Truncation of MalF Results in Lactose Transport via the Maltose Transport System of Escherichia coli*

Gonzalo Merino and Howard A. ShumanDagger

From the Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The active accumulation of maltose and maltodextrins by Escherichia coli is dependent on the maltose transport system. Several lines of evidence suggest that the substrate specificity of the system is not only determined by the periplasmic maltose-binding protein but that a further level of substrate specificity is contributed by the inner membrane integral membrane components of the system, MalF and MalG.

We have isolated and characterized an altered substrate specificity mutant that transports lactose. The mutation responsible for the altered substrate specificity results in an amber stop codon at position 99 of MalF. The mutant requires functional MalK-ATPase activity and hydrolyzes ATP constitutively. It also requires MalG. The data suggest that in this mutant the MalG protein is capable of forming a low affinity transport path for substrate.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The maltose transport system of the Gram-negative bacterium Escherichia coli is responsible for the unidirectional uptake of maltooligosaccharides (alpha [1,4]-linked D-glucose polymers) (1). This system comprises five proteins. The LamB protein (also known as the maltoporin or lambda -receptor) in the outer membrane, the periplasmic maltose-binding protein (MBP),1 and the MalF, MalG, and MalK polypeptides that together (MalFGK2) form the inner membrane complex (2). The LamB protein, encoded by the lamB gene is responsible for the diffusion of maltooligosaccharides into the periplasm of the cell (3). MBP, a soluble periplasmic protein encoded by the malE gene, binds maltooligosaccharides with a high affinity (Kd = 0.1-1 µM) (4, 5) due primarily to a slow dissociation rate (6). MBP is absolutely required for the transport of maltose (7, 8). The three-dimensional structure of MBP has been determined in both the liganded and unliganded forms (9). The MalF and MalG proteins are integral membrane proteins that traverse the membrane eight and six times, respectively (10-12). MalK is an ATPase located on the cytoplasmic side of the inner membrane that is thought to be the energy-coupling component of the maltose transport system (3, 14). MalK shares strong sequence homology with other ATP-binding cassette transporter proteins (16). The stoichiomtry of the inner membrane complex is (MalFGK2) (17).

MBP has long been regarded as the prime determinant of substrate specificity for the maltose system. However, several observations suggest that the inner membrane complex plays an important role in determining substrate specificity. First, MBP-independent mutants of MalF and MalG have been isolated that transport maltose in the absence of MBP (8). Even in the absence of MBP, these mutants still retain substrate specificity for maltooligosaccharides, which suggests that the inner membrane complex alone is capable of maintaining the specificity of the system. These MBP-independent mutants share a similar Km for maltose transport of approximately 2 mM, but they each display a Vmax that varies from wild-type values to approximately 20-fold lower. Therefore, they all bind maltose equally well, which is suggestive of a similar substrate binding site, but they translocate with different efficiencies.

The observation that the ATP-binding cassette components of two members of the ABC superfamily, the Ugp and Mal transport systems, could be exchanged without any loss of substrate specificity suggests that this component does not play a role in determining the overall specificity of the system (21). The Ugp transport system of E. coli transports sn-glycerol-3-phosphate. The UgpC and MalK proteins of these systems are highly homologous and both couple energy via ATP-hydrolysis (21). Thus, the two other components of the maltose transport system inner membrane complex, MalF and MalG, must be contributing to the specificity of the system.

Mutants of MalF have been isolated that alter the range of substrates transported by the maltose transport system (22). The various mutations map to the malF gene and cause alterations in transmembrane domains 6, 7, and 8 of MalF. The mutants could only recognize either maltose or longer maltodextrins, but not both. The mutations cluster along the transmembrane helices, and suppressor mutations in neighboring helices of MalF suggest a physical interaction.

Studies on other members of the ABC superfamily, such as P-glycoprotein and the HlyB transporter, have also suggested that the transmembrane helices play an important role in determining substrate specificity (23-27).

In the present study, we describe a mutant of the maltose transport system (MalF540) that transports lactose efficiently. The MalF540 mutant we isolated carries a mutation in the malF gene that changes a glutamine codon at position 99 of MalF to an amber stop codon. This mutant suggests a novel mechanism for altering substrate specificity. The isolation and characterization of this mutant with respect to transport efficiency, maltose transport components required, and substrate specificity is described.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Media and Genetic Techniques-- Rich media (LB) and minimal media (M63) were prepared as described previously (30). Standard genetic procedures were performed by the method of Miller (30) unless otherwise noted. Maltose and lactose were obtained from Pfanstiehl Laboratories, Inc. All other sugars were obtained from Sigma. The following antibiotics were used at the indicated concentration unless otherwise noted: 100 µg/ml carbenicillin, 100 µg/ml ampicillin, 50 µg/ml kanamycin, 25 µg/ml chloramphenicol, 20 µg/ml tetracycline.

Bacterial Strains-- Bacterial strains are listed in Table I. The recombination defective recA1 strain, GM1418, was constructed by P1vir transduction using a lysate prepared from HS3078 containing the srl::Tn10 mutation closely linked to the recA1 allele. Tetracycline-resistant transductants of LH1375 were selected and screened for UV sensitivity.

