Subunit interactions in ABC transporters: aconserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits
1997; Springer Nature; Volume: 16; Issue: 11 Linguagem: Inglês
10.1093/emboj/16.11.3066
ISSN1460-2075
Autores Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoArticle1 June 1997free access Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits Michaël Mourez Michaël Mourez Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25 rue du Dr Roux, F75645 Paris, cedex 15, France Search for more papers by this author Maurice Hofnung Maurice Hofnung Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25 rue du Dr Roux, F75645 Paris, cedex 15, France Search for more papers by this author Elie Dassa Elie Dassa Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25 rue du Dr Roux, F75645 Paris, cedex 15, France Search for more papers by this author Michaël Mourez Michaël Mourez Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25 rue du Dr Roux, F75645 Paris, cedex 15, France Search for more papers by this author Maurice Hofnung Maurice Hofnung Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25 rue du Dr Roux, F75645 Paris, cedex 15, France Search for more papers by this author Elie Dassa Elie Dassa Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25 rue du Dr Roux, F75645 Paris, cedex 15, France Search for more papers by this author Author Information Michaël Mourez1, Maurice Hofnung1 and Elie Dassa1 1Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25 rue du Dr Roux, F75645 Paris, cedex 15, France The EMBO Journal (1997)16:3066-3077https://doi.org/10.1093/emboj/16.11.3066 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The cytoplasmic membrane proteins of bacterial binding protein-dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA---G---------I-LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose-negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane-bound, as in wild-type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose-negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain. Introduction In bacteria, high affinity uptake of nutrients is achieved by complex substrate binding protein-dependent transport systems. These multi-component permeases consist of one periplasmic substrate-binding protein, two hydrophobic cytoplasmic membrane proteins and two subunits of a peripheral cytoplasmic membrane protein that displays ATP-binding motifs. The cytoplasmic membrane proteins form a complex with a stoichiometry of two ATP-binding subunits and two integral membrane subunits that mediates the ATP-dependent translocation of the substrates into the cytoplasm (Bishop et al., 1989; Davidson and Nikaido, 1991). Such a characteristic organization is an intrinsic property of the superfamily of ABC (ATP Binding Cassette) transporters (Higgins, 1992) or Traffic ATPases (Ames and Lecar, 1992) widely distributed among living organisms. The maltose transport system of Escherichia coli is one of the most studied bacterial ABC transporters and its functional mechanism is known to some extent (see Boos et al., 1996 for a review). Genetic, and more recently biochemical approaches, have led to a functional model that is summarized below. Maltose and maltodextrins enter the periplasm by facilitated diffusion through a specific outer membrane porin coded for by the lamB gene, which is specifically required for maltose transport at sub-micromolar concentrations and for maltodextrins at all concentrations (Szmelcman and Hofnung, 1975). In the periplasm, maltose-binding protein (MBP) binds substrates at high affinity (KD = 1 μM). Upon binding of substrates, MBP undergoes a conformational change (Szmelcman et al., 1976) and interacts with a cytoplasmic membrane complex made of MalF, MalG and two subunits of MalK (MalFGK2), and very likely with hydrophobic membrane proteins MalF and MalG (Hor and Shuman, 1993) that carry also a substrate binding site(s) (Treptow and Shuman, 1988). Recent results obtained with a proteoliposome-reconstituted transport system suggest that MBP transmits through MalF and MalG a signal to MalK allowing it to hydrolyze ATP (Davidson et al., 1992). An important step in understanding maltose transport mechanism is the analysis of protein–protein interactions. The existence of mutations allowing maltose transport in the absence of MBP led to the identification of sites on MalF and MalG thought to be important for the recognition of MBP (Covitz et al., 1994). We are interested in the identification of regions of hydrophobic membrane proteins involved in the interaction with the MalK