JOURNAL OF BACTERIOLOGY, Apr. 1991, p. 2180-2186
Vol. 173, No. 7
0021-9193/91/072180-07$02.00/0 Copyright © 1991, American Society for Microbiology
The Activities of the Escherichia coli MalK Protein in Maltose Transport, Regulation, and Inducer Exclusion Can Be Separated by Mutations SABINE KUHNAU,1 MORAIMA REYES,2 AMANDA SIEVERTSEN,2 HOWARD A. SHUMAN,2 AND WINFRIED BOOS'* Department of Biology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany,' and Department of
Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 100322 Received 5 November 1990/Accepted 23 January 1991
The maltose regulon consists of several genes encoding proteins involved in the uptake and utilization of maltose and maltodextrins. Five proteins make up a periplasmic binding-protein-dependent active transport system. One of these proteins, MalK, contains an ATP-binding site and is thought to couple the hydrolysis of ATP to the accumulation of substrate. Beside its function in transport, MalK has two additional roles: (i) it negatively regulates mal regulon expression and (ii) it serves as the target for regulation of transport activity by enzyme IIIGIc of the phosphotransferase system. To determine whether the three functions of MalK are separable, we have isolated and characterized three classes of malK mutations. The first type (class I) exhibited constitutive mal gene expression but still allowed normal transport of maltose; the second type (class II) lacked the ability to transport maltose but retained the ability to repress the mal genes. Class I mutations were localized in the last third of the gene, at amino acids 267 (Trp to Gly) and 346 (Gly to Ser). Mutations of class H were found at the positions 137 (Gly to Ala), 140 (AGln Arg), and 158 (Asp to Asn). These mutations are near or within the region of MalK that exhibits extensive homology to the B site of an ATP-binding fold. In addition, site-directed mutagenesis was used to add or remove one amino acid in the A site of the ATP-binding fold. Plasmids carrying these mutations also behaved as class II mutants. The third class of maiK mutations resulted in resistance to the enzyme IHlGlc_mediated inhibitory effects of a-methylglucoside. These mutations did not interfere with the regulatory function of MalK. One of these mutations (exchanging a serine at position 282 for leucine) is located in a short stretch of amino acids that exhibits homology to a sequence in the Escherichia coli Lac permease in which a-methylglucoside-resistant mutations have been found.
The Escherichia coli maltose transport system is a typical periplasmic binding-protein-dependent transport system (1, 17, 34). Maltose and maltodextrins are bound by the periplasmic maltose-binding protein, which in turn is recognized by the two integral membrane proteins, MalF and MalG, forming the substrate translocation complex (17, 34). The energy needed for accumulation of the substrate was shown to be mediated by the hydrolysis of ATP, presumably by the MalK protein (11, 24). The MalK protein (35) contains an ATP-binding fold and exhibits sequence homology to the corresponding proteins of other binding-protein-dependent transport systems as well as to a variety of other proteins of procaryotic and eucaryotic origin thought to couple ATP hydrolysis to energy-consuming cellular functions (15, 18, 19, 38). From its amino acid sequence, it appears that the MalK protein is a soluble protein (10, 16), but localization studies have shown that it is a peripheral membrane protein. The membrane association of MalK requires MalG, one of the integral membrane-bound proteins of the system (35). The genes for maltose transport and maltose utilization are induced by maltose or maltodextrins in the growth medium. The expression of all mal genes requires the presence of the MalT regulator protein that is activated by the inducer (9, 27, 30). In vitro transcription studies using purified MalT protein have shown that of all linear a(1-4)-linked maltodextrins, only maltotriose is able to activate MalT (26). Thus, the induction by external maltodextrins, including maltose,
*
Corresponding author. 2180
should result from the formation of internal maltotriose. At present, it is unclear which enzyme(s) produces maltotriose.