                              
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Table I
Bacterial strains, plasmids, and phage

The malG-amber strain, GM1408, was constructed by conjugating the strain carrying F' KLF10 malGamV67, HS3670, with the malTp1Tp7 strain, HS3238. The mating was plated on minimal glucose plates that select only for HS3238. Recipients of the F' were selected and screened by sensitivity to VCS-M13 phage. Independent overnight cultures of the transconjugants in LB were plated on tetrazolium maltose plates, and Mal- colonies were selected. Some of these should represent a gene conversion event between the malGamV67 on the episome and malG on the chromosome. The malTp1Tp7 allele is easily lost, so the Mal- colonies were screened for the presence of malTp1Tp7 by testing the ability to hydrolyze pNPG2 when lysed with chloroform. This is due to the overproduction of MalZ in response to the elevated levels of MalT. The presence of the malGamV67 was further confirmed by complementation of the Mal- phenotype with the plasmid pMR24, which carries wild-type malG, and by complementation with the phi 80SupF amber suppressor phage. The pAB1 and pLH22 plasmids were introduced into the resulting strain by transformation.

The malF deletion strains, GM1368 and GM1369, were constructed by conjugating the strain carrying F' KLF10 Delta malF argE::Tn10, HS3419, with the malTp1Tp7 strain, HS3238. F' recipients were selected on minimal glucose plates containing tetracycline. Independent overnight cultures of the transconjugants in LB containing tetracycline were plated on tetrazolium maltose plates, and Mal- colonies were selected. Some of these should represent a gene conversion event between the Delta malF3 on the F' and malF on the chromosome. Mal- colonies were then screened for the presence of malTp1Tp7. The presence of the Delta malF3 was further evaluated by complementation of the Mal- phenotype with the plasmid pMR28, which carries wild-type malF. The pAB1 and either the pGM7 or pLH22 plasmids were introduced into the resulting strain by transformation.

Plasmid Construction-- The plasmid pGM1, which carries the malE gene under control of the ptac promoter, was constructed by ligating the malE-containing EcoRI/StuI fragment from lambda NF630 (31) phage DNA, to EcoRI/SmaI-digested pMMB207 plasmid vector DNA. pGM7, which carries the malK and malG genes under the malB promoter, was constructed by ligating the malK-containing EcoRI/ScaI fragment from pHS4, to the malG-containing EcoRI/SnaBI fragment from pMR24. pLac2SM was constructed by digesting pLac2 with SacI and MfeI, thus deleting the portion of malF distal to the specificity mutation, "end-filling" and "chewing back" the respective overhangs with DNA polymerase I (Klenow) and T4 DNA-polymerase, and then ligating the resulting large DNA fragment to itself. pLH9SM was constructed in the same way except that the pLH9 plasmid was used. pLac2F' was constructed by ligating the PstI/BsmI fragment from pLac2 containing the 5'-end of the truncated malF540 gene including the mutation, to the PstI/BsmI fragment from pBR322 that contains the origin of replication. Pt-pLac2SM was constructed by ligating the PstI/BstEII fragment from pMR38 containing the Ptrc promoter and the 5'-end of the malF gene to the PstI/BstEII fragment from pLac2SM containing the 3'-end of truncated malF540 and malG.

Isolation of the Lactose Specificity Mutant-- A plasmid carrying the malF502 MBP-independent allele, pLH5, was mutagenized in the mutator strain KD1087 (32). This strain contains the mutD5 allele, which increases the frequency of mutational events 50-100 times above that observed in a wild-type strain (mut+). KD1087 was transformed with the pLH5 plasmid, and transformants were selected on LB plates containing carbenicillin. Individual transformants were then grown in LB containing carbenicillin at 37 °C overnight. Plasmid DNA was purified from these independent cultures and used to transform the lactose indicator strain GM1305. Transformants were plated on minimal 1% lactose plates containing carbenicillin, kanamycin, chloramphenicol, and 0.2% arginine. Those transformants that grew on the indicator media after 1-3 days of incubation at 37 °C were purified by passing three times on minimal 1% lactose plates. Plasmid DNA was purified from these isolates and used to retransform the lactose indicator strain to ensure that the mutation responsible for the Lac+ phenotype is linked to the pLH5 plasmid.

Nucleotide Sequence Determination-- The nucleotide sequence of malF and malG alleles carried on pLac2 and various amber mutant plasmids (pGM8-pGM12) was determined by using double-stranded DNA templates and 13 oligonucleotide primers (33). Double-stranded DNA templates were purified, denatured, and annealed with the primers as described (34). The Sanger dideoxynucleotide chain termination method (35) was used to determine the nucleotide sequences.

Transport Assays-- Maltose and lactose transport activity was estimated by measuring the uptake of [14C]maltose (360 mCi/mmol) or [14C]lactose (57 mCi/mmol) as described previously (7). When sugars were tested for their ability to inhibit radioactive sugar uptake, cells were incubated in the presence of inhibitor sugar for 5 s prior to the addition of radioactive substrate. [14C]Maltose was obtained from Moravek Biochemicals, Inc. [14C]Lactose was obtained from Amersham International, plc.

Assay of ATPase Activity in Inside-out Membrane Vesicles-- To measure the ATPase activity of the mutant maltose systems, the respective proteins must be overproduced. We used the strain HS3309, which carries the pMR11 plasmid that has the malK gene under the Ptac promoter. This strain was transformed with either pBR322 as a vector control; pNT11, which carries the malF502 MBP-independent allele under Ptrc; or pt-Lac2SM, which carries the malF540 allele under Ptrc. Inside-out membrane vesicles were prepared from transformants induced with isopropyl-1-thio-beta -D-galactopyranoside, and ATPase activity was measured as described (14, 36). Protein concentration was determined using the BCA protein assay reagent kit from Pierce.

