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Journal of Bacteriology, June 2002, p. 3069-3077, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3069-3077.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The N Terminus of the Escherichia coli Transcription Activator MalT Is the Domain of Interaction with MalY

Anja Schlegel,¹ Olivier Danot,² Evelyne Richet,² Thomas Ferenci,³ and Winfried Boos^1*

Department of Biology, University of Konstanz, 78457 Konstanz, Germany,¹ Unité de Génétique Moléculaire, Institut Pasteur, FRE CNRS 2364, 75724 Paris Cedex 15, France,² Department of Microbiology G08, University of Sydney, Sydney, New South Wales 2006, Australia³

Received 26 December 2001/ Accepted 1 March 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Escherichia coli maltose system consists of 10 genes encoding proteins dedicated to the uptake and the metabolism of maltose and maltodextrins (4). These genes are under the control of MalT, a specific transcriptional activator of 901 amino acids (aa). MalT belongs to a class of bacterial transactivators, the MalT or LAL family (11, 42). They are large proteins (>90 kDa), possess an ATP binding site near their N terminus, and share homology with LuxR near their C terminus. MalT binds and activates its target promoters (29) only in the presence of ATP (34) and the inducer maltotriose (28). MalT consists of four structural domains (11): domain 1 (DT1, aa 1 to 241) binds ATP, domains 2 (DT2, aa 250 to 436) and 3 (DT3, aa 437 to 806) bind the inducer, and domain 4 (DT4, aa 807 to 901) harbors the DNA binding site (11, 43). MalT exists in an equilibrium between a monomeric (inactive) and a monomeric (active) form and is prone to multimerize. This equilibrium is shifted to the active form by the inducer maltotriose, which triggers a conformational change involving DT1, -2, and -3 and the linkers in between. The conformational change, which also requires ATP, is a step towards the formation of a high-order oligomer, the transcriptionally competent form of the protein (11, 36). Point mutations in malT (malT^c) have been isolated that confer a constitutive expression of the maltose regulon when maltodextrin is not present in the growth medium (12, 13) The in vitro analysis of two corresponding MalT^c proteins revealed that, in contrast to the wild-type MalT, they could activate transcription from a MalT-dependent promoter in the absence of maltotriose but could still be stimulated further by maltotriose. The binding affinity for maltotriose was increased in these MalT^c proteins (12).

The E. coli maltose system is unusual for its diverse regulatory input. In addition to the positive effectors, maltotriose and ATP, three proteins are known to be able to interact with MalT, curbing its activity as a transcriptional activator. The physiologically most straightforward is MalK, the energy-providing ABC subunit of the high-affinity and binding protein-dependent maltose/maltodextrin transporter. The working model for its function predicts that, in the absence of transport, under conditions when ATP is bound to but not hydrolyzed by transport complex-engaged MalK, MalT is likely to be sequestered by MalK in monomeric (inactive) form. When substrate is transported, ATP is hydrolyzed. This, in turn, will release MalT, allowing its activation by inducer, and will lead to transcriptional activity (3). The most straightforward support for such a model is the observation that a MalK-LacZ fusion monitoring mal gene expression is low in a merodiploid malK⁺ genetic background but is 20-fold higher (and nearly constitutive) in a background lacking MalK function (6). Thus, when a wild-type strain is growing in the absence of an exogenous inducer, the "uninduced mal gene expression" actually represents strong repression by MalK. The fact that MalK can interact with MalT has been demonstrated biochemically (25). Also, the notion that repression by MalK is governed by transport has been inferred by the phenotypes of malK as well as malF mutants (25).

The second protein interacting with MalT is MalY. This protein was discovered by the isolation of mutations (in malI) that cause repression of a malK-lacZ fusion (31). Repression is caused by the MalY protein whose level is increased in malI mutants. MalY is an enzyme with cystathionase activity. This activity is not required for repression; a mutant lacking the enzymatic activity still shows repressor activity (44). On the other hand, mutants can be isolated that exhibit normal cystathionase activity but are defective in their repressor activity (10). MalY is a negative effector of MalT that competes with maltotriose binding, thereby inhibiting its transcriptional activity. MalY most likely stabilizes MalT in its inactive (monomeric) form (37). The three-dimensional structure of the MalY dimer has been determined, revealing its interaction site with MalT (10).

