via the melibiose transport system but also via the lactose, maltose, or glycerol uptake systems is regulated (inhibited) by the PTS. It has been revealed that this inhibition of transport is caused by binding of one of the PTS proteins, dephosphorylated IIIGIc, to the transport carriers or glycerol kinase (4). IIIGIC is an important component of the glucose PTS (4), and its phosphorylation and dephosphorylation are crucial not only for activation or inactivation of the carriers but also for inactivation or activation of adenylate cyclase (7), which is involved in the synthesis of cyclic AMP (8,9). Binding of IIIG1" to the lactose carrier of E. coli has been investigated (10, 11). Mutants possessing an altered lactose carrier resistant to the binding of IIIG" (therefore resistant to inhibition by the PTS) have been isolated (12). Replacement of amino acid residues in such mutant carriers has been reported (13). PTS-resistant mutants of the maltose carrier and amino acid substitution in the carrier have been reported (14, 15).
Recently, we cloned the S. typhimurium melB gene that codes for the melibiose carrier, determined the nucleotide sequence, and deduced the amino acid sequence of the carrier (16). Thus it became possible to identify substituted amino acid residues in mutant carriers. We isolated mutants of 5' . typhimurium that showed PTS-resistant growth on melibiose and PTS-resistant transport of methyl-/3-D-thiogalactoside. We identified the substituted amino acid residues of the carrier of the mutants by cloning and sequencing of the mutated gene. Thus a region of the melibiose carrier that is important for I1Pc binding was identified.
For growth measurement, modified Tanaka medium (20) (Na+ salts in the original medium were replaced with K' salts) supplemented with 10 mM (or 5 mM where indicated) melibiose was used. When necessary, 5 mM (or 10 mM where indicated) 2-deoxyglucose or methyl-a-glucoside (these sugars are nonmetabolizable substrates of the glucose PTS) was added. Cells were grown at 37 "C under aerobic conditions. Growth was measured turbidimetrically at 650 nm. For transport assays, cells were grown in modified Tanaka medium supplemented with 10 mM melibiose and 1% Tryptone (Difco). Cells were harvested at the exponential phase of growth, washed with modified Tanaka medium, and suspended in the same medium. For DNA propagation, L broth (21) supplemented with ampicillin (50-100 rg/ml) was used.
Isolation of Mutants-A mutant PP2098 was isolated in two steps (22): (i) introduction of cysA1539:TnlO (from NK1186) into LT2 by phage P22 transduction, selecting for resistance to tetracycline (resuiting strain is PP2092, cysA1539:TnlO); (ii) introduction of ptsZl7 from SB1476 into PP2092 by P22 transduction, selecting for growth in the absence of cysteine and no growth on mannitol (resulting strain is PP2098). This strain is hypersensitive to PTS regulation. Cells of PP2098 were unable to grow on melibiose in the presence of 2deoxyglucose or methyl-a-glucoside. Cells of PP2098 were spread on agar plates containing 10 mM melibiose and 5 mM 2-deoxyglucose and incubated at 37 "C for a few days. Spontaneous mutants grown on the plates were isolated. All mutants used were independent isolates. Each colony obtained was checked for growth on maltose or glycerol in the absence or presence of 2-deoxyglucose. We isolated mutants that grew on melibiose but not on maltose or glycerol in the presence of 2-deoxyglucose. Thus we isolated PP2102, PP2103, PP2104, PP2105, PP2106, PP2107, PP2155, and PP2158.
Preparation of DNA-Chromosomal DNA (23) and plasmid DNA (24, 25) were prepared by published procedures. When necessary, DNA was digested with restriction endonucleases and separated by agarose (or polyacrylamide) gel electrophoresis (26).
Plasmids-Plasmids pBR322 and pBluescriptIIKS(+) were used as cloning vectors. Plasmid pTKKl was constructed by ligating a SmaI-SmaI fragment (6 kbp) containing the melB region of S. typhirnurium LT2 to the EcoRV site of the pBR322. Three deletion derivatives of pTKKl were constructed that each lacked a part of the rnelB gene and were used to clone various parts of the mutant melB gene. pTKKll lacks the central part of melB (EcoRV-EcoRV fragment), pTKK12 lacks the downstream region of melB (NruI-NruI fragment; the second NruI site is in the vector pBR322), and pTKK13 lacks the upstream region of rnelB (KpnI-KpnI fragment).