Construction of the Various malF-amber Mutants-- The plasmids carrying mutations in malF resulting in amber stop codons in place of amino acids Glu39, Tyr55, Glu130, Lys275, and Tyr317 were constructed by site-directed in vitro mutagenesis (37). The following oligomers were used in this experiment: GM1, 5'-GGCGAACAGGTACTACCCTTGTGCG-3', Glu39 to amber stop; GM2, 5'-CGATTGGCGAAAATCTACAGCCCCG-3', Tyr55 to amber stop; GM3, 5'-CGCCAGTTGCCACTAATCGCCCGC-3', Glu130 to amber stop; GM4, 5'-GGCGAGGAACGGCTACTGAATGCC-3', Lys275 to amber stop; GM5, 5'-GCAGGACGCTAGACCGGCTTTGC-3', Tyr317 to amber stop.

The underlined bases are those that are changed in the mutants. Single-stranded plasmid DNA was made from a VCS-M13 phage lysate of the dut ung strain CJ236 carrying the pLH9 (wild-type malF and malG) plasmid. The oligomers were phosphorylated and annealed to the template as described (38). Double-stranded pDNA was then synthesized in vitro by Klenow fragment and circularized by T4 DNA ligase. An aliquot of the reaction mixtures was used to transform DH5alpha . Plasmid DNA was purified from individual transformants and analyzed by enzymatic digestion, and the nucleotide sequence of the region of interest was determined to verify the presence of the amber mutations.

Protein Gel Electrophoresis-- Gel electrophoresis was carried out by the method of Laemmli (39). Samples were diluted in sample buffer containing 5% (v/v) 2-mercaptoethanol and heated in a boiling water bath for 5 min. Electrophoresis was carried out in 12% polyacrylamide gels. Protein was visualized by staining the gels with 0.2% Coomassie Brilliant Blue R-250 in methanol-acetic acid-water (5:1:5) and destaining in 7.5% acetic acid, 5% methanol.

Membrane Protein Solubilization-- We examined the ability of Triton X-100 to solubilize the MalFGK proteins as described previously (14) with some alterations. 250 µg of protein from the inside-out membrane vesicles was incubated in 1.0 ml of 50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 5 mM MgCl containing 2% (v/v) Triton X-100 for 1/2 h. The samples were centrifuged at 14,000 × g in a microcentrifuge for 20 min. The supernatant was carefully removed, and the pellet was resuspended in 50 µl of 100 mM Tris-HCl; 5 µl of this sample was loaded on gels for Coomassie staining and 15 µl for Western blots after boiling for 4 min along with 3 µl of 50% glycerol and 10 µl of sample buffer. The supernatant was added to 2.0 ml of cold ethanol and placed on dry ice for 1 h. The samples were centrifuged at 12,000 × g in a microcentrifuge for 30 min at 4 °C. The ethanol supernatant was removed, and the percipitated proteins were resuspended in 40 µl of 100 mM Tris-HCl, pH 7.5. 6 µl of this sample was loaded on gels for Coomassie staining and 17 µl for Western blots after boiling for 4 min along with 3 µl of 50% glycerol and 10 µl of sample buffer.

Immunodetection by Western Blotting-- Proteins were transferred from SDS-polyacrylamide gels to sheets of nitrocellulose (BAS 0.45-µm pore size, Schleicher & Schuell) by electroblotting (20 V, overnight) as described elsewhere (40). The nitrocellulose sheets were blocked with 5% (w/v) nonfat powdered milk in TBST for 1 h and then incubated with the appropriate dilution of anti-MalK or anti-MalF rabbit antibody. After extensive washing with TBST, goat anti-rabbit IgG coupled to peroxidase was added for 1 h. The resulting blot was then developed using the ECL-Western blotting kit from Amersham Life Science.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of the Lactose Specificity Mutant-- The indicator strains used to detect altered substrate specificity mutants of the maltose transport system that transport lactose share some common attributes. They all have a deletion of the chromosomal lactose operon but carry the lacZ gene on a plasmid or F'-episome. Thus, these strains can metabolize intracellular lactose but are incapable of transporting it into the cell. The strains also carry the malTp1Tp7 allele (41) ensuring constitutive mal gene expression in all growth media.

Plasmids carrying either wild-type malF and malG or MBP-independent alleles of these genes under the control of the malB promoter on the pBR322 replicon were mutagenized in the mutD5 mutator strain KD1087 (32). The lactose indicator strains were transformed with the mutagenized plasmids, and mutants were selected for the ability to utilize lactose as a sole carbon source on minimal lactose plates. To confirm that the Lac+ phenotype is linked to the plasmid, prospective mutant plasmids were retransformed into the lactose indicator strains and evaluated on minimal lactose plates.

Although many plasmid-linked mutants were isolated by this technique, only one mutant displayed a strong Lac+ phenotype. This mutant allele, malF540, is carried on the plasmid pLac2. The pLac2 plasmid was derived from the mutagenized pLH5 parent plasmid (42) that carries the MBP-independent allele malF502 (8). It was isolated as an allele that is dominant to wild-type malF and malG because the indicator strain used (GM1305) had all of the chromosomal maltose transport genes intact (Table II).