The last protein known to interfere with MalT function is Aes, an enzyme with acyl esterase activity (20, 26). Cells harboring a plasmid encoding this enzyme under its own promoter are repressed in their mal gene expression (26). Like MalY, Aes is a negative effector of MalT that interferes with maltotriose binding (N. Joly and E. Richet, personal communication).

From their common repression-resistant phenotype in malT^c mutants (26, 30, 32), it appeared likely that the mechanism by which MalK, MalY, and Aes interfere with mal gene expression might be similar. Thus, they might all interact with MalT and keep it in the monomeric (inactive) form. It is not known where in MalT the interaction with these proteins takes place. Here we show that one region in MalT that interacts with MalY is contained in the N-terminal domain, DT1, as defined by Danot (11). Yet this domain alone is not responsible for the interaction with MalK. We analyzed a set of 26 independent mutations in malT that had been isolated for their elevated mal gene expression in a MalK⁺ strain in the absence of exogenous maltodextrins. We found that the binding site for Aes is also likely to be contained in the N-terminal portion of MalT encompassing DT1 and DT2.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains, plasmids, and genetic methods. The bacterial strains and plasmids used in this study are described in Table 1. pACS20 to -24 were constructed by amplifying N-terminal fragments of malT on pOM2 using flanking primers containing an NcoI cleavage site at the 5'end and a HindIII cleavage site at the 3'end. The products were digested by NcoI and HindIII and were ligated into the corresponding sites of pTrcTrx165-189, creating fusions of trxA and six histidine residues (His tag) to the N-terminal site of the malT fragments, with the His tag between trxA and malT⁰. pACS2 was constructed by digesting pRP11 with HindIII and BspHI and by ligating the 2,447-bp fragment into the corresponding sites of pACYC184. Plasmid preparations were carried out with the Qiagen kit (Qiagen GmbH, Hilden, Germany) transformations according to Inoue et al. (18) and Chung et al. (9). Restriction enzymes were used and ligations were done as described by Sambrook et al. (35).

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TABLE 1. Bacterial strains and plasmids used in this study

Strain pop7169 (MC4100 malB107 trp::[KanR-malEp92-lac]_op malT220) was constructed by P1 transductions. malB107 deletes most, if not all, of the malK gene (17). malT220 deletes the entire malT gene (37). The transcriptional malEp92-lac fusion was obtained by inserting the EcoRI-EcoRI malEp92 DNA fragment (29) into the EcoRI site of pRS551 (39), upstream of the lac operon. The fusion was transferred into the trp locus of strain TE2680 as described by Elliott (15) and then brought by P1 transduction into an MC4100 context. The wild-type and the several mutated malT alleles in BW2952 as well as the malT^c genes from HS3018 and RP151-158 were transfered to pop7169 by P1_vir transductions (22). To ensure that the tester strain (pop7169) would obtain the respective malT allele by transduction, the following strategy was used: a P1 lysate of a strain containing ompR::Tn10 (cotransducible with malT) and malT::cam (chloramphenicol resistance) was transduced into all of the above strains harboring a malT allele as well as the wild-type malT. Selection was for tetracycline resistance. Transductants were screened for chloramphenicol sensitivity as well as high ß-galactosidase activity. Subsequently, a P1 lysate was made of the correct transductant and was used to transduce the reporter strain pop7169. This strain has a deletion of malT, lacks MalK, and harbors malE-lacZ as a reporter for mal gene expression. The selection was for tetracycline resistance, while the screen was for high expression of malE-lacZ (blue colonies on 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal]-containing indicator plates). This procedure ensured the presence of the respective malT allele in the tester strain.

Growth conditions. Strains were grown at 37°C in Luria-Bertani broth, tryptone broth, NZA medium (10 g of NZ-amine [Sheffield Products Inc.], 5 g of yeast extract, and 7.5 g of NaCl, all per liter), or minimal medium A (22) supplemented with 0.4% glycerol and 20.5 µg of tryptophan/ml. Ampicillin and chloramphenicol were added at final concentrations of 200 and 30 µg/ml, respectively. X-Gal was used in solid media at a final concentration of 40 µg/ml. Genes under inducible promoters were induced by adding 100 µM isopropyl-ß-D-thiogalactopyranoside (IPTG).