Amylomaltase, the first enzyme in maltose metabolism, is not necessary for this induction, since mutants lacking the enzyme are still inducible by maltose even though they exhibit increased uninduced levels of mal gene expression. Mutants lacking MalK are unable to transport maltose or maltodextrins and no longer utilize these sugars as carbon sources. However, these mutants express the remaining mal genes at 30 to 40% of the fully induced level in the absence of external inducer. It is clear that this constitutive expression in malK mutants is not caused by the lack of transport per se because mutations in malE or in malF coding for the other subunits of the transport system do not result in constitutive mal gene expression (6, 20, 33). Although the MalT protein is necessary for malK-dependent constitutivity to occur, the mechanism of the regulatory function of MalK is still unclear. Previously, we explained the apparent constitutivity of malK mutants by proposing the existence of an internal inducer, different from maltotriose, that is recognized by MalT and is inactivated by MalK (14). Consistent with this model, overexpression of MalK prevents the expression of the maltose genes in malT+ but not in malT (Con) strains (29). We have isolated a mutation in a gene termed malI that results in decreased constitutive mal gene expression in malK mutants. The analysis of mall has revealed that it codes for a typical repressor protein controlling, beside its own expression, expression of an adjacent and divergently oriented gene, malX (28). Since loss of malX together with
VOL. 173,
1991
SEPARATION OF E. COLI MalK ACTIVITIES BY MUTATIONS
mall results in high expression of malK, we concluded that MalX must also be an enzyme inactivating or degrading the internal inducer. The nature of the hypothetical internal inducer and the mode of its synthesis are still unclear. If, as we propose, the internal induction is controlled by the concentration of a sugar molecule, this inducer is likely to be a glucose-containing compound but not of the maltodextrin type (14). It would not be degraded by amylomaltase, and its synthesis would not require ADP-glucose. The level of internal inducer would be responsible not only for the high expression of the mal genes in malK mutants but also for the basal-level expression of the mal genes in wild-type strains. In both cases, the level of endogenous induction is dependent on the osmolarity of the medium, being high only at low osmolarity and being turned off at high osmolarity (6). In contrast, in strains carrying a mutation in malQ, the gene encoding amylomaltase, the expression of malK-lacZ is less sensitive to high osmolarity, and expression under these conditions occurs even in malI mutants. This malI-resistant and largely osmolarity-insensitive constitutivity of mal gene expression can be observed only when the cells are able to synthesize glycogen. We have demonstrated that cells accumulate a(1-4)-linked maltodextrins, including maltotriose, under these conditions, particularly when grown at high
osmolarity (14).
At present, it is unclear how MalK inactivates endogenous inducers. One possibility is via phosphorylation. In this respect, the regulatory function of the phosphotransferase system (PTS) is relevant: enzyme IIIGIc (EIIIGlC) of the PTS affects not only the expression of the mal genes by regulating the activity of adenylate cyclase (catabolite repression) but also maltose transport (inducer exclusion) by directly interacting with the MalK protein (12, 22). Thus, the interaction of MalK with EIIIGIC may be relevant to the regulatory function of MalK. In this study, we genetically dissected the three different functions of MalK. In particular, we explored whether the function of MalK in energizing maltodextrin transport is required to inactivate the internal inducer. We demonstrate that this is not the case. We describe the isolation of mutations in malK that no longer transported maltose but were still able to abolish internal induction; similarly, we were able to obtain mutations that were strongly reduced in the ability to repress mal genes but that still transported maltose normally. We isolated mutations in malK that are insensitive to the inhibition by EIIIGIC. These mutations do not abolish the regulatory function of MalK. Thus, it appears that MalK can carry out all three functions independently.
MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains and plasmids used are described in Table 1. Strains were grown in Luria broth (LB) or in minimal medium A (MMA) or M63 (23) with 0.4% carbon source. For determining ,-galactosidase activity after growth at different osmolarities, cells were grown in 1/6 MMA containing 50, 125, or 375 mM NaCl and 0.4% glycerol as a carbon source. Indicator media were used as described by Miller (23). Chloramphenicol and kanamycin were added to the media to final concentrations of 25 and 50 ,ug/ml, respectively. Genetic techniques. Plvir transduction was done as described by Miller (23). Other standard genetic techniques such as restriction endonuclease analysis, cloning, transformation, and plasmid preparation were done as described by Maniatis et al. (21). For mutagenesis of pMR11, the plasmid
2181
TABLE 1. Bacterial strains and plasmids Strain or
plasmid Strains MC4100
Relevant genotype
F- araD A(argF-lac)U169
rpsL150 relAl deoCI
Origin Casadaban (7)
BRE1162
ptsF25 flbB5301 rbsR LE392 A(argF-lac)U169 mutD5 MC4100 'F[(maIK::lacZ)]
HS3628 LJ143 ME347
LJ143 AmalK zjb-729::TnlO ptsH315 MC4100 FD(ma1K-1acZ)347
This study Saier et al. (31) Ehrmann and Boos
ME422
BRE1162 mdoA malQ7
REI7 SK1180 SK1138 SK1248 SK1280 SK1278 SK1800 LE392
BRE1162 mall malX (Mal') ME347 recA ME347 malT(Con) recA ME422 recA BRE1162 recA REI7 recA UE15 glpD recA F- supF supE hsdR galK trpR metB lacYfhuA
Ehrmann and Boos (14) Reidl et al. (28) This study This study This study This study This study This study Silhavy et al. (36)
BD25
hyblll3
Laboratory collection Bremer et al. (4)
(14)
Plasmids pACYC184
Chang and Cohen (8) Chang and Cohen (8) This study Reyes and Shuman (29) pMR11 malK916 (W267G) This study pMR11 malK922 (G346S) This study pMR11 malK941 (G137A) This study pMR11 malK954 (140 AQR) This study pMR11 malK936 (D158N) This study pMR11 malK800 (AG36, P37A) This study pMR11 malK801 (G36, +R) This study pHS4 malK903 (S322F) This study pHS4 malK908 (R228C) This study pHS4 malK911 (G302D) This study pHS4 malK913 (E119K) This study pHS4 malK926 (S282L) This study
pACYC177 pACYC177 malK+ pHS4 pACYC184 Ptrc-malK+ pMR11 pSK16 pSK22 pSK41 pSK54 pSK36 pMR800 pMR801 pMR903 pMR908 pMR911 pMR913 pMR926
was transformed into the mutator strain BD25 mutDS, and several samples were grown separately overnight at 37°C in LB containing 25 ,ug of chloramphenicol per ml (13). Plasmid DNA was prepared and used to transform strains SK1180 and SK1138, which were then screened for high expression of malK-lacZ or the ability to metabolize maltose. The a-methylglucoside (oaMG)-resistant mutations were induced by hydroxylamine or UV (36) treatment of pHS4 carrying the malK gene with its own promoter (29). Strain HS3628 was transformed with the mutagenized plasmid and screened for the ability to metabolize maltose in the presence of 1 mM aMG but the inability to utilize lactose, arabinose, or glycerol under these conditions. To create a deletion and an insertion within the ATPbinding domain of malK, we took advantage of the Avall site within the A site of the binding fold. After digestion with Avall, we either trimmed the 5' overlapping ends by nuclease S1 or filled in the 3' gap with Klenow enzyme and then ligated the blunt ends. For DNA sequencing, the dideoxynucleotide chain termination method of Sanger et al. (32) as modified by Biggin et al. (2) was used. For annealing reactions, we used malK-specific primers kindly provided by
2182
KUHNAU ET AL.
J. BACTERIOL.
TABLE 2. Effects of malK mutations on mal gene expression and maltose transporta
Si-Galactosidase activity
Maltose uptake
Strain
(,umol of ONPG/ min/mg of protein)
nMoltose10 cetake (nmolmin/10 cells)
pACYC184 pMR11 Class I mutants pSK16 pSK22 Class II mutants pSK41 pSK54
1.461 0.008
0 0.16
0.689 0.370
0.17 0.15
0.007 0.007 0.005 0.025 0.018
0 0 0 0 0
pSK36 pMR800 pMR801
a The effect of plasmid-encoded MalK mutants on the expression of malK-lacZ was measured in SK1180 (malTl). The effect on transport activity was measured in SK1138 [malT(Con)], since malT(Con) strains are insensitive to regulatory effects of MalK. The general low transport activity is due to the absence of X receptor. ONPG, o-Nitrophenyl-,-D-galactopyranoside.
