The maltose system is responsible for the uptake and efficient catabolism of (14)-linked glucose polymers (maltodextrins) up to 7 to 8 glucose units. This system has turned out to be a far richer source of interesting molecules and regulatory phenomena than anyone might have anticipated 30 years ago when the malA and malB regions were mapped on the E. coli chromosome (239). For example, in the course of studying the receptor activity of E. coli responsible for  attachment, Randall and Schwartz discovered the  receptor (LamB) (207) and, in collaboration with Hofnung, Szmelcman, and one of us (W.B.), showed that this outer membrane protein constituted the channel for the passage of sugars, especially maltodextrins, across the outer membrane (263, 265). This information focused attention on the possibility of porins as specific channels and culminated in the solution of the three-dimensional structure of the maltoporin channel associated with malto-oligosaccharide ligands (231). Together with a large body of information about the functional sites within LamB, the structure represents one of the most complete descriptions of a membrane transport protein.

From the earliest time that the maltose system was studied, transcriptional control was a major focus because of the interest in a positively controlled system. The MalT protein, which is the activator at all mal promoters (112, 206, 214), and the sites at which it binds (MalT boxes) have provided an important alternative view to the lac operon about how transcription is activated. Although the precise mechanism of how MalT interacts with and stimulates RNA polymerase is still unknown, there is considerable information about the structural requirements for MalT binding to DNA as well as its ability to form a nucleoprotein complex with the catabolite activator protein (CAP).

The periplasmic binding protein-dependent ATP binding cassette (ABC) transporter of the maltose system has been studied to understand the mechanism of transport and the mechanism of protein localization to different cellular compartments (e.g., inner membrane, outer membrane, and periplasm) (21, 156, 173, 250), as well as the determinants of membrane protein topology. The periplasmic maltose binding protein (MBP) has been characterized structurally (257), and a variety of functional sites within its structure for interacting with the chemosensory apparatus and the ABC transporter have been identified. The actual membrane transporter (MalFGK₂), made up of two integral membrane proteins (MalF and MalG) and two copies of the ATP-hydrolyzing subunit (MalK), has been characterized biochemically, and there is some information about functional sites that are important for substrate recognition, ATP binding, and subunit contacts and interactions with MBP. The ability to manipulate the transporter genetically offers a rare opportunity to dissect the mechanism of transport by a combined approach of biochemistry and genetics. The direct participation of the transporter in transcriptional regulation indicates that gene regulation in bacteria may depend on the rates of substrate entry in addition to the physical presence of the substrate itself.

Reviews on the maltose system of E. coli have appeared (22, 241). In this review, we attempt to summarize what is currently known about this system. We have focused on three areas: (i) transport of maltodextrins into the cell via an outer membrane porin and a periplasmic binding protein-dependent ABC transporter, (ii) metabolism of the internalized sugars, and (iii) transcriptional regulation of the mal genes. We attempt to clarify what we believe are significant functional relationships between transport activity and transcriptional control. Also, we discuss the interconnections between the maltose-degrading enzymes and those of glucose catabolism and gluconeogenesis from the perspective of how endogenous inducers of the mal system are produced. Because the initial breakdown of maltodextrins results in both glucose and glucose-1-phosphate, these enzyme activities must be coordinated with those for glycogen synthesis and breakdown. Finally, other less obvious connections with phosphate metabolism and trehalose metabolism are also discussed. We will not consider aspects of secretion (169), folding (272), or assembly (151, 223) of the components of the maltose system.

Table 1 summarizes all known mal genes in E. coli. Their definition is based on the function of MalT. Thus, all genes regulated by MalT belong to the maltose regulon, while the expression of malT itself is independent of MalT. malP and malQ encode essential enzymes for maltose and maltodextrin metabolism, whereas malS and malZ encode nonessential maltodextrin-metabolizing enzymes. malEFG and malK lamB encode the binding protein-dependent ABC transporter. Some mal genes are organized in clusters. The malA region at 76.5 min contains two divergently orientated operons, with malT transcribed clockwise and malPQ transcribed counterclockwise (65, 126, 200). Likewise, all maltose transport genes are clustered at 91.4 min in the malB region with two divergently organized operons: malEFG in the counterclockwise orientation and malK lamB malM in the clockwise orientation (203, 254).