ß-Galactosidase assays. The activities of the lacZ fusions were determined by the method of Miller (22). For measurements of the activities of ME429 and BRE1219 with plasmids encoding MalT fragments, freshly transformed cells were washed from tryptone broth plates and resuspended in Z buffer (22). The activities of pop7169 with different mutations in malT were measured using overnight cultures grown on minimal medium A-glycerol. Strains harboring the malK-encoding plasmid express MalK constitutively and strains harboring malY under tac promoter control were grown in the presence of 100 µM IPTG to fully induce MalY, whereas plasmid-encoded aes was expressed from its natural promoter.

Overexpression of His-tagged MalT fragments. Cells were grown in NZA medium to an optical density at 578 nm of about 0.6, induced with IPTG, and grown for a further 3 h. Before and after induction, samples were taken for sodium dodecyl sulfate-12% polyacrylamide gel electrophoreis (SDS-PAGE) (21). Cells were disrupted by passage through a French pressure cell and were centrifuged at 12,000 x g for 30 min. Samples were taken from the pellet and the supernatant and were loaded on SDS gels. Gels were run in parallel for Western blotting and immunostaining with antibodies against the His tag according to the method of Harlow and Lane (16), modified according to Towbin et al. (41).

Gel filtration. Gel filtration was performed in a Smart system (Amersham Pharmacia Biotech). Thirty-microliter samples containing either MalY (20 µM; a generous gift of T. Clausen) or DT1H (24 µM; purified as described earlier [11]) or both in a Tris-HCl, pH 7.7, buffer (containing 0.2 M KCl, 1 mM MgCl₂, 0.1 mM EDTA, 0.1 mM 5'-adenylylimidodiphosphate, 10 µM pyridoxal 5'-phosphate, and 10% sucrose) were incubated for 20 min at 0°C. Twenty microliters of the mixture was injected through a Superose 12 column (PC 3.2/30; Amersham Pharmacia Biotech) equilibrated with the same buffer without sucrose. Control experiments were carried out under the same conditions except that DT1H was replaced by DT3 (24 µM), purified as described earlier (11), and that no 5'-adenylylimidodiphosphate was present in the buffer. Filtration was performed at 4°C at a flow rate of 40 µl/min. Fifty-microliter fractions were collected when needed. Markers used to calibrate the column were aldolase (158 kDa; Boehringer Mannheim, Mannheim, Germany), bovine serum albumin (66 kDa; Sigma Aldrich, Deisenhofen, Germany), carbonic anhydrase (29 kDa, Sigma Aldrich), and cytochrome c (12.5 kDa; Boehringer Mannheim).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The N-terminal 250 aa of MalT are sufficient for the interaction with MalY in vivo. Plasmids expressing five different N-terminal MalT fragments were constructed. They encode the first 325, 250, 200, 150, and 100 N-terminal aa of MalT. For better stabilization, they were fused to the TrxA protein, and an N-terminal His tag was attached for easy purification. Expression was monitored by SDS-PAGE followed by Western blots using anti-His-tag antibodies. With all plasmids, low-level expression occurred without the addition of IPTG, resulting in similar amounts of protein. Induction by IPTG led to strong overexpression (Fig. 1). Crude extracts obtained after passage through a French pressure cell were centrifuged and submitted to SDS-PAGE. The gels showed that IPTG induction caused the formation of inclusion bodies (data not shown), and it was therefore avoided.

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FIG. 1. Western blots of cells overproducing MalT fragments performed with anti-His-tag antibodies. Strain ME429 was grown with or without the addition of 100 µM IPTG. M, prestained protein standards. (A) Lanes 1 and 2, 325-aa fragment, lanes 3 and 4, 250-aa fragment; lanes 5 and 6, 250-aa fragment; and lanes 7 and 8, 200-aa fragment. Lanes 2, 4, and 6 are from IPTG-induced samples. (B) Lanes 7 and 8, 150-aa fragment; lanes 9 and 10, 100-aa fragment; and lanes 11 and 12, vector. Lane 8, 10, and 12 are from IPTG-induced samples. The numbers on the left and right of the blots indicate the sizes of the marker proteins in kilodaltons.