Boehringer Mannheim or purchased from Operon Technologies (Berkeley, Calif.). P-Galactosidase assays were done by the method of Miller (23). Transport assay. Cells lacking the X receptor (see Table 2) were grown in LB in the presence of antibiotics overnight at 37°C. They were washed in MMA and adjusted to an optical density at 600 nm of 7.0. The high cell density was necessary since lamB strains are deficient in the specific diffusion of maltose and maltodextrins through the outer membrane, particularly at low substrate concentrations. The lack of LamB increases the apparent Km of maltose uptake by a factor of 100 (37). For the transport assay, 20 ,ul of ["4C]maltose (0.14 ,uCi) was added to 1.8 ml of cells to a final concentration of 10 ,uM, and 0.3-ml samples were filtered after 20, 40, 60, 180, and 540 s. For measuring maltose transport in plasmid-encoded malK mutants resistant to aMG (see Table 5), we introduced into the chromosome AmalK with zjb-729: :TnJO. This TnJO is located between malK and lamB and provides a malindependent expression of lamB that is about fourfold lower than fully maltose-induced levels (3). Cells were grown in M63 with 0.4% glycerol or maltose as a carbon source and resuspended for the transport assay to an optical density at 600 nm of 0.5 for glycerol-grown cells or 0.1 for maltosegrown cells. [14C]maltose was added to 1 p.M (final concentration) in the presence or absence of 1% aMG, and 180-pd samples were filtered at 30, 60, 90, and 120 s. RESULTS I Isolation of class and class II mutations in malK. Mutations in malK were obtained on plasmid pMR11, in which the malK gene is under the control of the Ptrc promoter (29). After in vivo mutagenesis, plasmid DNA was prepared and transformed into recA strains carrying a malK-lacZ fusion. To obtain mutations specifically affecting regulation, we used a recipient strain that carried a wild-type malT gene (SK1180). Introduction of pMR11, containing a wild-type malK gene, resulted in very low P-galactosidase activity (Table 2) and a Mal- phenotype. (29). Transformants in which MalK was no longer able to down-regulate mal gene expression were identified as darker blue on plates contain-
ing 5-bromo-4-chloro-3-indolyl-i-D-galactoside (X-Gal). Candidates were then tested for the ability to utilize maltose
as a carbon source. Mal' clones were tested quantitatively for ,B-galactosidase activity. About 50% of the transformants exhibited activity only 10- to 20-fold higher than that in strains containing the wild-type plasmid and were not analyzed. Three plasmids (class I mutations) exhibited high activity ranging from 25 to 50% of the activity seen in the malK-lacZ strain carrying only the vector plasmid (Table 2). When these plasmids were transformed into a malK mutant carrying a malT(Con) allele and maltose transport was measured, no transport defect could be detected (Table 2). To obtain mutations specifically affecting transport, the mutagenized plasmid DNA was transformed into a recipient strain that carried a malT(Con) allele (strain SK1138). Transformants were screened for loss of the ability to utilize maltose as a carbon source. The plasmid DNA of these candidates was transformed into a malK-lacZ strain carrying the malT+ allele, and the expression of P-galactosidase was assayed. Three candidates that appeared white on X-Galcontaining plates were further analyzed. Their 3-galactosidase activities in malT+ strains and transport activities in malT(Con) strains are shown in Table 2. Even though they were fully active in repressing mal gene expression, they no longer transported maltose (class II mutations). Sequencing of malK mutations. Prior to sequencing, restriction fragments of the mutated plasmids were exchanged with the corresponding fragments of the wild-type malK DNA. In this way it was found that the class I mutations were located at the 3' portion of the gene, downstream of the unique DraIII site, while all class II mutations were located between DraIII and HpaI. After cloning in M13, the corresponding fragments were sequenced by the dideoxynucleotide termination method. Class I mutations had occurred at positions 267 (Trp to Gly) (twice isolated) and 346 (Gly to Ser), while class II mutations had occurred at positions 137 (Gly to Ala), 140 (AGln-Arg), and 158 (Asp to Asn) (Fig. 1). These class II mutations are located near or within the B site of the nucleotide-binding fold of MalK, and they had occurred in amino acids that are conserved in the corresponding polypeptide chains of other binding-protein-dependent transport systems as well as other proteins thought to couple ATP hydrolysis to energy-requiring cellular processes (Fig. 2). Amino acid alterations in the A site of the ATP-binding fold exhibit a class II phenotype. The observation that mutations of the class II phenotype were located in the B site of the ATP-binding fold prompted our attempts to specifically induce mutations in the A site and to analyze their phenotypes. We replaced glycine and proline at positions 36 and 37 with one alanine and inserted arginine between glycine and proline at the same position. Both mutations resulted in the complete loss of transport activity but were