ATPase. By comparing the sequences of hydrophobic membrane proteins from several binding protein-dependent transporters, we identified a conserved sequence (EAA---G---------I-LP) located at a distance of ∼100 residues of their C-terminal ends (Dassa and Hofnung, 1985b). This 20 amino acid sequence (called thereafter EAA region) is hydrophilic and lies in a loop facing the cytosol in all proteins of known topology (Saurin et al., 1994). It has been proposed that it might constitute a recognition site for a ligand or a partner common to binding protein-dependent permeases (Dassa and Hofnung, 1985a), which could be the most conserved component in such permeases, namely the ATP-binding proteins (Kerppola and Ames, 1992). This EAA region is probably of functional importance since several mutations have been characterized within that reduce or abolish transport in the maltose (Dassa, 1990, 1993), the iron (III) hydroxamate (Köster and Böhm, 1992) and the phosphate (Webb et al., 1992) uptake systems. To investigate the physiological relevance of the conserved EAA region, we generated substitution mutations in the corresponding regions of MalF and MalG. The most conserved residues were modified and we analyzed the consequences of these changes on maltose and maltodextrins uptake, and on the expression of the proteins. The membrane association of MalK was altered in some uptake-defective mutants. We isolated suppressor mutations in malK that restore maltose uptake and MalK membrane association in these mutants. We discussed a model suggesting that EAA regions constitute a recognition site for the MalK subunits. Results The experimental system We constructed three plasmids to generate easily site-directed mutations within EAA regions. Plasmids pTAZFQ and pTAZGQ carry the malF and malG genes respectively under the control of the tac promoter. Plasmid pTAZFGQ carrying these two genes, was constructed by inserting the BamHI–HindIII fragment from pTAZGQ into pTAZFQ. Each plasmid contains a copy of the lacIQ gene in order to control the expression of cloned genes (Figure 1). pTAZGQ, pTAZFQ and pTAZFGQ were able to complement mutations in PMED34 (MalG−), DHB4 (MalF−) and ED170 (MalF−G−) respectively. To evaluate the expression of cloned genes, particulate fractions were prepared from strain MM140 (ED170 transformed with pTAZFGQ), analyzed by SDS–PAGE, and proteins were revealed by immunoblotting. We compared this amount of protein with the level of expression of MalF and MalG in a total extract of the fully induced maltose transport positive strain MC4100. We found that a concentration of 10 μM isopropyl β-D-thiogalactopyranoside (IPTG) was enough to ensure the production of 110 and 78% of the induced chromosomal levels for the MalF and MalG proteins respectively, as judged from the scanning of Western blots. We analyzed the cellular localization of MalF and MalG (Figure 2). The proteins partitioned in a particulate fraction, from which they could be partially extracted by a buffer containing 1% Triton X-100. As shown previously, a substantial amount of MalF and MalG is still not extracted by the detergent (Dassa, 1990; Panagiotidis et al., 1993). Triton X-100 is a non-ionic detergent known to specifically solubilize cytoplasmic membrane proteins (Diedrich et al., 1977), and has been used to show the membrane localization of the MalFGK2 complex. Thus, we interpret this result to indicate that, under our expression conditions, the bulk of the proteins is correctly assembled in the membrane. Figure 1.Plasmids used in this work. Three compatible replicons have been used to produce simultaneously MalE, MalF and MalG, and MalK. The genes were placed under the control of the ptac or the ptrc promoters. Symbols represent the restriction enzymes used to generate these plasmids (for details see Materials and methods). Download figure Download PowerPoint Figure 2.Cellular localization of MalF and MalG proteins produced by plasmid pTAZFGQ. Strains MM140 and ED170 were grown in LB medium. Plasmid genes were induced using 10 μM IPTG. Cell fractionation was performed as described in Materials and methods and particulate fractions were extracted using 1% Triton X-100. Ten micrograms total protein of the different fractions were separated by SDS–PAGE and transferred onto nitrocellulose membranes. MalF (top) and MalG (bottom) proteins were revealed using polyclonal antibodies, horseradish peroxidase conjugate and ECL detection reagents. Only the relevant part of the membrane is shown. Lane 1, total cellular extract of ED170; lanes 2–6, fractions of