Strain ME429 was transformed with these plasmids. This strain carries a malK-lacZ fusion to monitor mal gene expression. Normally, in an otherwise wild-type background, the expression of the malK-lacZ is nearly constitutive due to the very low uninduced levels of MalY and Aes (26, 44) and to the absence of a functional MalK protein. Here ME429 carries a Tn10 insertion in malI, the gene coding for the repressor of the malX malY operon, leading to elevated levels of MalY. Thus, the activity of the malK-lacZ fusion is low in ME429 due to MalY-MalT interaction. The plasmid-encoded N-terminal MalT fragments were tested for their ability to interfere with the MalY-MalT interaction, thus lifting malK-lacZ activity. ß-Galactosidase activities with the uninduced plasmids were recorded (Table 2). Strong derepression was observed with the 325-aa fragment compared to the result with the vector only, indicating titration of MalY. The 250-aa fragment also interacted with MalY, but the effect was much weaker. The 200-aa fragment had no effect, whereas the two smallest fragments even reduced the activity (Table 2). The increase in MalK-LacZ activity caused by the 325- and the 250-aa MalT fragments was not observed in a strain that does not express MalY and contains wild-type MalK (Table 3). Also, the largest and smallest fragments of MalT did not change or alter the expression of a malK-lacZ transcriptional fusion in BRE1162, which lacks MalK (data not shown). These data suggested that the N-terminal 250 to 300 aa of MalT are interacting with MalY.

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TABLE 2. Expression of malK-lacZ in ME429 with plasmids encoding MalT fragments

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TABLE 3. Expression of malK-lacZ in BRE1219 with plasmids encoding MalT fragmentsa

Domain 1 (aa 1 to 241, DT1) of MalT coelutes with MalY during gel filtration. The only structural domain of MalT contained in all fragments causing derepression is DT1. To show that the titration effect observed in vivo is actually due to a direct physical interaction between DT1 and MalY, purified MalY and DT1H (a C-terminally His-tagged version of DT1 [11]) were subjected to gel filtration (Fig. 2), either separately or in a 1:1.2 mixture. DT1H alone (28 kDa) elutes as a globular protein of 20 kDa, while MalY alone elutes as two peaks with apparent molecular masses of approximately 150 and 72 kDa, corresponding to the tetrameric and dimeric forms of the protein. The pattern obtained with the mixture is different from the sum of the patterns of the proteins alone. All peaks are shifted towards lower elution volumes (for instance, the MalY dimer peak now shows an apparent molecular masses of 92 kDa), and the DT1H peak is smaller and has a different shape. The simplest explanation for these observations is that MalY and DT1H form a complex that slowly dissociates during the filtration; hence, the remaining DT1H peak. This conclusion is corroborated by the finding that the 92-kDa peak indeed contains DT1H in addition to MalY, as shown by analysis of the fractions collected during the filtration (Fig. 2, insert). This DT1H-MalY interaction is specific since no complex could be observed when DT1H was replaced by DT3, another domain of MalT, at the same molar concentration (data not shown). Along with the in vivo results, this clearly demonstrates that DT1 contains part of the interaction site for MalY.

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FIG. 2. Molecular sieve chromatography of N-terminal MalT fragments with MalY. Samples were preincubated and injected on a Superose 12 column as described in Materials and Methods. The two dotted lines correspond to injections of MalY alone or DT1H alone, as indicated. The solid line corresponds to the filtration of the mixture of the two proteins. Ve stands for elution volume, Vo for exclusion volume, and Vt for the total volume of the column. Insert: Coomassie blue-stained SDS-PAGE gel loaded with 50-µl fractions from Ve = 0.85 ml to Ve = 1.7 ml and put to scale with respect to the graph.

The N terminus of MalT does not titrate MalK. Plasmids pACS20 to -24 harboring the N-terminal fragments of MalT were transformed into BRE1219. This strain is malK⁺ but carries in addition a malK-lacZ fusion to monitor mal gene expression. In contrast to a malK strain, the MalK-LacZ activity of BRE1219 (malK⁺) is low due to the inhibition of MalT by chromosomal levels of MalK. Interference of the N-terminal fragments of MalT with the MalK-MalT interaction should easily be detected by an increase in MalK-LacZ activity. However, this was not observed with any of the MalT fragments (Table 3). This indicates that the targets of MalK and MalY on MalT are different and that DT1 does not contain major determinants of the MalK binding site.