                              
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Table II
Lactose phenotype in various indicator strains

Mapping of the Lactose Specificity Mutant-- The mutation responsible for the phenotype of the MalF540 mutant was mapped by performing a series of DNA fragment exchanges between the pLac2 plasmid, which carries the malF540 allele, and the pLH5 parent plasmid (Fig. 1). These exchanges suggest that the mutation is located proximal to the SacI site on the pLac2 plasmid, somewhere at the very beginning of malF. Not shown in Fig. 1 is the second restriction site used in these experiments, a PstI site located in the ampicillin resistance gene.


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Fig. 1.   Mapping of the lactose specificity mutation. Fragment exchanges between pLac2 and the parent MBP-independent plasmid pLH5 or the wild-type plasmid pLH9 are shown. The lactose phenotype was assayed as growth on minimal 1% lactose plates containing ampicillin after 2 days at 37 °C in strain GM1305.

The PstI-SacI DNA fragment was also used to perform exchanges between the pLac2 plasmid and the pLH9 plasmid (Fig. 1). pLH9 carries the wild-type malF and malG genes. A fragment containing the region proximal to the SacI site from pLac2 was sufficient to confer a Lac+ phenotype to pLH9. These exchanges indicate that the malF502, MBP-independent mutations are not necessary for the Lac+ phenotype observed with pLac2, these mutations being located distal to the SacI site.

The malF540 and malG+ genes of pLac2 were sequenced using the dideoxy method (see "Materials and Methods"). A guanine to adenine mutation was found. This mutation causes a glutamine codon to be converted to an amber stop codon (Q99(Am)). Thus, in the pLac2 plasmid, only a small portion of MalF containing the first three transmembrane helices is being translated (Fig. 2).


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Fig. 2.   Topological map of the MalF and MalG proteins. This model is based on the results obtained with malF-phoA and malG-phoA hybrids. The positions of the amino acid residues altered in the lactose specificity mutant MalF540 (Q99Amber) and the MBP-independent mutant MalF502 (G338R and A502V) are indicated.

The Lactose Specificity Mutant Does Not Transport Maltose-- To find out if the mutation had any effect on the ability of cells to utilize maltose as a sole carbon and energy source, we evaluated the growth of a strain that makes wild-type MBP (GM1035) and a strain that does not (GM1042) on minimal maltose plates when transformed with pLac2 (Table III). These strains carry a chromosomal Tn5 insertion in malE that is polar on malF and malG but does not affect the intact chromosomal malK gene. A plasmid that carries the wild-type malF and malG genes (pLH9) conferred the ability to grow on maltose only in the strain background that contains MBP (GM1035). This was expected, since MBP is absolutely required for the wild-type transport of maltose. The pLH5 plasmid that carries the malF502 MBP-independent allele only permitted growth in the strain background that does not contain MBP (GM1042). MBP-independent mutants, including MalF502, were selected on the basis of being able to utilize maltose in the absence of MBP. MBP-independent mutants cannot utilize maltose in the presence of MBP at physiological concentrations due to a faulty interaction of MBP with the inner membrane complex (MalFGK2) (8). The pLac2 plasmid did not confer a Mal+ phenotype to either strain. It has thus lost the MBP-independent phenotype associated with its parent plasmid pLH5.

                              
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Table III
Effects of MBP on the maltose and lactose phenotypes and the requirement of MalK-ATPase activity for the lactose phenotype of the lactose specificity mutant

The Mal- phenotype associated with the pLac2 plasmid was suppressed by a tRNA amber suppressor, SupF (data not shown), indicating that the Q99(Am) mutation is responsible for the Mal- phenotype.

Lactose Transport by the Lactose Specificity Mutant Requires MalK-ATPase Activity but Does Not Require MBP-- We compared the lactose phenotype of MalF540 in both wild-type (GM1265) and Delta malB101 deletion (GM1191) strain backgrounds carrying the pLH22 plasmid that provides MalK. The Delta malB101 deletion covers all of the genes required for the transport of maltose across the cytoplasmic membrane. The plasmid carrying the malF540 allele and malG+, pLac2, conferred the ability to utilize lactose as a sole carbon source in both the GM1265 and GM1191 strains, as assayed by growth on minimal lactose plates (Table II). Since in both strains MalG and MalK are being provided from either a plasmid or from the chromosome, but only GM1265 produces MBP, this result suggests that MBP is not required for a Lac+ phenotype.

To further evaluate the effect of MBP on the ability of MalF540 to utilize lactose, we utilized a plasmid (pNT7) that carries the malE gene under the control of the malB promoter. The lactose phenotype of pLac2 was observed in a strain harboring the Delta malB101 deletion, the pLH22 plasmid that provides MalK, and either the pNT7 plasmid (GM1304) or the pSC101 vector control (GM1306). The pLac2 plasmid conferred the ability to utilize lactose in the presence or absence of MBP (Table III). Therefore, the MalF540 mutant does not require MBP to transport lactose.

To determine if MalF540 requires MalK-ATPase activity for lactose transport, we utilized a plasmid (pSN1) that carries the malK804 allele (14). This allele has a mutation that causes a lysine to arginine amino acid change at position 42 of MalK. This mutation renders the resulting MalK protein unable to function as an ATPase but still allows formation of stable inner membrane complex. As a positive control, we used a plasmid that carries the wild-type malK gene (pHS4). The lactose phenotype of pLac2 was observed in a strain harboring the Delta malB101 deletion, and either the pSN1 plasmid (GM1308) or the pHS4 plasmid (GM1307). The pLac2 plasmid conferred the ability to utilize lactose only in the strain making a functional MalK protein (Table III), suggesting that MalK ATPase activity is essential for lactose transport by this mutant.