MM140; lane 2, cell debris; lane 3, particulate fraction; lane 4, Triton insoluble fraction; lane 5, Triton soluble fraction; lane 6, cytoplasmic fraction. Arrows indicate the positions of MalF and MalG proteins. Stars indicate cross-reacting proteins. Download figure Download PowerPoint Site-directed mutagenesis of conserved residues in the EAA region Starting from the assumption that residues are conserved because they play an important functional role, we mutagenized the most conserved residues in the EAA regions of MalF and MalG. We modified residues located at positions 1 (E), 3 (A or S), 7 (G) and 20 (P) of the consensus of EAA motifs (Saurin et al., 1994). In the absence of structural information on MalF and MalG, we made several changes at each position, modifying the size, the charge and the side chain mobility of the amino acids (Figure 3). The mutations in malF and malG were generated on plasmids pTAZFQ and pTAZGQ respectively, sequenced and eventually recombined on plasmid pTAZFGQ as described above. Each mutant was identified by a set of two symbols separated by a slash. The first symbol describes the changes effected in malF and the second those in malG. A non-mutated gene was represented by a hyphen. Numbers between letters point to the relative position of the residue in the consensus sequence. For example, E1A/A3L means that the E residue in the EAA region of MalF was changed to A and that the A residue in position 3 of MalG was changed to L. Figure 3.Amino acid substitutions introduced in malF and malG genes. The sequence for the consensus of the EAA region of disaccharide uptake hydrophobic membrane proteins is shown on top with the relative positions of residues used to identify EAA mutations. The origin of the numeration is set on the E of the consensus sequence. The corresponding sequences of malF and malG are displayed below. Substituted amino acids in mutants are shown under the wild-type sequence. Download figure Download PowerPoint Twenty three plasmids were constructed, each carrying a substitution in MalG (nine mutants), in MalF (nine mutants) or in both proteins (five mutants). The growth rate of mutants induced by 10 μM IPTG in glycerol-supplemented minimal medium and in ML medium (data not shown) was similar to that of strains MM141 (MalF−G−) and MM140 (MalF+G+). Hence, the expression of mutated proteins does not determine major growth defects in bacteria. Particulate fractions of mutants were separated on SDS–polyacrylamide gels and analyzed by immunoblotting (Figure 4 and Table I). Mutated proteins have electrophoretic mobilities similar to that of the wild-type. There are variations in protein amounts with a 2- to 3-fold difference between extreme values, with the exception of the MalG level in E1K/A3L, E1L/E1L and S3K/A3D mutants, where there is a 4-fold increase as compared to MM140. This higher level of expression was not investigated further. Each mutant protein was correctly inserted in the cytoplasmic membrane, as suggested by the fact that it was extracted from the particulate fraction by Triton X-100 as efficiently as wild-type proteins (data not shown). Figure 4.Immunodetection of mutated MalF and MalG proteins. Strain ED170 transformed with wild-type or mutated pTAZFGQ was grown in LB medium. Plasmid genes were induced using 10 μM IPTG and particulate fractions were recovered as described in Materials and methods. Approximately 5 μg of particulate fraction of each construction were separated on SDS–PAGE and transferred onto nitrocellulose membranes. MalF and MalG proteins were revealed as described in Figure 2. Mutants are identified using the conventions described in the text. Only the relevant part of the membrane is shown. Arrows indicate the positions of MalF and MalG proteins. The star indicates a cross-reacting protein. Download figure Download PowerPoint Table 1. Characteristics of EAA mutants Plasmidsa Phenotypeb Doubling timec Initial rate of maltose uptaked Relative amount of proteine MalF MalG pTAZFGQ +++ 238 210 ± 20 1.00 1.00 pTZ18R − >700 700 2±0.3 1.44 2.17 −/G7P − >700 4±0.6 1.21 2.05 E1K/E1K − 305 2±0.2 1.30 2.34 E1K/A3L − >700 700 1±0.1 1.62 4.09 S3K/A3D − >700 ND 1.83 4.53 All experiments are performed in strain ED170. a Mutant plasmids are designated with the conventions described in the text. b Maltose and maltodextrins utilization is assayed on MacConkey plates containing 2% maltose or 1% maltodextrins. No difference was found in the utilization of these sugars. +++, deep red colonies; ++, red colonies; +, pink colonies; −, white colonies. c Doubling