Other gram-negative enteric bacteria have additional genes, not present in E. coli, that are under the control of MalT. For instance, Klebsiella pneumoniae harbors a battery of malT-controlled pul genes that are necessary for the biosynthesis and the secretion of pullulanase (71), an extracellular enzyme that degrades pullulan to maltotriose (14, 285). Pullulan, first isolated from Pullularia pullulans (13), consists of (16)-glucosidically linked maltotriose units. The MalT-dependent pulA gene, encoding the lipoprotein pullulanase (163), is positioned divergently from a series of genes, also dependent on MalT, that are necessary for the export of pullulanase through the outer membrane (193, 195). Likewise, Klebsiella oxytoca harbors a series of cym genes encoding proteins for the uptake and metabolism of cyclic dextrins (89). The expression of these genes may also directly or indirectly depend on malT. The expression of the maltose regulon in Vibrio cholerae affects the virulence of this organism, and mutations in malQ and malF render it less virulent (149). The products of all the MalT-dependent E. coli genes have been identified, but the function of one, the periplasmic MalM protein (104, 222), is still unclear. The observation that MalM is also present in Salmonella typhimurium may suggest that this periplasmic protein has an important function (234).

Table 1 contains many more genes that do not belong to the maltose regulon but whose encoded proteins affect the metabolism of maltodextrins or the regulation of mal gene expression at different levels. Dextrin-metabolizing or synthesizing enzymes affect the level of internal maltotriose, the inducer of the system which is recognized by MalT; cyclic AMP (cAMP)/CAP, as well as the product of the mlc gene, controls the expression of malT itself; and the levels of MalK, the ATP-hydrolyzing subunit of the transport system, as well as of MalY (a C-S lyase) and of Aes (an esterase with homology to lipases) affect the expression of the maltose transport genes. There are some more genes which, when mutated, have a subtle effect on mal gene expression. Among them is asuE, encoding a tRNA-modifying enzyme, and treR, encoding a repressor for the trehalose-utilyzing system in E. coli. These aspects will be discussed in detail below.

POSITIVE TRANSCRIPTIONAL ACTIVATOR, MALT

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malT, Its Product, and Regulation of Its Expression

An early genetic approach identified malT as a gene that appeared essential for the expression of all maltose-inducible functions (112, 113, 126). Since all maltose-inducible operons require malT and since malT fails to repress the operons in the absence of inducer (67, 126), it was clear that the protein encoded by malT is a purely positive regulator that is activated by an inducer and stimulates transcription by activating RNA polymerase (48). The malT gene was cloned (204) and sequenced (40), and its product, MalT, was purified and characterized (214). The protein appears monomeric in dilute solution, contains 901 amino acids, and exhibits a molecular weight of 103,000. It binds ATP (K_D, 0.4 µM) (215) and maltotriose (K_D, about 20 µM) (49), both of which are necessary for transcriptional activation, even though ATP hydrolysis is not required, since the binding of ADP or nonhydrolyzable ATP analogs also stimulates transcription (215).

Mutations in malT [malT(Con)], resulting in the constitutive expression of all mal genes, have been isolated (49, 67). These mutations center around amino acids 243 and 358 of the polypeptide chain. Whereas in an in vitro assay the wild-type MalT protein is inactive in the absence of maltotriose, the MalT(Con) proteins are active in the absence of maltotriose but still bind maltotriose with an increased affinity. All mutant proteins can still be stimulated further in their transcriptional activity by maltotriose (49). The last C-terminal 95 amino acids of MalT constitute the DNA-binding domain. In this area, MalT exhibits homology to a number of prokaryotic transcriptional activators (280) of the UhpA-LuxR family of regulatory proteins (260). Truncated forms of MalT lacking its C-terminal DNA binding domain are negatively dominant over the function of the wild-type protein, indicating that the protein interacts with itself when binding to DNA (40).