Cloning and Subcloning of Mutated melB-The procedure developed by Comeau and Inouye (27) for cloning of a mutated gene was used. Plasmid pTKKl was used as host plasmid. The plasmid possessing the melB region derived from PP2102 is pTKK2102, and so on. Each part of the mutant melB (EcoRV-EcoRV fragment, NruI-NruI fragment, and KpnI-KpnI fragment) was cloned using pTKK11, pTKK12, and pTKK13 as vector. Plasmids carrying each of the three parts derived from pTKK2102 were designated pTKKll-2102, pTKK12-2102, and pTKK13-2102, and so on.
DNA Sequencing-The nucleotide sequences were determined by the dideoxy chain termination method (28, 29).
Southern Blot Analysis-Southern blot analysis was performed by a published method (30), and DNA was detected using the ECL DNA detection kit (Amersham Corp.). An EcoRV-HaeIII fragment derived from the melB gene was used as a probe.
Assays-Activity of a-galactosidase was measured as described previously (31). Transport of Na' elicited by melibiose influx (32) or transport of ["CITMG (33) was measured as described previously. Protein contents were determined by the method of Lowry et at. (34).
Materials-Restriction endonucleases were from Nippon Gene Co., Takara Shuzo Co., or New England Biolabs. [ c x -~~S J~C T P and [a-'"PIdCTP were from Amersham Japan Co., ["CITMG was from Du Pont-New England Nuclear, and Sequenase (a sequencing kit) was from U. S. Biochemical Corp. Plasmid pBluescriptIIKS(+) was from Stratagene Inc., and KS and SK primers were synthesized by a DNA synthesizer. In some cases, sequencing primers were synthesized based on the nucleotide sequence of wild type melB of S. typhirnurium. that is resistant to such PTS sugars. We tested the effects of the nonmetabolizable PTS sugars on the growth of the mutants. In fact, PP2098 was unable to grow on melibiose as a sole source of carbon in the presence of 2-deoxyglucose ( Fig.  1) or methyl-a-glucoside (data not shown). On the other hand, mutant PP2102 grew on melibiose even in the presence of 2deoxyglucose or methyl-a-glucoside. It is noteworthy that the growth of both PP2098 and PP2102 on maltose or glycerol was strongly inhibited by both 2-deoxyglucose and methyl-aglucoside (data not shown). We obtained similar results with other mutants, PP2103, PP2104, PP2105, PP2106, PP2107, PP2155, and PP2158 (data not shown). Therefore, the release of growth inhibition in these mutants is specific for melibiose. This suggests that the mutations in these mutants are localized in the melibiose operon but not in the genes of components of the PTS, such as crr encoding IIIG". It should be pointed out that S. typhimurium does not possess the lactose system, and the melibiose system is the sole system for transport of melibiose.

Properties
Although our results indicate that mutations in the mutant PP2102 exist in the melibiose operon, as described above, two types of mutations in the operon seem to be possible. One type is a mutation in the regulatory region of the operon. If overexpression of the operon takes place, a large amount of the melibiose carrier could be synthesized, and the limited amount of IIIG" present in cells would not be able to inhibit all of the melibiose carrier. In this case, we should detect elevated activities of a-galactosidase (me@ product) and the melibiose carrier (rnelB product). In fact, we obtained a number of these mutations in E. coL2 The second type of mutation is in the melB gene. In this case, normal levels of a-galactosidase activity and melibiose transport activity and PTSresistant transport would be observed. We are interested in the latter type of mutants.
We measured the a-galactosidase activity of the mutants and found that the activity in all of the mutants was almost the same as that of the parent (PP2098) (data not shown). Thus it seemed that the mutations were not in the regulatory region of the operon, but in the melB gene.