still fully active in repressing mal gene expression (Table 2). As expected, plasmids carrying either mutation were unable to complement a malT(Con) AmalK strain to Mal'. Endogenous low- and high-osmolarity inducers are metabolized by MalK. In the framework of our hypothesis, elevated expression of mal genes in malK mutants can be mediated by two types of unidentified inducers which are present in distinct physiological conditions. One type is present at high levels in malK mutants at low osmolarity. The other type is derived from glycogen and is found at both high and low osmolarity in strains missing amylomaltase (malQ) (14). It was therefore of interest to determine whether the overproduction of MalK inactivated both types of inducers and whether this function was similarly defective in class I mutants. Table 3 shows the 0-galactosidase activities of the malK-lacZ fusion in a malQ strain carrying
SEPARATION OF E. COLI MalK ACTIVITIES BY MUTATIONS
VOL. 173, 1991 A-region---->
Vol. 173, No. 7
0021-9193/91/072180-07$02.00/0 Copyright © 1991, American Society for Microbiology
The Activities of the Escherichia coli MalK Protein in Maltose Transport, Regulation, and Inducer Exclusion Can Be Separated by Mutations SABINE KUHNAU,1 MORAIMA REYES,2 AMANDA SIEVERTSEN,2 HOWARD A. SHUMAN,2 AND WINFRIED BOOS'* Department of Biology, University of Konstanz, D-7750 Konstanz, Federal Republic of Germany,' and Department of
Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 100322 Received 5 November 1990/Accepted 23 January 1991
The maltose regulon consists of several genes encoding proteins involved in the uptake and utilization of maltose and maltodextrins. Five proteins make up a periplasmic binding-protein-dependent active transport system. One of these proteins, MalK, contains an ATP-binding site and is thought to couple the hydrolysis of ATP to the accumulation of substrate. Beside its function in transport, MalK has two additional roles: (i) it negatively regulates mal regulon expression and (ii) it serves as the target for regulation of transport activity by enzyme IIIGIc of the phosphotransferase system. To determine whether the three functions of MalK are separable, we have isolated and characterized three classes of malK mutations. The first type (class I) exhibited constitutive mal gene expression but still allowed normal transport of maltose; the second type (class II) lacked the ability to transport maltose but retained the ability to repress the mal genes. Class I mutations were localized in the last third of the gene, at amino acids 267 (Trp to Gly) and 346 (Gly to Ser). Mutations of class H were found at the positions 137 (Gly to Ala), 140 (AGln Arg), and 158 (Asp to Asn). These mutations are near or within the region of MalK that exhibits extensive homology to the B site of an ATP-binding fold. In addition, site-directed mutagenesis was used to add or remove one amino acid in the A site of the ATP-binding fold. Plasmids carrying these mutations also behaved as class II mutants. The third class of maiK mutations resulted in resistance to the enzyme IHlGlc_mediated inhibitory effects of a-methylglucoside. These mutations did not interfere with the regulatory function of MalK. One of these mutations (exchanging a serine at position 282 for leucine) is located in a short stretch of amino acids that exhibits homology to a sequence in the Escherichia coli Lac permease in which a-methylglucoside-resistant mutations have been found.
The Escherichia coli maltose transport system is a typical periplasmic binding-protein-dependent transport system (1, 17, 34). Maltose and maltodextrins are bound by the periplasmic maltose-binding protein, which in turn is recognized by the two integral membrane proteins, MalF and MalG, forming the substrate translocation complex (17, 34). The energy needed for accumulation of the substrate was shown to be mediated by the hydrolysis of ATP, presumably by the MalK protein (11, 24). The MalK protein (35) contains an ATP-binding fold and exhibits sequence homology to the corresponding proteins of other binding-protein-dependent transport systems as well as to a variety of other proteins of procaryotic and eucaryotic origin thought to couple ATP hydrolysis to energy-consuming cellular functions (15, 18, 19, 38). From its amino acid sequence, it appears that the MalK protein is a soluble protein (10, 16), but localization studies have shown that it is a peripheral membrane protein. The membrane association of MalK requires MalG, one of the integral membrane-bound proteins of the system (35). The genes for maltose transport and maltose utilization are induced by maltose or maltodextrins in the growth medium. The expression of all mal genes requires the presence of the MalT regulator protein that is activated by the inducer (9, 27, 30). In vitro transcription studies using purified MalT protein have shown that of all linear a(1-4)-linked maltodextrins, only maltotriose is able to activate MalT (26). Thus, the induction by external maltodextrins, including maltose,
*
Corresponding author. 2180
should result from the formation of internal maltotriose. At present, it is unclear which enzyme(s) produces maltotriose.
