Binding protein-dependent ABC transport system for glycerol 3-phosphate of Escherichia coli.
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ATP-Binding Cassette Transporters↗
Biomedical subjects
W Boos
Publications and source records attributed to W Boos.
Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc.
The maltose regulon consists of 10 genes encoding a multicomponent and binding protein-dependent ABC transporter for maltose and maltodextrins as well as enzymes necessary for the degradation of these sugars. MalT, the transcriptional activator of the system, is necessary for the transcription of all mal genes. MalK, the energy-transducing subunit of the transport system, acts phenotypically as repressor, particularly when overproduced. We isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK. The affected gene was sequenced and identified as mlc, a known gene encoding a protein of unknown function with homology to the Escherichia coli NagC protein. The loss of Mlc function led to a threefold increase in malT expression, and the presence of mlc on a multicopy plasmid reduced malT expression. By DNasel protection assay, we found that Mlc protected a DNA region comprising positions +1 to +23 of the malT transcriptional start point. Using a mlc-lacZ fusion in a mlc and mlc+ background, we found that Mlc represses its own expression. As Mlc also regulates another operon (manXYZ, see pages 369-379 of this issue), it may very well constitute a new global regulator of carbohydrate utilization.
ATP-Binding Cassette Transporters↗
The ABC maltose transporter.
Bacterial ATP-binding cassette (ABC) transporters and their homologues in eukaryotic cells form one of the largest superfamilies known today. They function as primary pumps that couple substrate translocation across the cytoplasmic membrane to ATP hydrolysis. Although ABC transporters have been studied for more than three decades, the structure of these multi-component systems is unknown, and the mechanism of transport is not understood. This article reviews one of the most widely studied ABC systems, the maltose transporter of Escherichia coli. A first structural model of the transport channel allows discussion of possible mechanisms of transport. In addition, recent experimental evidence suggests that regulation of gene expression and transport activity is far more complex than expected.
ATP-Binding Cassette Transporters↗
The ATP-binding cassette subunit of the maltose transporter MalK antagonizes MalT, the activator of the Escherichia coli mal regulon.
Transcription of the mal regulon of Escherichia coli K-12 is regulated by the positive activator, MalT. In the presence of ATP and maltotriose, MalT binds to decanucleotide MalT boxes that are found upstream of mal promoters and activates transcription at these sites. The earliest studies of the mal regulon, however, suggested a negative role for the MalK protein, the ATP-binding cassette subunit of the maltose transporter, in regulating mal gene expression. More recently, it was found that overexpression of the MalK protein resulted in very low levels of mal gene transcription. In this report we describe the use of tagged versions of MalT to provide evidence that it physically interacts with the MalK protein both in vitro and in vivo. In addition, we show that a novel malK mutation, malK941, results in an increased ability of MalK to down-modulate MalT activity in vivo. The fact that the MalK941 protein binds but does not hydrolyse ATP suggests that the MalK941 mutant protein mimics the inactive, ATP-bound form of the normal MalK protein. In contrast, cells with high levels of MalK ATPase show a reduced ability to down-modulate MalT and express several mal genes constitutively. These results are consistent with a model in which the inactive form of MalK down-modulates MalT and decreases transcription, whereas the active form of MalK does not. This model suggests that bacteria may be able to couple information about extracellular substrate availability to the transcriptional apparatus via the levels of ATP hydrolysis associated with transport.
ATP-Binding Cassette Transporters↗
The periplasmic cyclodextrin binding protein CymE from Klebsiella oxytoca and its role in maltodextrin and cyclodextrin transport.
Klebsiella oxytoca M5a1 has the capacity to transport and to metabolize alpha-, beta- and gamma-cyclodextrins. Cyclodextrin transport is mediated by the products of the cymE, cymF, cymG, cymD, and cymA genes, which are functionally homologous to the malE, malF, malG, malK, and lamB gene products of Escherichia coli. CymE, which is the periplasmic binding protein, has been overproduced and purified. By substrate-induced fluorescence quenching, the binding of ligands was analyzed. CymE bound alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin, with dissociation constants (Kd) of 0.02, 0.14 and 0.30 microM, respectively, and linear maltoheptaose, with a Kd of 70 microM. In transport experiments, alpha-cyclodextrin was taken up by the cym system of K. oxytoca three to five times less efficiently than maltohexaose by the E. coli maltose system. Besides alpha-cyclodextrin, maltohexaose was also taken up by the K. oxytoca cym system, but because of the inability of maltodextrins to induce the cym system, growth of E. coli mal mutants on linear maltodextrin was not observed when the cells harbored only the cym uptake system. Strains which gained this capacity by mutation could easily be selected, however.