times, in min, are measured in synthetic medium 63 supplemented with 0.4% maltose. The turbidity at 600 nm is measured every 30 min or every hour depending of the growth rate and the resulting plots are fitted on an exponential equation. d Maltose uptake is measured at 4 μM final concentration of maltose as described in Materials and methods on strains transformed by the indicated plasmid. Values expressed in pmol maltose/min/108 bacteria are from single experiments. Errors represent the error estimated by fitting to a linear equation the amount of radioactivity incorporated during the experiment. In some cases three independent experiments were made and the standard error of the mean was <15%. e Films of the immunoblots in Figure 4 were scanned using a MasterScan Interpretive densitometer (Scanalytics), quantities of MalF and MalG were normalized to the quantity present in MM140. ND, not determined. Phenotypes of mutants Table I shows the characteristics of mutants. Their phenotypes were scored on MacConkey plates supplemented with maltose or maltodextrins. No Mal+ Dex− or Dex+ Mal− mutants were found. Phenotypes were characterized by measuring growth rates on maltose minimal medium and by determining maltose uptake rates at a substrate concentration of 4 μM. Mutants were classified according to decreasing maltose uptake rates. Class I mutants have initial velocities ranging from 240 to 150, doubling times ranging from 220 to 240 min and they form deep red colonies on MacConkey plates. Class II mutants have initial velocities ranging from 60 to 145, doubling times comprised between 230 and 300 min and they form red to pink colonies. Class III mutants form white colonies, do not grow on minimum maltose medium and do not transport maltose. These three parameters are in general well correlated, with few exceptions that could be explained by the difference of maltose concentration used in phenotypic determinations, in growth rate measurements and in transport assays. Since the mutated proteins are not found in significantly lesser amounts than the wild-type proteins, the defects in maltose-uptake-deficient mutants are most probably not due to a reduced protein expression, nor to a defective localization. E at position 1 is conserved in 50% of EAA sequences and is substituted by D in 12% sequences. Position 3 (A) is conserved in 75% of EAA sequences or is substituted by S, T or C in 20% sequences (Saurin et al., 1994). Our data show that any change affecting the E residue in MalF or in MalG leads to a reduction by 2- to 3-fold in transport rates. Changes made to the third residue of the region (A or S) promote a reduction in transport rates that is related to the nature of the side chain of the replacing residue. A change to a charged residue (K or D) is more detrimental for transport than a change to a short side-chain polar (S) residue. The G residue, invariant in EAA motifs, could be changed to A without incidence on transport. This is in agreement with earlier observations made on the FhuB protein (Köster and Böhm, 1992) and establishes that conserved EAA motifs play similar roles in binding protein-dependent transport systems. The highly conserved P residue at position 20 is not essential for transport. This residue is predicted to be in a transmembrane segment in MalF and in MalG (Boyd and Beckwith, 1989; Dassa and Muir, 1993). It may be concluded that transport is not dependent on a peculiar property of this residue such as a cis–trans isomerization. Substitutions made at the same positions in malG or in malF affect MalG more severely than MalF. At position 3, a substitution to D has a more pronounced effect in MalG (transport defective) than in MalF (reduced transport rate). At position 7, a change to P leads to a completely defective MalG protein while MalF is unaffected. All double mutants having a mutation in malF and in malG, except G7A/G7A, are unable to carry out transport (Class III). However, the single mutants from which the double mutants are made have a transport activity of ∼50–30% of the wild-type (Class II). Cytoplasmic localization of MalK in maltose–uptake-defective mutants The EAA region is located in a cytoplasmic hydrophilic loop in all hydrophobic membrane proteins with a known topology (Saurin et al., 1994). This location makes it a candidate for an interaction site with the ATPase subunit MalK. We reasoned that the phenotypes of our mutants might be explained by a defective or a non-productive interaction with MalK. To test this hypothesis, we analyzed the cellular localization of MalK in