We then tested the effects of the nonmetabolizable PTS sugars on transport activity of the melibiose carrier by two methods. In the first method, we measured the uptake of ["C] TMG (Table I). The uptake of TMG was inhibited about 70% by 5 mM 2-deoxyglucose in PP2098 (parent), whereas the TMG uptake in the mutants (PP2102-PP2106, PP2155, and PP2158) was very resistant (0-15% inhibition). In the second method, we measured uptake of Na+, elicited by the addition  of melibiose to a cell suspension, using an Na+-selective electrode. Since the melibiose carrier mediates cotransport of Na+ and melibiose, influx of melibiose into cells elicits the Na+ uptake (35). The Na+ uptake was inhibited 75% by 2deoxyglucose in PP2098, whereas it was resistant in PP2102-PP2106, PP2155, and PP2158 (5-25% inhibition) (data not shown). Thus, all of the mutants we tested seem to possess mutations in the melB gene and are suitable for our purpose and for further analysis.
Cloning of the Mutated melB Gene-For cloning of the mutated melB gene, we used a procedure developed for the cloning of the mutated envZ gene (27) (Fig. 2). Chromosomal DNA of mutant PP2102 was digested with the restriction endonuclease SmaI, and vector pBR322 was digested with EcoRV. Plasmid pTKK1, which carries the wild type melB gene, was digested with BglII and dephosphorylated. These DNA fragments were mixed, denatured by high temperature, and reannealed. Competent cells of E. coli RE16r (melA+B-) were transformed with the DNA mixture and spread onto agar plates containing L broth and ampicillin (50 pg/ml).
Ampicillin-resistant colonies were then checked for growth on melibiose. Plasmids were prepared from the cells that were able to grow on melibiose, and the size of the plasmids was checked. Plasmids of proper size were then introduced into competent cells of E. coli PPA172 (ptsl), and growth of the transformants on melibiose in the absence or presence of 2deoxyglucose was tested on agar plates. Plasmids were isolated from cells that showed 2-deoxyglucose-resistant growth, and designated pTKK2102. Plasmid pTKK2102 is expected to possess the melB gene derived from PP2102. Plasmids pTKK2103, pTKK2104, pTKK2105, pTKK2106, pTKK2107, pTKK2155, and pTKK2158, which carry the melB region of PP2103 to PP2158, respectively, were similarly constructed. Fig. 3 shows growth of PPA172/pTKK1 and PPA172/ pTKK2102 on melibiose in the absence or presence of 2deoxyglucose. Cells of PPA172/pTKK2102 grew on melibiose even in the presence of 2-deoxyglucose, whereas cells of PPA172/pTKK1, which carry the wild type melB gene, did not. Thus it seems clear that pTKK2102 carries the melB gene derived from the mutant PP2102. Similar results were obtained with cells harboring plasmids carrying the mutated melB gene of other mutants (data not shown).
We tested the effects of 2-deoxyglucose on the activity of the melibiose carrier derived from the pTKK2102. Because PPA172 possesses its own melibiose carrier, pTKK2102 was introduced into E. coli strain DW2 that lacks the melibiose carrier. The carrier activity was tested by measuring Na+ uptake elicited by melibiose influx. The Na+ uptake in DW2/ pTKK2102 was not inhibited significantly, whereas that in DW2/pTKKl was strongly inhibited (85% inhibition). Thus we confirmed that the 2-deoxyglucose-resistant growth of PPA172/pTKK2102 was due to the melB gene carried by the plasmid pTKK2102. Similar results were obtained with cells of DW2 harboring a plasmid carrying the other mutated melB gene (data not shown).
Next we tried to do intracistronic mapping of the mutations in the melB gene of the mutants. Three types of plasmids (for example, pTKKll-2102, pTKK12-2102, and pTKK13-2102), each carrying a different segment of the mutant melB gene, were constructed (Fig. 4). Competent cells of PPA172 were transformed with the plasmids, and growth of PPA172/ pTKKll-2102, PPA172/pTKK12-2102, and PPA172/  pTKK13-2102 on melibiose was tested in the absence or presence of 2-deoxyglucose. Only PPA172 cells harboring pTKK12-2102 grew in the presence of 2-deoxyglucose (Fig.  4). Thus the mutation of PP2102, responsible for the resistance to the PTS sugar, is localized in the NruI-NruI fragment of the melB region, which corresponds to the COOH-terminal portion of the melibiose carrier. We obtained the same result with all other mutants tested (data not shown).
Sequence Analyses-We determined the nucleotide sequences of the NruI-NruI DNA fragment derived from mutated melB of PP2102. We found only one nucleotide replacement in the DNA region. The replacement was T with A a t position 1,334 starting from A of initiation ATG of the melB gene (16). This replacement results in the amino acid substitution of Ile-445 with Asn in the melibiose carrier.