Bacterial Proteins↗
Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis.
We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.
ATP-Binding Cassette Transporters↗
Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation.
The maltose system of Escherichia coli offers an unusually rich set of enzymes, transporters, and regulators as objects of study. This system is responsible for the uptake and metabolism of glucose polymers (maltodextrins), which must be a preferred class of nutrients for E. coli in both mammalian hosts and in the environment. Because the metabolism of glucose polymers must be coordinated with both the anabolic and catabolic uses of glucose and glycogen, an intricate set of regulatory mechanisms controls the expression of mal genes, the activity of the maltose transporter, and the activities of the maltose/maltodextrin catabolic enzymes. The ease of isolating many of the mal gene products has contributed greatly to the understanding of the structures and functions of several classes of proteins. Not only was the outer membrane maltoporin, LamB, or the phage lambda receptor, the first virus receptor to be isolated, but also its three-dimensional structure, together with extensive knowledge of functional sites for ligand binding as well as for phage lambda binding, has led to a relatively complete description of this sugar-specific aqueous channel. The periplasmic maltose binding protein (MBP) has been studied with respect to its role in both maltose transport and maltose taxis. Again, the combination of structural and functional information has led to a significant understanding of how this soluble receptor participates in signaling the presence of sugar to the chemosensory apparatus as well as how it participates in sugar transport. The maltose transporter belongs to the ATP binding cassette family, and although its structure is not yet known at atomic resolution, there is some insight into the structures of several functional sites, including those that are involved in interactions with MBP and recognition of substrates and ATP. A particularly astonishing discovery is the direct participation of the transporter in transcriptional control of the mal regulon. The MalT protein activates transcription at all mal promoters. A subset also requires the cyclic AMP receptor protein for transcription. The MalT protein requires maltotriose and ATP as ligands for binding to a dodecanucleotide MalT box that appears in multiple copies upstream of all mal promoters. Recent data indicate that the ATP binding cassette transporter subunit MalK can directly inhibit MalT when the transporter is inactive due to the absence of substrate. Despite this wealth of knowledge, there are still basic issues that require clarification concerning the mechanism of MalT-mediated activation, repression by the transporter, biosynthesis and assembly of the outer membrane and inner membrane transporter proteins, and interrelationships between the mal enzymes and those of glucose and glycogen metabolism.
Amino Acid Sequence↗
Characterization of TreR, the major regulator of the Escherichia coli trehalose system.
The pathway of trehalose utilization in Escherichia coli is different at low and high osmolarity. The low osmolarity system takes up trehalose as trehalose 6-phosphate which is hydrolyzed to glucose and glucose 6-phosphate. treB and treC, the genes for the enzymes involved, form an operon that is controlled by TreR (encoded by treR), the repressor of the system, for which trehalose 6-phosphate is the inducer. We have cloned and sequenced treR. The protein contains 315 amino acids with a molecular weight of 34,508. TreR was purified and shown to bind as a dimer trehalose 6-phosphate and trehalose with a Kd of 10 and 280 microM, respectively. The conformations of the protein differ from each other with either one or the other substrate-bound. Protease treatment removed the DNA-binding domain from the intact protein leaving the dimerization domain (a 29-kDa carboxyl-terminal fragment) intact. Nuclease protection experiments revealed a palindromic sequence located directly upstream of the -35 promoter sequence of treB that functions as the operator of the system.
Amino Acid Sequence↗
Quantitative analysis of binding protein-mediated ABC transport systems.
We present a mathematical proof for the applicability of the Michaelis-Menten theory to the analysis of binding protein-dependent transport systems. Fitting the rate equation for transport of substrate to experimental data obtained with the Escherichia coli maltose system under conditions where the concentrations of the binding protein was varied, it was possible to determine without any further assumptions: (1) K1, the affinity constant of the binding protein towards its substrate; (2) K3, the affinity constant of the unloaded binding protein towards the membrane components; and (3) R, the concentration of the membrane component. This analysis allows determination of the alteration of KM and Vmax of the system at varying concentrations of binding protein.