Class II and III mutants. As in MM140 and its mutant derivatives the malK gene is chromosomal and under the control of the pmal promoter, maltose-uptake-negative mutants would have reduced to undetectable levels of protein MalK. To overcome this problem, we constructed plasmid pACYK that carries the malK gene under the control of the trc promoter. We found that in MM142 (ED170 transformed with pTAZFGQ and pACYK), a 10 μM IPTG concentration determines an amount of MalK 77% of that in maltose-induced MC4100. In these conditions the amount of MalF is 90% of that in 10 μM IPTG-induced MM140. The presence of pACYK does not alter the phenotype of MM142, since maltose uptake rates at 4 μM maltose and growth rates on maltose minimal medium are identical to those of MM140 (data not shown). We transformed with pACYK a selected set of Class II and Class III mutants and we used strains MM142 and MM143 as controls. We found that particulate fractions of mutants and controls have relative amounts of MalF and MalG as described in Table II. These relative amounts are somewhat different to what was observed without pACYK but remain in the same range of a 2- to 3-fold difference between extreme values. Table 2. Relative amounts of MalG and MalF proteins in presence of pACYK Plasmids WT E1L/− −/E1L E1K/− −/A3L E1L/E1L E1K/A3L −/A3D −/G7P MalF 1.00 0.97 0.66 1.16 0.66 1.15 1.23 0.56 1.28 MalG 1.00 1.20 0.82 1.27 1.02 1.54 1.67 0.51 1.10 Particulate fractions of strain ED170 bearing pACYK and wild-type or mutated pTAZFGQ plasmids were analyzed as in Figure 4. Films of the immunoblots were scanned using a MasterScan Interpretive densitometer (Scanalytics), quantities of MalF and MalG were normalized to the quantity in MM142. WT, wild-type genes. The cellular localization of MalK was determined as described in Materials and methods (Figure 5). In MM142 (MalF+G+), 80% of MalK is found in the particulate fraction and it is efficiently extracted by Triton X-100. In MM143 (MalF−G−), ∼60% of MalK is found in a soluble fraction. The residual MalK protein present in the particulate fraction is hardly extracted by Triton X-100, but the Triton-insoluble pellet could be solubilized by using 6 M urea. By comparison, the Triton-insoluble fraction of MalG is insoluble in urea, consistent with its membrane association (data not shown). This suggests that the Triton-insoluble MalK fraction might constitute aggregates that sediment artefactually with the membranes as has been previously observed by other groups (Reyes and Shuman, 1988; Walter et al., 1992a). Thus we interpret our results as follows: the amount of MalK found in the Triton X-100 soluble fraction (Figure 5, Ts) represents the fraction correctly associated in the membrane, whereas the amount of protein found in the soluble fraction (Figure 5C) and in the Triton insoluble–urea soluble fraction (Figure 5, Us) represents the fraction of MalK which is not associated with the membrane and is consequently located in the cytoplasm. The Triton insoluble–urea insoluble fraction (Figure 5, Ui) contains membrane proteins not extractable by Triton X-100 and most notably outer membrane proteins (data not shown). Figure 5.Cellular localization of MalK. Strain ED170 transformed with pACYK and wild-type or mutated pTAZFGQ were grown in LB medium, plasmid genes were induced using 10 μM IPTG. Cell fractionation, Triton X-100 and urea solubilizations were performed as described in Materials and methods. Equal volumes of the different fractions, corresponding to ∼20 μg of total protein, were separated on SDS–PAGE and transferred onto nitrocellulose membranes. MalK protein was revealed using a polyclonal antibody and the ECL detection kit. The amount of protein in each fraction was evaluated using a MasterScan Interpretive densitometer (Scanalytics), and the percentage of protein located in each fraction was deduced. The graph shows these deduced percentages in Class II and Class III mutants and control strains. Only the fractions recovered at the end of the fractionation procedure are shown. C, cytoplasmic fraction; P, particulate fraction; Ts, Triton X-100 soluble fraction of P; Ti, Triton X-100 insoluble fraction of P; Us, urea soluble fraction of Ti; Ui, urea insoluble fraction of Ti. MalF+G+, MM142; MalF−G−, MM143; MalF+G−, MM144; MalF−G+, MM145. Download figure Download PowerPoint We analyzed the cellular localization of MalK in strains MM144 (MalF−G+) and MM145 (MalF+G−). In both cases, the bulk of MalK was present in the soluble and in the Triton insoluble–urea soluble membrane fraction. The roles of MalF