Analysis of mutations in malT leading to elevated mal gene expression. We screened all available mutations in malT conferring elevated expression of the maltose regulon in the absence of external maltodextrins to find out whether mutations could be identified that specifically altered the MalK binding site of MalT. For this purpose we transduced wild-type malT as well as all different malT alleles into the indicator strain pop7169. This strain lacks MalK and harbors a malEp92-lacZ transcriptional fusion for monitoring MalT-dependent mal gene expression (Table 4). malEp92 is a truncated variant of the malEp promoter whose full activation requires a higher concentration of MalT than does malEp⁺ (33) and which is expected to respond to any increase in the concentration of active MalT in an unrepressed context (i.e., free of MalK). pop7169 also does not contain Aes or MalY, since these proteins are not induced under these conditions. The indicator strain has a very low level of malE-lacZ expression in the absence of MalT (0.005 U/mg of protein). With chromosomal wild-type malT, the ß-galactosidase activity (0.15 U/mg of protein) represents MalT-dependent gene expression in the absence of exogenous inducer and without repression by MalK, MalY, or Aes. Replacing chromosomal wild-type malT with the different chromosomal malT alleles, we found that all mutant alleles increased LacZ activity to various degrees, ranging from 3- (T38R) to 18-fold (R9S), over malT⁺ levels (vector column in Table 4; open bars in Fig. 3). These malT alleles had been isolated from glucose-limited chemostat cultures (24) where the selection was for higher expression of MalT-dependent lamB expression. Previously isolated malT^c mutations (12, 13) were included in this analysis. Here again, a similar range of 5- (malT^c1) to 11-fold (malT^c28) stimulation of LacZ activity over wild-type MalT-controlled mal gene expression was observed. Thus, all 26 available mutant malT alleles caused elevated mal gene expression in the absence of MalK, MalY, or Aes. A mutation in MalT leading exclusively to a loss of MalK interaction should have shown wild-type MalT behavior (0.15 U/mg of protein) in this test. Such a mutation was not present among the 26 mutants tested.

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TABLE 4. ß-Galactosidase activity (U mg-1) of pop7169 with wild-type and mutant MalT proteins in the presence and absence of plasmid-encoded MalK, MalY, and Aesb

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FIG. 3. Sensitivity of wild-type (wt) and mutant MalTs for the three repressors MalK, MalY, and Aes. The data contained in Table 4 are shown. mal gene expression in the absence of MalK, MalY, and Aes (empty bars) or in the presence of plasmid-encoded MalK (bars with slanted stripes) or plasmid-encoded MalY (black bars) or plasmid-encoded Aes (bars with horizontal stripes) is given. (A) class 1 mutants; (B) class 2a mutants; (C) class 2b mutants. The positions of the mutations in the primary sequence as well as the exchanged amino acids are indicated.

Since MalT is known to interact not only with MalK but also with MalY and Aes, we tested in vivo the sensitivity of these mutant MalT proteins towards the three repressor proteins. We introduced the genes encoding these proteins on plasmids into the indicator strain pop7169, harboring the various malT alleles, and analyzed the effect of plasmid-encoded MalK, MalY, and Aes on the expression of the chromosomal reporter gene malE-lacZ. Table 4 and Fig. 3 show the data. Under the chosen conditions, mal gene expression controlled by wild-type MalT was inhibited 7.5-fold by MalK, whereas the elevated mal gene expression caused by the MalT mutant proteins was inhibited by MalK from 3.3- (T38R) to 52-fold (R242C) (pmalK column in Table 4). The inhibition by plasmid-encoded MalY was 7.5-fold in the case of wild-type MalT and ranged from 2.9- (A240E) to 21.5-fold (T38R) with the mutant MalTs (pmalY column in Table 4). With plasmid-encoded Aes, mal gene expression controlled by wild-type MalT was inhibited more than 7.5-fold, whereas several MalT variants were hardly affected (only one- to twofold inhibition; paes column in Table 4). However, with a few malT alleles, such as P10Q or I37L or S358I, mal gene expression was reduced more than 20-fold. Thus, these mutant MalT proteins were as sensitive to Aes as wild-type MalT or even more sensitive (>7.5- versus 20-fold inhibition). The data are presented in graphic form in Fig. 3. Depicted in this figure is malE-lacZ expression controlled by the different malT alleles in the absence of repressors (open bars) and in the presence of plasmid-encoded MalK, MalY, or Aes (differently shaded bars).