The Lactose Specificity Mutant Does Not Require Full-length MalF to Transport Lactose-- To determine if the region of malF distal to the amber stop codon of MalF540 is required and to evaluate the possibility of a restart protein playing a role in the mutant phenotype, the remaining portion of malF was deleted from the plasmid pLac2, and the resulting plasmid, pLac2SM, was assayed for a lactose phenotype on minimal lactose plates (Table II). The region deleted was from a SacI site located 300 base pairs distal to the amber stop codon, to an MfeI site at the very end of malF. The pLac2SM plasmid still displays a Lac+ phenotype comparable with the pLac2 plasmid when introduced into the lactose indicator strains GM1305 and GM1191. The fact that the pLac2SM plasmid displays a Lac+ phenotype in the deletion strain GM1191 indicates that full-length MalF is not required in conjunction with the amber mutant to transport lactose. To ensure that the deletion itself does not cause a Lac+ phenotype, the same deletion was performed on the wild-type plasmid pLH9. The resulting plasmid, pLH9SM, did not display a Lac+ phenotype.

To directly address the need for a full-length MalF protein, we constructed a strain, GM1368, that has a deletion of the malF gene in the chromosome. Thus, the only MalF being made in the strain will be derived from the plasmid-encoded alleles of the malF gene. This strain was capable of growing on minimal lactose plates when transformed with either the pLac2 or pLac2SM plasmids (Table II), suggesting that MalF is not required.

The Lactose Specificity Mutant Requires MalG to Transport Lactose-- To ascertain if MalG is required for the transport of lactose by MalF540, we took a small fragment containing the malB promoter and the beginning of malF540 carrying the Q99(Am) mutation from pLac2 (up to the BsmA1 site, which is 300 base pairs distal to the mutation) and cloned it into pBR322. The resulting plasmid, pLac2F', did not display a Lac+ phenotype when introduced into the strain GM1191, which is deleted for the chromosomal malB region and has MalK provided off of a plasmid (Table II). However, the plasmid pLac2SM, which carries a similar portion of malF540 and malG+, displays a Lac+ phenotype in the same strain, suggesting that MalG is required for the Lac+ phenotype observed with the pLac2 plasmid.

The Lac+ phenotype in the GM1191 strain is relatively weak, so we decided to address this point in a more direct fashion. We constructed a strain, GM1408, that carries the malGamV67 mutation. This strain makes only the first 129 amino acids of MalG. This strain was capable of utilizing lactose as a sole carbon source, as assayed on minimal lactose plates, when transformed with the pLac2 plasmid but not when transformed with the pLac2F' plasmid (Table II). Only the pLac2 plasmid carries the malG gene, suggesting that full-length MalG is required for a Lac+ phenotype.

Interestingly, enough MalG must be provided relative to the amount of MalK being produced by the cell to observe a strong Lac+ phenotype with MalF540. The pLac2F' plasmid conferred a weaker Lac+ phenotype (+) on strains GM1305 and GM1368 than the pLac2 plasmid (++) (Table II). Both of these strains carry multiple copies of the malK gene. We constructed isogenic strains (GM1361 and GM1369, respectively) that carry an extra copy of the malG gene on the plasmid pGM7. The plasmid pLac2F displays a strong Lac+ phenotype (++) in these strains that is comparable with that observed with pLac2. Furthermore, GM1380 is a Delta malF strain that carries no plasmid copies of malK or malG, so that the only copies of malK and malG are those found on the chromosome. GM1380 transformed with either pLac2 or pLac2F displays a strong Lac+ phenotype.

Growth and Transport Properties of the Lactose Specificity Mutant-- To measure the ability of MalF540 to utilize lactose as a sole carbon and energy source, we determined the generation time of a strain carrying the pLac2 plasmid in M63 minimal lactose liquid media (Table IV). We also measured the ability of the mutant to transport [14C]lactose (Table IV).

                              
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Table IV
Growth and transport properties of the lactose specificity mutant

The pLac2 plasmid conferred shorter doubling times in M63 minimal lactose media than the plasmid pTE18, which carries the gene for lactose permease, lacY. This suggests that the specificity mutant is efficiently utilizing lactose. GM1305, carrying either the wild-type maltose transport genes (malF and malG), on the pLH9 plasmid, or the malF502 MBP-independent allele, on the pLH5 plasmid, did not yield a measurable doubling time.

We compared the kinetics of lactose transport by the lactose specificity mutant with that of the lactose permease system by measuring the initial velocity of [14C]lactose uptake as a function of external substrate concentration. The values obtained from a double-reciprocal plot of the initial velocity of [14C]lactose uptake versus the external concentration of lactose are shown in Table IV. The maximal velocity of lactose uptake (Vmax) conferred by the malF540 allele on the pLac2 plasmid is 250 pmol/min/108 cells. This value is not as high as that observed with lacY+ on the pTE18 plasmid, 330 pmol/min/108 cells, but it does fall within a comparable range. The concentration that results in half-maximal velocity (Km) is 1.3 mM for pTE18, while it is 1.7 mM for pLac2. The apparent contradiction that the doubling time for the strain carrying pTE18 is longer than that for the strain carrying pLac2, while the former transports lactose more efficiently, could be explained by the fact that overproduction of LacY by pTE18 might decrease the growth rate for reasons unrelated to transport activity.