The expression of malT is not autoregulated by MalT but is subject to catabolite repression and therefore requires the presence of the cAMP/CAP complex (35, 36, 65). According to the current view, the mechanism of catabolite repression is based mainly on the regulation of intracellular cAMP levels. This, in turn, is a function of adenylate cyclase, controlled by the enzyme IIA^Glc of the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS) in its phosphorylated (activating) and dephosphorylated (inactivating) states (191). Indeed, mutants lacking adenylate cyclase or CAP are unable to grow on maltose. It is rather informative to monitor mal gene expression in a wild-type strain during growth in rich media such as Luria-Bertani broth or tryptone broth, in the absence of external inducer. After inoculation (1:20 dilution) from an overnight culture, expression of a malK-lacZ fusion or maltose transport activity is high. It dramatically decreases (indicating a lack of new synthesis) as growth commences and reaches its lowest level during mid-log-phase growth. Since this phenomenon coincides with a cessation of cell division, it was once erroneously concluded that the synthesis of maltose binding protein (74) as well as galactose binding protein (246) might be connected to cell division. However, this phenomenon most probably reflects the concentration of internal cAMP (15), pointing once more to the importance of cAMP in catabolite repression.

Mutations in the control region of the malT gene (Fig. 1) have shown that its expression in the wild type is limited at both the transcriptional and translational levels. Mutations with increased malT expression have been isolated (malTp1, malTp7) (Fig. 1) (34). The use of deletions introduced upstream of the malT promoter revealed that the 120 bp upstream of the transcriptional start site, encompassing the binding sites for the polymerase and the cAMP/CAP complex, are sufficient for malT expression. However, deletion of DNA further upstream increased the expression of malT, indicating that a binding site for a protein reducing malT expression had been removed (200, 205). In addition, a Tn10 insertion (malT-P418::Tn10) (Fig. 1) that increases malT expression has been isolated. The position of this insertion is still upstream of the DNA region which, when deleted, increases expression (Fig. 1). Whether or not these sites are involved in the dominant negative effect of certain mutations in envZ (105, 286, 289), resulting in an overphosphorylated OmpR protein (2, 224, 291), is still unclear. Interestingly, the DNA upstream of the malT promoter whose deletion results in an increase in malT expression contains sequences that are similar to sequences identified as binding sites for phosphorylated OmpR (133, 155) (Fig. 1). Although ompR null mutants do not exhibit an increase in malT expression, mutations in envZ leading to uncontrolled phosphorylation of OmpR reduce the transcription of malT (33).

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Regulatory region upstream of malT. The unboxed shaded area represents the cAMP/CAP binding site for the malT promoter. The boxed shaded area represents the binding site of Mlc, the regulator of malT expression, as determined by footprint analysis. The arrows at malT +1 and +1 malP indicate the transcriptional startpoints of the malT and malP transcripts, respectively. The arrows upstream of the malP promoter show the single and tandem MalT boxes needed for malP expression. 10 and 35 regions are indicated for the malT promoter only. The changes of AT to GC at position 39 (malTp7) and of GC to TA at position 100 (malTp1) are mutations that increase malT expression at the translational and transcriptional levels, respectively. malTp1 renders malT expression independent of the cAMP/CAP complex (34). The extent of the deletions upstream of the malT promoter, 511, 512, 513, and 514, are indicated. Their malT expression relative to the wild type is increased by a factor of 2.8 (511), 2.2 (512), 1.1 (513), and 1.0 (514) (200). The Tn10(Cam) insertion at nucleotide 418 leads to a 2.5-fold increase in malT expression. Dotted lines indicate homology to sites that have been identified as OmpR binding sites. In particular, I and III indicate homology to site FII and II indicates site FI as defined in reference 155. Modified from reference 200 with permission of the publisher.