The following conclusions can be drawn from Table 4 and Fig. 3 and 4: all malT mutations were clustered in three regions (A to C) of the N-terminal third of MalT. A is situated at the very N terminus (aa 5 to 65), being in or near the ATP binding site (contained within DT1). Most mutations were found in B (aa 219 to 244), which corresponds to the C-terminal region of DT1 and the linker between DT1 and -2 (11). The remaining mutations cluster in C, within aa 302 to 359, i.e., in DT2, a domain known to be involved in the binding of maltotriose (11). No mutation was found in the linker region between DT2 and -3. Note that the previously isolated malT^c mutations (12, 13) fall within B and C, uniquely. No mutations were found in the last two-thirds of the protein.

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FIG. 4. The positions of 26 mutations in MalT along the primary sequence. All mutations are contained in the N-terminal third of the protein. Squares, class 1 mutations; diamonds, class 2a mutations; circles, class 2b mutations. Indicated are the four domains DT1, -2, -3, and -4, as well as the linker region. The positions of the mutations in the primary sequence as well as the exchanged amino acids are indicated.

The MalT mutant proteins showed different sensitivities to the three repressors allowing the distinction of two classes (Fig. 3). In the first class (Fig. 3A), MalT remained largely sensitive to all three repressors. In the second class (Fig. 3B and C) MalT became more resistant to Aes than the wild type. This class can be divided into those mutant MalTs that became more sensitive to MalK than to MalY (class 2a) and those that are more sensitive to MalY than to MalK (class 2b). It is noteworthy that no mutation was found that exhibited high resistance against all three repressors. Looking at the distribution of the mutations along the primary sequence of the protein (Fig. 4), it becomes clear that the different types of mutations do not coincide with the three clusters (A, B, and C) in which the mutations occur.

Top Abstract Introduction Materials and Methods Results Discussion References

 
MalT, the transcriptional activator of the E. coli maltose system, exhibits the peculiar property of interacting with five effectors that either increase or decrease its transcriptional activity. Three of these are proteins acting as negative effectors (repressors); they antagonize the action of maltotriose, the inducer, which activates the protein by promoting its multimerization. All of the available data suggest a model in which MalT exists in an equilibrium between an inactive form stabilized by repressor binding and an active form stabilized by maltotriose binding and prone to multimerization (37). In this work we attempted to learn more about the interaction between MalT and its different protein repressors. Where in MalT do the repressors bind? And are they bound by a common MalT/repressor interface?

The observation that a peptide containing the N-terminal 250 aa of MalT was able to interfere with the MalT-MalY interaction in vivo and that DT1, the N-terminal domain of MalT (aa 1 to 241), coelutes together with MalY during molecular sieve chromatography demonstrates that DT1 contains at least part of the interaction site with MalY. We observed that the fragment containing the first 325 aa of MalT was more potent in interfering with the MalT-MalY interaction than was the 250-aa fragment. This observation suggests a role for DT2 (aa 250 to 436) as an additional surface determinant for MalY binding.

In contrast, our attempts to observe interference of the N-terminal fragments of MalT with MalK-mediated repression failed. Our test system should easily have detected even a slight interference by these MalT fragments as a relief from MalK-mediated repression, since repression due to only basal levels of chromosomally encoded MalK was not saturating (Tables 2 and 3). Since no relief of repression was observed even with the longest fragment, the site of MalK interaction in MalT must differ from the MalY-MalT interaction site. The three-dimensional structure of E. coli MalK was recently modeled based on the established crystal structure of Thermococcus litoralis MalK (1). The positions of the amino acid residues in the regulatory domain of MalK, known to affect the interaction with MalT, did not resemble the patch in the crystal structure of MalY (a hydrophobic cone surrounded by a ring of charged residues) that represents the interaction site with MalT (10). This is consistent with the conclusion that MalK and MalY interaction sites with MalT are not identical.

In order to find out more about the interaction of MalT with its three repressors MalK, MalY, and Aes, we analyzed whether mutations in MalT leading to elevated mal gene expression have different sensitivities towards the three repressors. In addition, we tested whether mutations causing a constitutive phenotype exist that are solely based on a defective interaction with MalK. We constructed an indicator strain harboring a malE-lacZ fusion as the mal gene reporter. The strain lacked MalK and did not produce significant amounts of MalY or Aes. Fitting this strain with chromosomal levels of wild-type malT or with the different malT mutations, we used mal gene expression in the absence or presence of each individual plasmid-encoded repressor as a means to test the interaction of the three repressors with these mutant MalT proteins.