To discount the possibility that the transport of lactose observed is due to the release of cytosolic beta -galactosidase into the medium, we measured the levels of beta -galactosidase in the medium of cells used for transport with the colorimetric reagent ortho-nitrophenyl-beta -D-galactoside. Lactose could be metabolized to glucose by beta -galactosidase in the medium. The glucose units could then be taken up by the phosphoenolpyruvate phosphotransferase system of the cell. We observed similar low levels of beta -galactosidase in the medium of all strains used for measuring transport (data not shown). These low levels of beta -galactosidase are unlikely to account for the observed differences in their ability to transport lactose.

The Specificity of Transport by the Lactose Specificity Mutant-- We examined the specificity of lactose transport by MalF540 by evaluating the ability of different sugars to compete with [14C]lactose uptake (Fig. 3). A collection of 30 available monosaccharides, disaccharides, and trisaccharides was tested at 100 mM to see if any of the sugars inhibited the transport of 1 mM [14C]lactose (data not shown). Sugars that displayed the greatest inhibitory activity were then tested at 10 mM inhibitor concentration (Fig. 3). Only a small number of sugars strongly inhibited [14C]lactose transport at the lower concentration. Those sugars that were tested for inhibitory activity at 100 mM but did not noticeably inhibit included methyl glucopyranoside, arabinose, rhamnose, fructose, xylose, melibiose, raffinose, palatinose, cellobiose, gentiobiose, isomaltose, and fucose.


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Fig. 3.   Inhibition of [14C]lactose uptake by various sugars. Uptake of [14C]lactose by whole cells of the lactose specificity mutant MalF540 was measured at a [14C]lactose concentration of 0.5 mM in the GM1305 background. The concentration of competitor sugar was 10 mM.

Glucose displayed the greatest inhibitory activity. Inhibition by glucose suggests that the lactose specificity mutant is recognizing the reducing moiety of the disaccharide lactose (beta 1,4-glucose-galactose). Inhibition by glucose is unlikely to be the result of PTS factor IIAglu-mediated inhibition, because methyl glucopyranoside, a more potent mediator of IIAglu inhibition, had no measurable effect on lactose transport. ortho-Nitrophenyl-beta -D-galactoside inhibited [14C]lactose transport considerably (>80% inhibition). ortho-Nitrophenyl-beta -D-galactoside is a lactose analog that is used as a colorimetric reagent in assaying for the presence of beta -galactosidase. Lactose transport was also significantly inhibited by maltooligosaccharides, suggesting that the mutant complex can still bind these substrates although it is incapable of transporting them.

The Lactose Specificity Mutant Displays Constitutive ATPase Activity-- We were interested in characterizing the ATPase activity of the MalF540 mutant, because it is thought that MBP is responsible for triggering the ATPase activity of MalK and subsequent transport by the inner membrane complex (18). Since the MalF540 mutant does not require MBP for lactose transport, we speculated that this mutant might display constitutive ATPase activity in analogous fashion to the MBP-independent mutants described earlier.

We have shown that the lactose specificity mutant does not display a Lac+ phenotype when the only MalK provided is one that is ATPase-. To determine if the mutant displays ATPase activity and to ascertain if this activity is constitutive or dependent on the presence of substrate, we assayed the ATPase activity of MalF540. For this purpose, we used a strain background, CHP243, that lacks the F1Fo H+-translocating ATPase due to a deletion of the atp operon, has a deletion of the chromosomal maltose and lactose transport genes, and will allow us to overproduce the maltose transport proteins in an isopropyl-1-thio-beta -D-galactopyranoside-inducible manner. This strain was transformed with the pMR11 plasmid that has the malK gene under the Ptac promoter and pBR322-vector negative control, pNT11, or the pt-Lac2SM plasmid. The pNT11 plasmid served as the positive control, and it has the malF500 MBP-independent allele and malG+ genes under the Ptac promoter. MBP-independent mutants transport maltose in the absence of MBP and display constitutive ATPase activity. The pt-Lac2SM plasmid has the truncated amber malF540 gene and full-length malG+ (derived from pLac2SM) under the Ptac promoter. Everted membrane vesicles were made from these strains with either lactose or sucrose trapped inside of the vesicles. Sucrose was chosen as a sugar that we know has no measurable affinity for the lactose specificity mutant complex (Fig. 3). ATPase activity was quantitated using the Malachite-green ATPase assay method with vesicle concentrations corrected for the amount of total protein present. This assay accurately measures the release of phosphate from extraneously added ATP.

The results with lactose trapped inside of the inside-out membrane vesicles indicate that MalF540 displays ATPase activity that is well above the background level observed with the negative control (pBR322) but lower than the rate of hydrolysis displayed by the constitutive MBP-independent mutant (pNT11) (Table V). The ATPase activity of the lactose specificity mutant appears to be constitutive, since a comparable rate was observed when the vesicles with sucrose trapped inside were assayed (Table V).

                              
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Table V
ATPase activity of everted membrane vesicles

To determine if the amount of MalK that forms an inner membrane complex with the amber piece of MalF540 correlates with the low levels of constitutive ATPase activity we observed, we performed Western blots on the inside-out membrane vesicles used in the ATPase assays (Figs. 4 and 5).