ATP-Binding Cassette Transporters↗
Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity.
malQ mutants of Escherichia coli lacking amylomaltase cannot grow on maltose. They express the maltose system constitutively and are sensitive to maltose when grown on another carbon source. In an attempt to isolate a multicopy suppressor that would result in growth on maltose, we transformed a malQ mutant with a gene bank of E. coli DNA which had been digested with Sau3a and cloned in pBR322. We screened the transformants on MacConkey maltose plates. A colony was isolated that appeared to be resistant to maltose and was pink on these plates, but it was still unable to grow on minimal medium with maltose as the carbon source. The plasmid was isolated, and the gene causing this phenotype was characterized. The deduced amino acid sequence of the encoded protein shows homology to that of lipases and esterases. We termed the gene aes, for acetyl esterase. Extracts of cells harboring plasmid-encoded aes under its own promoter exhibit a fivefold higher capacity to hydrolyze p-nitrophenyl acetate than do extracts of cells of plasmid-free strains. Similarly, strains harboring plasmid-encoded aes are able to grow on triacetyl glycerol (triacetin) whereas the plasmid-free strains are not. The expression of plasmid-encoded aes resulted in strong repression of the maltose transport genes in malT+ strains (10-fold reduction), but not in a malT(Con) strain which is independent of the inducer. Also, overproduction of MalT counteracted the Aes-dependent repression, indicating a direct interaction between MalT and Aes.
Acetylesterase↗
Molecular characterization of glucokinase from Escherichia coli K-12.
glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced. Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000. The enzyme was purified, and its kinetic parameters were determined. Its Km values for glucose and ATP were 0.78 and 3.76 mM, respectively. Its Vmax was 158 U/mg of protein. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression. Under all conditions tested, only growth on glucose reduced the expression of glk by about 50%. A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR+ weakly decreased expression. A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk. Overexpression of glk interfered with the expression of the maltose system. Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system. It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer. This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system.
Amino Acid Sequence↗
The MalT-dependent and malZ-encoded maltodextrin glucosidase of Escherichia coli can be converted into a dextrinyltransferase by a single mutation.
malZ is a member of the mal regulon. It is controlled by MatT, the transcriptional activator of the maltose system. MalZ has been purified and identified as an enzyme hydrolyzing maltotriose and longer maltodextrins to glucose and maltose. MalZ is dispensable for growth on maltose or maltodextrins. Mutants lacking amylomaltase (encoded by malQ), the major maltose utilizing enzyme, cannot grow on maltose, maltotriose, or maltotetraose, despite the fact that they contain an effective transport system and MalZ. From such a malQ mutant a pseudorevertant was isolated that was able to grow on maltose. The suppressor mutation was mapped in malZ. The mutant gene was cloned. It contained a Trp to Cys exchange at position 292 of the deduced protein sequence. Surprisingly, the purified mutant enzyme was still unable to hydrolyze maltose as was the wild type enzyme, while both were able to release glucose from maltodextrins. However, the mutant enzyme had gained the ability to transfer dextrinyl moieties to glucose, maltose, and other maltodextrins. Thus, it had gained an activity associated with amylomaltase. It was the MalZ292-associated transferase reaction that allowed the utilization of maltose. In addition, we discovered that mutant and wild type enzyme alike were highly active as gamma-cyclodextrinases.
Bacterial Proteins↗
Improved method for the preparative synthesis of labeled trehalose of high specific activity by Escherichia coli.
We report an improvement of a published procedure using Escherichia coli to synthesize 14C-labeled trehalose from [14C]glucose (B. Brand and W. Boos, Appl. Environ. Microbiol. 55:2414-2415, 1989). Instead of inducing the expression of the trehalose-synthesizing enzymes encoded by the chromosomal genes otsAB by high osmolarity, we now induce their expression from a plasmid under normal growth conditions by the addition of IPTG (isopropyl-beta-D-thiogalactopyranoside). Instead of using a pgi zwf double mutant to prevent glucose utilization, we use a pgi::Tn10 insertion only. In addition to being defective in treA, which encodes a periplasmic trehalase, the strain is now also defective in treF, which encodes a newly discovered cytoplasmic trehalase. This strain is genetically stable; it has no growth defects; and after induction with IPTG, it will transform [14C]glucose to [14C]trehalose in minimal medium without any carbon source under aerobic conditions at a rate of 3 nmol/min/10(9) cells. With the improved method, the overall yield of trehalose from glucose is about 80% and the process takes place without dilution of the specific radioactivity of the glucose residues. The accumulated trehalose is extracted from the bacteria by 70% hot ethanol and can easily be purified radiochemically by chromatographic techniques.
DNA Transposable Elements↗
High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.
The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.
Archaea↗
Characterization of a cytoplasmic trehalase of Escherichia coli.
Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source. The pathway of trehalose utilization is different at low and high osmolarity. At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose. Glucose is then taken up by the phosphotransferase system. At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose. treF, the gene encoding this enzyme, was cloned under ara promoter control. The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein. The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency. TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA. The nonidentical amino acids of TreF are more polar and more acidic than those of TreA. The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor. Mutants producing 17-fold more TreF than does the wild type were isolated.