We tested a total of 26 independently isolated malT mutations. Some of the mutants (designated malT^c) were selected for an elevated expression of a malPQ-lacZ fusion (12, 13), while others were selected for faster growth on limiting glucose levels (24). In the latter case, the elevated expression of the receptor (catalyzing increased diffusion of glucose through the outer membrane) was presumably the principle of selection. All of the mutations were located in the N-terminal portion of the protein.

Among the 26 MalT mutations, there were none that exhibited wild-type mal gene expression in the absence of MalK. This suggests that, even if mutations in the MalK binding site are among these mutations, they will simultaneously shift MalT to its active, multimeric form. Considering the large number of mutations at hand, one is tempted to deduce that a mutation exclusively abolishing the interaction with MalK cannot exist. Thus, interaction with MalK might occur at a position in MalT that is engaged in the activation (multimerization) mechanism.

When one looks at the ability of mutant MalTs to be inhibited by the three repressors MalK, MalY, and Aes, the following conclusions can be drawn. One class of seven mutations (class 1 [Fig. 3A]) is still rather sensitive to all repressors, even though not always to the same extent. Thus, increased mal gene expression in these MalT mutants must have been caused solely by a change in the equilibrium towards the active form of MalT without altering appreciably the binding sites for any of the repressors. The mutants of the second class (classes 2a and -b [Fig. 3B and C]) are characterized by a high resistance to Aes. Among those, 8 were more sensitive to MalK than to MalY and 11 were more sensitive to MalY than to MalK. Among the class 2 mutations, several are highly resistant to Aes. These mutations occur at positions 38, 219, 220, 240, 242, 243, 244, 317, and 359. Among those, 38, 220, 243, and 359 are still sensitive to MalY. This indicates that at least the latter positions are part of the Aes binding site and not just causing conformational alterations of the N terminus. For one of the mutants (T38R), the participation in the Aes binding site has been verified by in vitro binding assays (19). These positions are in DT1 and DT2 and include the linker region between them (aa 240 to 244). This supports the notion that DT1 and DT2 are involved in inter- or intramolecular interactions taking place in the process of MalT activation and that these domain interactions are interfered by Aes. Since MalY also recognizes determinants in DT1 and -2, the interaction sites in MalT for MalY and Aes may overlap but cannot be identical.

It is worth noting that, with some mutations at the same positions, a class 1 or 2a (position 236) or a class 1 or 2b (position 242) phenotype was obtained depending on the amino acid substituted. These positions are close to or within the linker region between DT1 and -2. This suggests that in some cases the class 1 phenotype might simply be caused by an increased flexibility of the DT1-DT2 linker, which is known to play a key role in the transition between the active and the inactive states of MalT (11).

Recently, the crystal structure of DT3 of MalT was solved (40). In this domain, comprising aa 437 to 806, no mutation was found to confer elevated mal gene expression. Nevertheless, DT3 harbors the binding site for the inducer, whose affinity for maltotriose is counteracted directly or indirectly by the three repressor proteins. This has at least been shown for the interaction of MalT with MalY (37). Computer-aided fitting of the surface structure of MalK known to interact with MalT (1) onto the surface structure of DT3 (40) offered a possibility for a surprisingly tight fit between the two proteins, a starting point for future mutant analysis in search of the MalK-MalT interface.

 
One of us (W.B.) is indebted to Sandy Parkinson and his coworkers (Department of Biology, University of Utah, Salt Lake City), who were very helpful in the initial stages of this work during a sabbatical. We gratefully acknowledge the receipt of strains and plasmids from Michael Ehrmann, Ralf Peist, and Howard Shuman. We thank Tim Clausen for the kind gift of purified MalY.

Work in the Konstanz laboratory was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

 
* Corresponding author. Mailing address: Department of Biology, University of Konstanz, Konstanz, Germany. Phone: 49 7531 882658. Fax: 49 7531 883356. E-mail: Winfried.Boos{at}uni-konstanz.de.

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Journal of Bacteriology, June 2002, p. 3069-3077, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3069-3077.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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