The nucleotide sequence of the region where the mutation was found is AGATCC in the wild type and AGAACC in the mutant. A restriction endonuclease BstYI cleaves the AGATCC site, but not the AGAACC site. To test whether or not we can detect a difference in the digestion pattern between the melB region of wild type cells and the mutant cells, the chromosomal DNA from each strain was prepared, digested with BstYI, and analyzed by Southern blot hybridization. In fact, we detected cleavage of wild type melB at the expected site (Fig. 5) and DNA bands of 1.4 and 0.35 kbp. But the melB region of the mutant PP2102 was not cleaved, and we observed a DNA band of 1.75 kbp (Fig. 5). Surprisingly, we found the same digestion patterns in the melB regions derived from PP2103, PP2104, PP2105, PP2106, and PP2107 with BstYI. On the other hand, we observed the same digestion pattern in the melB region of PP2155 and PP2158 as that of the parent. These results indicate that PP2103-PP2107 have the same mutation in the melB gene as PP2102, and PP2155 and PP2158 have different mutation(s). Consistent results were obtained uhen plasmids carrying cloned melB regions derived from the mutants were propagated in GM33 (dam) cells, isolated, digested with MflI, which cleaves the same site as BstYI, and analyzed by agarose gel electrophoresis (data not shown).
In fact, we found the same replacement of nucleotide in the melB gene of PP2103-PP2107 and different replacements in PP2155 and PP2158. The nucleotide replacements were G with T at position 1,312 in PP2155 and C with A at position 1,321 in PP2158. Thus we identified amino acid substitutions Asp-438 with Tyr and Arg-441 with Ser, in addition to Ile-445 with Asn (Table 11).

DISCUSSION
E. coli and S. typhimurium are closely related microorganisms (36). We introduced the melB gene of S. typhimurium into E. coli cells; and the melB gene was functional in E. coli cells. The melibiose carrier of S. typhimurium synthesized in E. coli showed properties similar to those of the carrier synthesized in S. typhimurium cells. Furthermore, the wild type melibiose carrier of S. typhimurium was sensitive to the PTS of E. coli. Roseman and co-workers (37) demonstrated that the lactose carrier of E. coli reconstituted into membrane vesicles of S. typhimurium was sensitive to the PTS of S. typhimurium. In other words, 111' " of S. typhimurium is able to regulate the lactose carrier of E. coli. It is reported that the sequence homology in 111' ' ' of E. coli and S. typhimurium is 98% (38, 39).
We isolated mutants of S. typhimurium carrying the melibiose carrier that became resistant to inhibition via the PTS. The mutant cells grew on melibiose in the presence of 2deoxyglucose, a nonmetabolizable PTS sugar, although the parental cells did not. This means that the transport process for melibiose is crucial for cell growth in those cells under such conditions.
Since the melB genes, encoding the mutant melibiose carriers, were subsequently cloned on a multicopy plasmid, the resulting strains might become resistant to inhibition by PTS sugars due to overproduction of the melibiose carrier (40). However, from Fig. 3 it is clear that cells containing the pTKKl plasmid (encoding the wild type melibiose carrier) are still sensitive to inhibition by 2-deoxyglucose. In cells containing this plasmid, the activity of the melibiose carrier is about three times higher compared with control cells (data not shown). Clearly, this level of the melibiose carrier is not sufficient to escape from inhibition via 111"' ".
Recently, we determined the nucleotide sequences of the wild type melB gene of S. typhimurium (16). Thus it became possible to determine altered nucleotides in the melB gene of mutants. As a result of cloning and sequencing, we identified the replaced nucleotides in the melB gene of the mutants that became resistant to PTS sugars. The deduced amino acid substitutions in the melibiose carrier of the mutants were Asp-438 with Tyr, Arg-441 with Ser, and Ile-445 with Asn. ' Six mutants.