Expression of malT is controlled by a repressor called Mlc. This was detected by isolating a chromosomal insertion that increased malT expression. The insertion was found to be in mlc, a known gene whose product, when overproduced, impairs the utilization of glucose (131). The increase in malT expression could still be observed in mutants carrying the deletions as well as the insertion upstream of the malT promoter, excluding these regions as binding sites for Mlc. Mlc shows homology to NagC, a gene regulator functioning as a repressor for the divergent nag operons (encoding proteins for the uptake and degradation of N-acetyl-D-glucosamine) and as an activator as well as a repressor for the glmUS operon encoding enzymes for the biosynthesis of N-acetyl-D-glucosamine (188). By footprint analysis, it was shown that Mlc binds to malT DNA at a position 1 to 23 bp from the start of the malT transcript (Fig. 1). In mutants lacking Mlc, the expression of malT is increased by a factor of 2 to 3 when grown in glycerol. On the other hand, overexpression of Mlc from a multicopy plasmid strongly reduces malT expression (68). The identity of the effector for Mlc is still unclear. Internal free glucose or glucose derived from the metabolism of disaccharides such as trehalose (141) or even maltose (28) slightly induces malT expression in a Mlc-dependent fashion. However, free glucose, even 100 mM, does not prevent DNA binding of Mlc during footprint or band shift assays. Therefore, it is likely that a metabolic product of glucose could be the controlling compound for Mlc. Mlc not only regulates malT expression but also represses the man operon (259) when overproduced (189). Mlc may thus represent a new global regulator for sugar-metabolizing systems.

The first feature recognized for at least three MalT-dependent promoters was the lack of the usual 35 region, whereas the 10 region corresponds to those of constitutive promoters (47). Instead, the asymmetric hexanucleotide sequence 5'-GGA(G/T)GA-3', the so-called MalT box, was identified centered at bp 37.5 or at 38.5 upstream of the transcriptional start point (9, 10, 106, 201). In addition, DNA upstream of the 38 region was found to be essential for mal gene expression (106, 201), whereas the 30-bp sequence preceding the transcriptional start point contains only a few positions that are essential for promoter activity (64).

The second structural feature found to be essential for MalT-dependent mal gene expression was two additional MalT boxes in a direct repeat upstream of the 38 region (206, 279, 281). The analysis of these MalT boxes also led to an extension in the consensus sequence, which is now defined as 5'-GGGGA(T/G)GAGG-3' (281). In contrast to the orientation of the MalT box, which is proximal to the transcription initiation site and always oriented 5' to 3' in the direction of transcription, the orientation of the two repeats can be in either direction. This motif of MalT-dependent promoter structure with three MalT boxes has also been found by sequence analysis upstream of malS (235) and malZ (267), two MalT-dependent genes encoding maltodextrin-hydrolyzing enzymes in E. coli.

Figure 2 shows a schematic view of the arrangement of the MalT and cAMP/CAP-binding sites of the different E. coli mal gene promoters.

View larger version (7K): [in this window] [in a new window]   FIG. 2.   Scheme for the promoter structures of the different mal operons. Arrows indicate transcriptional start points. Solid large arrow-shaped bullets represent MalT binding sites (MalT boxes), and open rectangles represent cAMP/CAP binding sites. The open arrow-shaped bullet indicates a MalT box that has been identified by footprinting but seems dispensable for Mal-dependent transcriptional activation of malP (46). This site overlaps a CAP binding site (dashed cAMP/CAP binding site) as determined by footprint analysis. Again, mutation of this site does not affect the transcription of malP (46). Similarly, the cAMP/CAP binding site most proximal to the malK promoter (dashed rectangle) has been identified by footprint analysis but is not essential for transcription of malK (279). Having two MalT binding sites in a direct repeat is a recurrent feature of mal promoters and plays a crucial role in their activation (281). The dashed arrow in the malZ promoter indicates a second transcriptional start site that is independent of MalT and more frequently used at high osmolarity. The cAMP/CAP binding site in the malS promoter has not been tested for its functional importance. The three cAMP/CAP binding sites between malK and malE are essential for the transcription of both genes (279).