Amino Acid Sequence↗
The inhibition of maltose transport by the unliganded form of the maltose-binding protein of Escherichia coli: experimental findings and mathematical treatment.
Binding protein-dependent transport systems in Gram-negative enteric bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site. Usually, there are two integral membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur. Transport is against the concentration gradient and needs energization by an ATP hydrolizing polypeptide. Overall transport is considered mainly unidirectional due to the high energy of ATP hydrolysis coupled to transport. In the study reported here, maltose transport in membrane vesicles in the presence of varying concentrations of unliganded maltose-binding protein but with constant amounts of maltose was measured. The conditions were chosen such that the concentration of maltose was always smaller than that of the binding protein and the initial concentration of the liganded binding protein was essentially constant. It was found that the initial rate of transport went through a maximum with increasing amounts of binding protein and declined thereafter. This finding strongly supports the conclusion that both the liganded and the unliganded forms of the binding protein interact with the membrane components of the transport system. The mathematical treatment of the experimental data allowed the ratio of the affinities for the membrane components of the substrate loaded and unloaded binding protein to be estimated. Published data on the binding protein-dependent transport of histidine in membrane vesicles of Salmonella typhimurium were also used. The data allowed the ratio of the binding affinity of the membrane components to the substrate-loaded and free binding protein to be determined. In addition, the KM of transport to the KD of binding protein was approximated.
ATP-Binding Cassette Transporters↗
Mathematical treatment of the kinetics of binding protein dependent transport systems reveals that both the substrate loaded and unloaded binding proteins interact with the membrane components.
Binding-protein-dependent transport systems in Gram-negative bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site. Usually, there are two membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur. Transport is against the concentration gradient and needs energization by an ATP hydrolyzing polypeptide. Overall transport is considered mainly unidirectional owing to the high energy of ATP hydrolysis coupled to transport. We dissected the overall transport process into three individual steps: (i) reversible binding of substrate to the binding protein; (ii) reversible binding of the binding protein to the membrane components forming the translocation complex; (iii) irreversible transport of substrate through the membrane and dissociation of the binding protein from the complex. Two models were considered. In the first, only the substrate-loaded binding protein interacts with the membrane components, while in the second model both the loaded and the unloaded form of the binding protein interact with the membrane components. The mathematical analysis of the second model revealed that the substrate concentration KM at half-maximal rate of transport approaches KD of the binding protein when the last step of transport becomes low and when the concentration of binding protein in the periplasm becomes large. This is usually observed in real systems. Under the same conditions, in model 1 KM approaches zero and is hence considerably smaller than KD. This has never been observed in any real system. In addition, the dependence of the overall rate of transport on the concentration of binding protein in the periplasm follows a sigmoidal curve only when model 2 is considered. The sigmoidal behavior becomes more pronounced when the substrate concentration is low and it is less pronounced when the last step in overall transport is low. This phenomenon has been observed with the Escherichia coli maltose transport system. Thus, at least for the maltose transport system, it seems likely that both the loaded and the unloaded forms of the binding protein interact with the membrane components. We propose that this should generally be considered in binding-protein-dependent transport systems.
Adenosine Triphosphate↗
Molecular analysis of treB encoding the Escherichia coli enzyme II specific for trehalose.
A gene bank of partially Sau3A-digested Escherichia coli DNA ligated in plasmid pBR322 was screened for the ability to complement a mutant unable to metabolize trehalose at low osmolarity. The resulting plasmid was shown to contain the genes encoding transport (treB) and metabolic (treC) functions. The complementing DNA region was sequenced and shown to contain an operon of two genes, with treB as the promoter proximal gene and with treC as the promoter distal gene. The transcriptional start point was determined, and one major transcript was detected. The control region of the operon was found to contain consensus binding motifs for the cyclic AMP-catabolite activator protein complex and for a specific repressor protein whose gene, treR, is located immediately upstream of treB, being transcribed in the same direction as treB treC. The products of both genes could be expressed in minicells in which TreB revealed itself as a protein with an apparent molecular weight of 42,000. The gene product of treB consists of 485 amino acids with a calculated molecular weight of 52,308. It showed high homology to enzymes IIScr of enteric bacteria specific for the uptake of sucrose and encoded by plasmid pUR400 of enteric bacteria. Like enzyme IIScr, enzyme IITre belongs to the EIIBC domain type and lacks a covalently bound EIIA domain. Instead, enzyme IITre-mediated phosphorylation of trehalose requires the activity of enzyme IIAGlc, a component of the major glucose transport system.
Adaptation, Physiological↗