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Fig. 4.   The presence of MalK in whole cells used to make everted membrane vesicles. Equal amounts of whole cells of the strain CHP243 transformed with the indicated plasmid were subjected to electrophoresis on polyacrylamide gels containing SDS. Lanes 1 and 4, PBR322 control; lanes 2 and 5, pNT11 (malF500 MBP-independent mutant allele); lanes 3 and 6, ptac-Lac2s (malF540 lactose specificity mutant allele (Q99Amber)); lanes 4, 5, and 6, from a Western blot probed with anti-MalK antibodies.


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Fig. 5.   Presence of MalK in detergent-soluble fraction of everted membrane vesicles. Equal amounts, with respect to total protein concentration, of everted membrane vesicles of the strain CHP243 transformed with the indicated plasmid were solubilized with Triton X-100. Detergent-soluble fractions were subjected to electrophoresis on polyacrylamide gels containing SDS. Lanes 1, 4, and 7, pBR322 control; lanes 2, 5, and 8, pNT11 (malF500 MBP-independent mutant allele); lanes 3, 6, and 9, ptac-Lac2s (malF540 lactose specificity mutant allele (Q99Amber)). Lanes 4, 5, and 6 are from a Western blot probed with anti-MalF antibodies. Lanes 7, 8, and 9 are from a Western blot probed with anti-MalK antibodies.

We first performed blots on the cells used to make the everted membrane vesicles (Fig. 4). Equal amounts of cells (based on A600) were loaded on two identical SDS-polyacrylamide gels. One of the gels was Coomassie-stained, while the other was transferred onto nitrocellulose and probed with alpha -MalK antibodies. The Coomassie-stained gel indicates that equal amounts of cells were loaded with respect to the three samples analyzed: pBR322, pNT11, and pt-Lac2S. The Western blot with alpha -MalK indicates that all three samples contain the same amount of MalK; any discrepancies can be explained by slight differences in amounts loaded. This result is expected, since all strains carry the pMR11 plasmid that makes large quantities of MalK.

To determine the amounts of MalF and MalK that are associated with the membrane of the vesicles, we detergent-solubilized the samples with Triton X-100 and probed the soluble fractions with alpha -MalF and alpha -MalK antibodies (Fig. 5). Triton X-100 is known to solubilize proteins associated with the inner membrane. The alpha -MalF Western blots show that only the pNT11 sample displays a full-length MalF band, as expected, while the pLac2SM sample does not due to the amber mutation. The alpha -MalK Western blots indicate that the pt-Lac2S sample has some MalK associated with the inner membrane that is substantially more than the pBR322 negative control but substantially less than the pNT11 sample. The small band present in the pBR322 lane is probably representative of MalK-associated nonspecifically with the inner membrane.

Densitometry was performed on all samples on the Western blots (data not shown). Subtracting the lane of background levels of MalK associated with pBR322 from the other samples allowed us to calculate the ratio of MalK in the pt-pLac2S sample to the pNT11 sample as 1:5.4. Normalizing the levels of ATPase activity to the relative amounts of MalK associated with the inner membrane for each of the samples indicates that the MalK from MBP-independent mutant complex (the pNT11 sample) is 1.7 times more active than the MalK from the lactose specificity mutant complex (the pt-pLac2S sample).

Other Amber Mutants and Their Ability to Transport Lactose-- To map the extent of MalF protein that is necessary for promoting lactose transport, we constructed five amber mutants by performing site-directed mutagenesis on the plasmid pLH9, which carries the wild-type malF and malG genes (Fig. 6). E39(Am) contains an amber stop codon after the first transmembrane helix of MalF. Y55(Am) contains an amber stop codon after the second transmembrane helix of MalF. Q99(Am) contains an amber stop codon after the third transmembrane helix of MalF, and this is the original mutation responsible for the Lac+ phenotype conferred by the malF540 allele. E130(Am) contains an amber stop codon in the large periplasmic loop, after the third transmembrane helix of MalF. K275(Am) contains an amber stop codon after the large periplasmic loop of MalF. Y317(Am) contains an amber stop codon after the fourth transmembrane helix of MalF. We examined the maltose and lactose phenotypes of these amber mutants and measured their ability to transport [14C]lactose.


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Fig. 6.   Transport of [14C]lactose by various amber mutants in MalF. Uptake by whole cells was measured at a [14C]lactose concentration of 2 mM in the GM1305 strain background.

The ability of the amber mutants to utilize maltose as a sole carbon source was assayed by observing growth on minimal maltose plates in the maltose indicator strain GM1035 (data not shown). As expected, the plasmid that carries the wild-type malF and malG genes (pLH9) conferred the ability to grow on maltose. Neither the pLac2 plasmid nor the pGM13 plasmid, which carries the Q99(Am) mutation in a pLH9 wild-type background, conveyed any ability to grow on maltose, as expected. All of the plasmids carrying the amber mutations also exhibited a Mal- phenotype.

The ability of the amber mutants to utilize lactose as a sole carbon source was assayed by observing growth on M63 minimal lactose plates in the lactose indicator strain GM1305 (data not shown). The wild-type plasmid pLH9 did not allow growth on lactose. Plasmid pTE18, which carries the lacY gene encoding lactose permease, conferred the ability to grow on lactose. Plasmids pGM13 and pLac2 both conferred the ability to grow on lactose. This was anticipated, since they both carry the Q99(Am) mutation in malF. All of the amber plasmids imparted an ability to utilize lactose that is better than that of the negative wild-type control (pLH9). However, none of the amber plasmids supported growth on lactose comparable with the original lactose specificity plasmid pLac2.