All of these residues are in the COOH-terminal region of the carrier. Surprisingly, we found the same amino acid substitution (Ile-445 with Asn) in six mutants that were isolated independently. This suggests that Ile-445 is very important for the regulation of the carrier by 1IIG''. Since  are all located in the COOH-terminal region of the carrier, it is very likely that this COOH-terminal region is involved in IIIG1" binding. Judging from the positions of the identified residues (438, 441, and 445)) it seemed that these residues could be present on the same side if this region is in an a-helix form. In fact, calculation by the method of Chou and Fasman (41) predicted an a-helix structure in this region (data not shown). A vertical view of the a-helix of this region showed that Asp-438, Arg-441, and Ile-445 are really on the same side of the a-helix (Fig. 6). Therefore, it is likely that these 3 residues are involved in interaction with IIIGIC. It would be interesting to substitute Arg-452, which is on the same side of the a-helix as , with another residue by site-directed mutagenesis. Among the 3 identified residues, 2 residues possess a charge (Asp and Arg) that might be important for binding (or interaction) with IIIG'".
We proposed a topological model of the melibiose carrier of E. coli based on several types of analysis (42). Since the sequence homology between the melibiose carriers of E. coli and of S. typhimurium is very high (16)) the topology of the two carriers would be very similar. Fig. 7 shows the topological model of S. typhimurium melibiose carrier. According to this model, the COOH-terminal portion of the melibiose carrier of S. typhimurium is on the cytoplasmic side. Since IIIGLc is a soluble cytoplasmic protein, this model is convenient for understanding the interaction between the IIIG1' and the carrier. Saier and co-workers (14) identified substituted amino acid residues in the MalK protein of PTS-resistant mutants. Two of the substitutions are Gly-278 and Gly-284. They pointed out that this region of the MalK protein showed some similarity of amino acid sequence to the central loop portion of the melibiose carrier of E. coli. The amino acid sequences of the melibiose carriers of S. typhimurium and of E. coli are highly homologous, and the sequences of the central loop portions of the two melibiose carriers are very similar (92% homology including conservative changes) (16). Thus, it is likely that the central loop portion of the melibiose carrier of S. typhimurium is involved in the binding of IIIG1". The central loop portion is on the cytoplasmic side according to our topological model (Fig. 7). It has been reported that a central loop portion of the E. coli lactose carrier is important for binding of IIIG'" (13). Perhaps the COOH-terminal portion and the central loop portion of the melibiose carrier are in close contact and form a binding site for IIIG". In the case of the lactose carrier, it seems that the NH2-terminal portion is also involved in the IIIGLc binding (43). Analysis of more mutants will clarify domains that are involved in IIIG1" binding.
We also found sequence similarity between the COOHterminal region that contained Ile-445 of the melibiose carrier and a region of PTS-resistant MalK in which amino acid substitution (Ala-124 with Thr) was found (14) (Fig. 8). Thus it seems reasonable to assume that these regions are really involved in regulation by the PTS, namely binding of IIIGIC.
Both the melibiose carrier and the lactose carrier mediate the cotransport of H+ and melibiose (and other galactosides).
The two carriers are similarly regulated by the PTS. However, no significant sequence similarity was found between the two carriers (44, 45). Interestingly, hydropathy patterns and topological models of the two carriers are similar and suggest 12 transmembrane segments (42,46). Although the sequence similarity is low, the three-dimensional structure of the binding site for IIIG" in the two carriers could be similar.
The hydrophilic COOH-terminal part of the melibiose carrier of S. typhimurium (16) and E. coli (44) is almost 30 residues longer than that of the lactose carrier (45). It was not clear whether this long COOH-terminal portion of the melibiose carrier is really necessary for the function of the carrier. It has been reported that introduction of a termination codon in position 434, 440, or 446 of the melibiose carrier of E. coli (original length is 469 residues) resulted in a reduction of activity to 25% of that of the intact carrier (47). However, truncation at position 460 or 456 caused no significant reduction in transport activity. These results indicate that the 14 COOH-terminal residues of the carrier are not necessary for carrier activity and that the residues at positions 15-36 from Numbers from 1 to 12 indicate the 12 putative membrane-spanning regions of the melibiose carrier. The substituted residues identified in PTS-resistant melibiose carrier are in the COOH-terminal portion, which is on the cytoplasmic side of the membrane. Two regions (Z and ZZ) surrounded by the dotted line are regions that showed sequence similarity to suggested sequences for IIIG1" binding in MalK, Lacy, and GlpK (region I ) (14) or in MalK (region ZZ) (see Fig. 8). Identical residues (*) and conservative changes ( * ) are indicated.