Amylomaltase, the first enzyme in maltose metabolism, is not necessary for this induction, since mutants lacking the enzyme are still inducible by maltose even though they exhibit increased uninduced levels of mal gene expression. Mutants lacking MalK are unable to transport maltose or maltodextrins and no longer utilize these sugars as carbon sources. However, these mutants express the remaining mal genes at 30 to 40% of the fully induced level in the absence of external inducer. It is clear that this constitutive expression in malK mutants is not caused by the lack of transport per se because mutations in malE or in malF coding for the other subunits of the transport system do not result in constitutive mal gene expression (6, 20, 33). Although the MalT protein is necessary for malK-dependent constitutivity to occur, the mechanism of the regulatory function of MalK is still unclear. Previously, we explained the apparent constitutivity of malK mutants by proposing the existence of an internal inducer, different from maltotriose, that is recognized by MalT and is inactivated by MalK (14). Consistent with this model, overexpression of MalK prevents the expression of the maltose genes in malT+ but not in malT (Con) strains (29). We have isolated a mutation in a gene termed malI that results in decreased constitutive mal gene expression in malK mutants. The analysis of mall has revealed that it codes for a typical repressor protein controlling, beside its own expression, expression of an adjacent and divergently oriented gene, malX (28). Since loss of malX together with
VOL. 173,
1991
SEPARATION OF E. COLI MalK ACTIVITIES BY MUTATIONS
mall results in high expression of malK, we concluded that MalX must also be an enzyme inactivating or degrading the internal inducer. The nature of the hypothetical internal inducer and the mode of its synthesis are still unclear. If, as we propose, the internal induction is controlled by the concentration of a sugar molecule, this inducer is likely to be a glucose-containing compound but not of the maltodextrin type (14). It would not be degraded by amylomaltase, and its synthesis would not require ADP-glucose. The level of internal inducer would be responsible not only for the high expression of the mal genes in malK mutants but also for the basal-level expression of the mal genes in wild-type strains. In both cases, the level of endogenous induction is dependent on the osmolarity of the medium, being high only at low osmolarity and being turned off at high osmolarity (6). In contrast, in strains carrying a mutation in malQ, the gene encoding amylomaltase, the expression of malK-lacZ is less sensitive to high osmolarity, and expression under these conditions occurs even in malI mutants. This malI-resistant and largely osmolarity-insensitive constitutivity of mal gene expression can be observed only when the cells are able to synthesize glycogen. We have demonstrated that cells accumulate a(1-4)-linked maltodextrins, including maltotriose, under these conditions, particularly when grown at high
osmolarity (14).
At present, it is unclear how MalK inactivates endogenous inducers. One possibility is via phosphorylation. In this respect, the regulatory function of the phosphotransferase system (PTS) is relevant: enzyme IIIGIc (EIIIGlC) of the PTS affects not only the expression of the mal genes by regulating the activity of adenylate cyclase (catabolite repression) but also maltose transport (inducer exclusion) by directly interacting with the MalK protein (12, 22). Thus, the interaction of MalK with EIIIGIC may be relevant to the regulatory function of MalK. In this study, we genetically dissected the three different functions of MalK. In particular, we explored whether the function of MalK in energizing maltodextrin transport is required to inactivate the internal inducer. We demonstrate that this is not the case. We describe the isolation of mutations in malK that no longer transported maltose but were still able to abolish internal induction; similarly, we were able to obtain mutations that were strongly reduced in the ability to repress mal genes but that still transported maltose normally. We isolated mutations in malK that are insensitive to the inhibition by EIIIGIC. These mutations do not abolish the regulatory function of MalK. Thus, it appears that MalK can carry out all three functions independently.
MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains and plasmids used are described in Table 1. Strains were grown in Luria broth (LB) or in minimal medium A (MMA) or M63 (23) with 0.4% carbon source. For determining ,-galactosidase activity after growth at different osmolarities, cells were grown in 1/6 MMA containing 50, 125, or 375 mM NaCl and 0.4% glycerol as a carbon source. Indicator media were used as described by Miller (23). Chloramphenicol and kanamycin were added to the media to final concentrations of 25 and 50 ,ug/ml, respectively. Genetic techniques. Plvir transduction was done as described by Miller (23). Other standard genetic techniques such as restriction endonuclease analysis, cloning, transformation, and plasmid preparation were done as described by Maniatis et al. (21). For mutagenesis of pMR11, the plasmid
2181
TABLE 1. Bacterial strains and plasmids Strain or
plasmid Strains MC4100
Relevant genotype
F- araD A(argF-lac)U169
rpsL150 relAl deoCI
Origin Casadaban (7)
BRE1162
ptsF25 flbB5301 rbsR LE392 A(argF-lac)U169 mutD5 MC4100 'F[(maIK::lacZ)]
HS3628 LJ143 ME347
LJ143 AmalK zjb-729::TnlO ptsH315 MC4100 FD(ma1K-1acZ)347
This study Saier et al. (31) Ehrmann and Boos
ME422
BRE1162 mdoA malQ7
REI7 SK1180 SK1138 SK1248 SK1280 SK1278 SK1800 LE392
BRE1162 mall malX (Mal') ME347 recA ME347 malT(Con) recA ME422 recA BRE1162 recA REI7 recA UE15 glpD recA F- supF supE hsdR galK trpR metB lacYfhuA
Ehrmann and Boos (14) Reidl et al. (28) This study This study This study This study This study This study Silhavy et al. (36)
BD25
hyblll3
Laboratory collection Bremer et al. (4)
(14)
Plasmids pACYC184
Chang and Cohen (8) Chang and Cohen (8) This study Reyes and Shuman (29) pMR11 malK916 (W267G) This study pMR11 malK922 (G346S) This study pMR11 malK941 (G137A) This study pMR11 malK954 (140 AQR) This study pMR11 malK936 (D158N) This study pMR11 malK800 (AG36, P37A) This study pMR11 malK801 (G36, +R) This study pHS4 malK903 (S322F) This study pHS4 malK908 (R228C) This study pHS4 malK911 (G302D) This study pHS4 malK913 (E119K) This study pHS4 malK926 (S282L) This study
pACYC177 pACYC177 malK+ pHS4 pACYC184 Ptrc-malK+ pMR11 pSK16 pSK22 pSK41 pSK54 pSK36 pMR800 pMR801 pMR903 pMR908 pMR911 pMR913 pMR926
was transformed into the mutator strain BD25 mutDS, and several samples were grown separately overnight at 37°C in LB containing 25 ,ug of chloramphenicol per ml (13). Plasmid DNA was prepared and used to transform strains SK1180 and SK1138, which were then screened for high expression of malK-lacZ or the ability to metabolize maltose. The a-methylglucoside (oaMG)-resistant mutations were induced by hydroxylamine or UV (36) treatment of pHS4 carrying the malK gene with its own promoter (29). Strain HS3628 was transformed with the mutagenized plasmid and screened for the ability to metabolize maltose in the presence of 1 mM aMG but the inability to utilize lactose, arabinose, or glycerol under these conditions. To create a deletion and an insertion within the ATPbinding domain of malK, we took advantage of the Avall site within the A site of the binding fold. After digestion with Avall, we either trimmed the 5' overlapping ends by nuclease S1 or filled in the 3' gap with Klenow enzyme and then ligated the blunt ends. For DNA sequencing, the dideoxynucleotide chain termination method of Sanger et al. (32) as modified by Biggin et al. (2) was used. For annealing reactions, we used malK-specific primers kindly provided by
2182
KUHNAU ET AL.
J. BACTERIOL.
TABLE 2. Effects of malK mutations on mal gene expression and maltose transporta
Si-Galactosidase activity
Maltose uptake
Strain
(,umol of ONPG/ min/mg of protein)
nMoltose10 cetake (nmolmin/10 cells)
pACYC184 pMR11 Class I mutants pSK16 pSK22 Class II mutants pSK41 pSK54
1.461 0.008
0 0.16
0.689 0.370
0.17 0.15
0.007 0.007 0.005 0.025 0.018
0 0 0 0 0
pSK36 pMR800 pMR801
a The effect of plasmid-encoded MalK mutants on the expression of malK-lacZ was measured in SK1180 (malTl). The effect on transport activity was measured in SK1138 [malT(Con)], since malT(Con) strains are insensitive to regulatory effects of MalK. The general low transport activity is due to the absence of X receptor. ONPG, o-Nitrophenyl-,-D-galactopyranoside.