To further differentiate the ability of the amber mutants to transport lactose, we measured [14C]lactose uptake (Fig. 6). The MalF540 mutant transported [14C]lactose at a rate that is comparable with that observed with lactose permease (lacY). The wild-type control transported [14C]lactose at a very low rate. The Q99(Am) mutant, which carries the same mutation as that found in MalF540, transported [14C]lactose at the highest rate of all the amber mutants. Those ambers located proximal to the Q99(Am) transported [14C]lactose much better than those located distally. It appears that inclusion of the large periplasmic loop, found in the E130(Am), K275(Am), and Y317(Am) mutants, greatly reduced the ability to transport [14C]lactose.

These results show that a single transmembrane helix of MalF together with MalG and MalK is sufficient to allow lactose uptake. Inclusion of the second or third transmembrane helices increased, while inclusion of the periplasmic loop greatly decreased, the rate of [14C]lactose uptake.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have isolated a mutant of the maltose transport system that alters its substrate specificity. The wild-type system is incapable of transporting lactose, but the mutant transports lactose and has lost the ability to transport maltose. A Q99(Am) mutation in the MalF protein is responsible for the Lac+ phenotype. In addition, two sugars that do not inhibit transport by the wild-type or MBP-independent transporters do inhibit lactose transport by the MalF540 complex (4, 8).

The fact that the lactose specificity mutant does not require MBP and the first three transmembrane helices of MalF, MalG, and MalK are sufficient to transport lactose, while still retaining substrate specificity, suggests that the integral membrane components of the mutant alone are sufficient to form a substrate site and determine substrate specificity. Recent experiments on the P-glycoprotein (P-gp) system, a eukaryotic ABC transporter, have provided additional evidence that the integral membrane components play an important role in determining the substrate specificity of the system. These experiments include the genetic analysis of chimeric molecules constructed from different members of the P-gp family (43), the isolation and construction of discrete mutations in P-gp (27), epitope mapping and cross-linking studies with substrates and substrate analogs of P-gp (24, 25, 44), as well as energy transfer experiments (23).

[14C]Lactose transport experiments with a collection of amber mutants at strategic locations along MalF indicate that the first transmembrane segment of MalF along with MalG (malF541; E39(Am)) and MalK is sufficient to transport lactose (~67% of malF540) (Fig. 6), while MalG and MalK alone cannot transport lactose (data not shown). Solubilization studies have indicated that MalF and MalK can form a stable complex in the inner membrane but that MalG and MalK do not (14). Others have shown that the first transmembrane helix of MalF can be deleted without seriously affecting the transport of [14C]maltose (~60% of wild type) (45). Since the first helix is not essential for maltose transport, yet the lactose specificity mutant requires this helix for lactose uptake, it is highly unlikely that this helix is directly involved in the transport of substrate. Rather, it is more likely that the helix is allowing MalG to assume the correct conformation in the membrane that is solely responsible for the recognition and transport of lactose. Very similar results have been observed with LacY. A large portion (N-terminal 22 amino acids) of the first transmembrane helix of LacY was found not to be obligatory for lactose transport activity (46). However, a deletion construct of LacY, which contains only the first N-terminal transmembrane helix and the last six C-terminal transmembrane helices, was found to transport lactose efficiently (~80% of wild type) and specifically (47). Furthermore, with over 95% of the residues of LacY mutagenized, the four charged residue that have been shown to be mandatory for lactose transport are located in transmembrane domains of the C-terminal half of the protein (48). Much as in our lactose specificity mutant, a pathway for lactose transport in LacY can be formed solely by the last six putative transmembrane helices.

A mutant of another member of the ABC transporter superfamily, the cystic fibrosis transmembrane conductance regulator, only contains the first six transmembrane helices, the first nucleotide binding domain, and the regulatory domain, yet it is still able to form a regulated ion channel in HeLa cells (49). Sucrose gradient sedimentation studies indicate that the "half molecule" truncation mutant forms homodimers. This mutant is similar in structure to our lactose specificity mutants, in that it only has half of the wild-type transport apparatus, and suggests a possible model for its activity. A subunit composed of the MalG protein and the stabilizing small portion of MalF may form a homodimer with another such subunit, thus forming a transport path for substrate.

The results with the MalF amber mutants indicate that the ability to transport lactose is compromised once a portion of the periplasmic loop between the third and fourth transmembrane helices is included in the construct (Fig. 6). Inclusion of this loop might block the access of the transport path by lactose, or the loop might simply not allow the MalG protein to assume the correct conformation in the membrane due to a steric constraint. Alternatively, the loop might compromise the constitutive ATPase activity of the MalK subunit. Although the periplasmic loop has been shown to be essential for the transport of maltose,2 no specific function has ever been assigned to this domain of the transport apparatus.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Microbiology, College of Physicians and Surgeons, 701 West 168th street, Columbia University, New York, NY 10032. Tel.: 212-305-6913; Fax: 212-305-1468; E-mail: shuman{at}cuccfa.ccc.columbia.edu.

1 The abbreviation used is: MBP, maltose-binding protein.

2 M. Reyes and H. A. Shuman, unpublished results.

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Abstract
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