and MalG in the membrane association of MalK are controversial. Earlier observations showed that MalK was found in a cytoplasmic fraction in a mutant defective in the malG gene (Shuman et al., 1980). More recently, the same group showed that MalF alone was able to direct MalK to the cytoplasmic membrane (Panagiotidis et al., 1993). Our results support the idea that both MalF and MalG are needed for the correct membrane association of MalK. The cellular localization of MalK in mutants was also evaluated. Two of the five Class III mutants tested, −/A3D and −/G7P, behaved as MalF−G− deficient strains although they expressed these proteins. One Class III mutant, E1K/A3L, and most of the Class II mutants, E1K/−, E1L/− and −/E1L behaved as the wild-type strain. In mutants E1L/E1L, −/A3L of Class III and Class II respectively, MalK had an intermediate distribution. The fact that one of the Class III mutants behaved as the wild-type strain suggests that there are at least two different types of defects introduced by mutations in the EAA region, one that results in the cytoplasmic localization of MalK and one that maintains the correct membrane association of MalK. It is noticeable that the two mutations that completely dislocate MalK are substitutions in the EAA region of MalG. This is consistent with the observation that MalG is important for the constitution of the membrane complex. We asked whether substitutions located outside the EAA region might have the same effects. We analyzed the cellular localization of MalK in a strain carrying a derivative of pTAZFGQ, having a substitution D262K affecting a residue located in the last transmembrane segment of MalG (P.Lambert and E.Dassa, unpublished). Despite the fact that this strain is completely defective for maltose uptake, the cellular localization of MalK is not affected. Mutations in the malK gene restore transport in maltose-negative mutants We then asked whether mutations of the malK gene would restore maltose uptake. Plasmid pACYK was mutagenized in vitro by hydroxylamine and was used to transform ED170 harboring maltose-negative mutants −/G7P, E1K/A3L and E1L/E1L. These strains carry a chromosomal wild-type copy of malK. We decided to isolate mutants in such a background for the following reasons. First, mutant −/G7P has a wild-type allele of malF that might not function in the presence of a mutated allele of malK able to correct the defect present in malG. Second, as observed above, mutations in malF produce proteins able to support maltose transport to some extent by contrast with malG mutations that lead to completely defective proteins. We therefore suspected that in double mutants E1K/A3L and E1L/E1L, mutated MalF proteins would not accommodate malK mutations able to correct defects of malG mutations. Nineteen maltose-positive colonies, scored on MacConkey maltose medium, appeared after 2 days incubation on strains −/G7P, E1K/A3L and E1L/E1L. pACYK DNA was purified from these clones and transformed again into strain ED170 carrying the respective pTAZFGQ mutated plasmids. Eleven transformants became maltose positive in these conditions, indicating that the phenotype was indeed linked to the mutagenized pACYK plasmid. We replaced the wild-type gene of pACYK by malK genes from the mutated plasmids and transformed the corresponding pTAZFGQ mutants with these constructs. Eight subclones were maltose positive, thereby demonstrating that a mutation(s) in malK was responsible for the phenotypic change. The three remaining subclones malK301, malK306 and malK308, all isolated in a E1L/E1L background, formed white colonies on MacConkey maltose plates. This is probably due to the presence of a mutation outside of malK in the original plasmid. To assess if such a mutation could be sufficient to restore maltose transport, we replaced the malK allele of these plasmids by a wild-type copy from pACYK. The resulting phenotype was white. These experiments strongly suggest that a mutation in malK and an additional mutation(s) elsewhere on the plasmid are needed to determine a maltose-positive phenotype in these clones. Such a mutation would probably raise the cellular level of the mutated MalK proteins, either by increasing the transcriptional level of the gene or the plasmid copy number. Indeed, we found that the expression of MalK in these three alleles was at least twice the normal level (Table III). DNA sequencing of the mutants (see below) revealed that no mutation was found in the ptrc promoter of malK. Table 3. Identificati
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