Boehringer Mannheim or purchased from Operon Technologies (Berkeley, Calif.). P-Galactosidase assays were done by the method of Miller (23). Transport assay. Cells lacking the X receptor (see Table 2) were grown in LB in the presence of antibiotics overnight at 37°C. They were washed in MMA and adjusted to an optical density at 600 nm of 7.0. The high cell density was necessary since lamB strains are deficient in the specific diffusion of maltose and maltodextrins through the outer membrane, particularly at low substrate concentrations. The lack of LamB increases the apparent Km of maltose uptake by a factor of 100 (37). For the transport assay, 20 ,ul of ["4C]maltose (0.14 ,uCi) was added to 1.8 ml of cells to a final concentration of 10 ,uM, and 0.3-ml samples were filtered after 20, 40, 60, 180, and 540 s. For measuring maltose transport in plasmid-encoded malK mutants resistant to aMG (see Table 5), we introduced into the chromosome AmalK with zjb-729: :TnJO. This TnJO is located between malK and lamB and provides a malindependent expression of lamB that is about fourfold lower than fully maltose-induced levels (3). Cells were grown in M63 with 0.4% glycerol or maltose as a carbon source and resuspended for the transport assay to an optical density at 600 nm of 0.5 for glycerol-grown cells or 0.1 for maltosegrown cells. [14C]maltose was added to 1 p.M (final concentration) in the presence or absence of 1% aMG, and 180-pd samples were filtered at 30, 60, 90, and 120 s. RESULTS I Isolation of class and class II mutations in malK. Mutations in malK were obtained on plasmid pMR11, in which the malK gene is under the control of the Ptrc promoter (29). After in vivo mutagenesis, plasmid DNA was prepared and transformed into recA strains carrying a malK-lacZ fusion. To obtain mutations specifically affecting regulation, we used a recipient strain that carried a wild-type malT gene (SK1180). Introduction of pMR11, containing a wild-type malK gene, resulted in very low P-galactosidase activity (Table 2) and a Mal- phenotype. (29). Transformants in which MalK was no longer able to down-regulate mal gene expression were identified as darker blue on plates contain-
ing 5-bromo-4-chloro-3-indolyl-i-D-galactoside (X-Gal). Candidates were then tested for the ability to utilize maltose
as a carbon source. Mal' clones were tested quantitatively for ,B-galactosidase activity. About 50% of the transformants exhibited activity only 10- to 20-fold higher than that in strains containing the wild-type plasmid and were not analyzed. Three plasmids (class I mutations) exhibited high activity ranging from 25 to 50% of the activity seen in the malK-lacZ strain carrying only the vector plasmid (Table 2). When these plasmids were transformed into a malK mutant carrying a malT(Con) allele and maltose transport was measured, no transport defect could be detected (Table 2). To obtain mutations specifically affecting transport, the mutagenized plasmid DNA was transformed into a recipient strain that carried a malT(Con) allele (strain SK1138). Transformants were screened for loss of the ability to utilize maltose as a carbon source. The plasmid DNA of these candidates was transformed into a malK-lacZ strain carrying the malT+ allele, and the expression of P-galactosidase was assayed. Three candidates that appeared white on X-Galcontaining plates were further analyzed. Their 3-galactosidase activities in malT+ strains and transport activities in malT(Con) strains are shown in Table 2. Even though they were fully active in repressing mal gene expression, they no longer transported maltose (class II mutations). Sequencing of malK mutations. Prior to sequencing, restriction fragments of the mutated plasmids were exchanged with the corresponding fragments of the wild-type malK DNA. In this way it was found that the class I mutations were located at the 3' portion of the gene, downstream of the unique DraIII site, while all class II mutations were located between DraIII and HpaI. After cloning in M13, the corresponding fragments were sequenced by the dideoxynucleotide termination method. Class I mutations had occurred at positions 267 (Trp to Gly) (twice isolated) and 346 (Gly to Ser), while class II mutations had occurred at positions 137 (Gly to Ala), 140 (AGln-Arg), and 158 (Asp to Asn) (Fig. 1). These class II mutations are located near or within the B site of the nucleotide-binding fold of MalK, and they had occurred in amino acids that are conserved in the corresponding polypeptide chains of other binding-protein-dependent transport systems as well as other proteins thought to couple ATP hydrolysis to energy-requiring cellular processes (Fig. 2). Amino acid alterations in the A site of the ATP-binding fold exhibit a class II phenotype. The observation that mutations of the class II phenotype were located in the B site of the ATP-binding fold prompted our attempts to specifically induce mutations in the A site and to analyze their phenotypes. We replaced glycine and proline at positions 36 and 37 with one alanine and inserted arginine between glycine and proline at the same position. Both mutations resulted in the complete loss of transport activity but were still fully active in repressing mal gene expression (Table 2). As expected, plasmids carrying either mutation were unable to complement a malT(Con) AmalK strain to Mal'. Endogenous low- and high-osmolarity inducers are metabolized by MalK. In the framework of our hypothesis, elevated expression of mal genes in malK mutants can be mediated by two types of unidentified inducers which are present in distinct physiological conditions. One type is present at high levels in malK mutants at low osmolarity. The other type is derived from glycogen and is found at both high and low osmolarity in strains missing amylomaltase (malQ) (14). It was therefore of interest to determine whether the overproduction of MalK inactivated both types of inducers and whether this function was similarly defective in class I mutants. Table 3 shows the 0-galactosidase activities of the malK-lacZ fusion in a malQ strain carrying
SEPARATION OF E. COLI MalK ACTIVITIES BY MUTATIONS
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