By using the decanucleotide 5'-GGGGAGGAGG-3', the MalT-binding consensus box, and nucleotide sequences present in the polylinker of a vector plasmid as connecting sequences, Danot and Raibaud constructed several functional semisynthetic promoters and tested them for MalT-dependent gene expression (45). These studies confirmed the importance of the structural motif formed by two MalT-binding sites in a direct repeat. This motif is involved in promoter activation either alone or in conjunction with a third MalT-binding site proximal to the transcription start site. In this configuration, the promoters are active irrespective of the orientation of the repeat. Provided that the alignment along the axis of the helix is retained, the distance of the repeat to the proximal MalT-binding site can be varied to some extent. This analysis defines the inducible MalT-dependent promoter that does not require the cAMP/CAP complex (45). The role of these sites in the contact and activation of RNA polymerase by MalT has been assessed by mutating each site and measuring the contribution of the remaining sites (46).

Of the five known MalT-dependent operons in E. coli, two (malPQ and malZ) are independent of cAMP/CAP and two, malK lamB malM and malEFG, depend on it. The upstream region of the last mal gene, malS, does contain a sequence resembling a cAMP/CAP binding site, but whether it is functional is unclear. malK lamB malM and malEFG are oriented divergently from each other. Their transcription start sites are 271 bp apart. Located between these start sites is a 210-bp regulatory region which comprises two series of MalT boxes (three MalT boxes, including the repeat, in front of malK, and two MalT boxes, the repeat, in front of malE) separated by three cAMP/CAP binding sites. The entire control region is required for the full expression of both operons. Sequential removal of the two distal MalT boxes reduces but does not abolish the expression of malE, whereas the removal of any MalT box abolishes the expression of the malK operon (206). This suggested that multiple copies of MalT and cAMP/CAP form a unique nucleoprotein structure at this regulatory region that is required for maximal activity of both promoters. The observation that the function of these sites is sensitive to the phase of the DNA helix has led to a model in which the DNA is wrapped around the complex composed of MalT and cAMP/CAP (199, 206). The state of supercoiling also appears to be important for the effectiveness by which the two operons are transcribed. Not only is relaxed DNA less efficient in transcription initiation, but also the sites occupied by the activator complex are shifted in supercoiled relative to relaxed DNA (216).

By a series of elegant experiments, it was found that binding of cAMP/CAP in the intergenic regulatory region between malE and malK results in a repositioning of MalT binding. In the absence of cAMP/CAP, MalT binds with high affinity to the three MalT boxes upstream of the malK transcriptional start site (identified previously). In the presence of cAMP/CAP, MalT binding is shifted by three nucleotides toward the Pribnow box of the malK promoter. The cAMP/CAP effect requires the malKp-distal MalT binding sites (218). The repositioning is caused by DNA bending, which can be mimicked by replacing cAMP/CAP with the integration host factor (217). The role of the CAP and MalT binding sites in transcriptional regulation of the two divergently oriented promoters continues to be the subject of extensive studies (213).

Recently, it has been observed that Lrp, the leucine-responsive protein (32), also affects the transcription of malT as well as of some malT-dependent genes (268). By using operon fusions to malT, malE, and malK in the genetic background of strain MC4100, this Lrp dependence could not be observed in the laboratory of W.B. It has also been reported that the expression of the malK lamB malM and malEFG operons, but not of the malT gene, is controlled by the pH of the medium, being elevated at high pH (121). The mediator of this pH-dependent control is unknown.

MALTOSE/MALTODEXTRIN TRANSPORT SYSTEM

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Periplasmic Substrate Recognition Site and Its Interaction with Membrane Components

The maltose/maltodextrin transport system is a member of the family of multicomponent and periplasmic binding protein-dependent ABC high-affinity transport systems of gram-negative enteric bacteria (21, 57, 116, 173, 249, 250). The substrate recognition site of the system is determined primarily by the soluble binding protein with a high affinity for maltose and maltodextrins (K_D around 1 µM) (138, 265) that is located in the periplasm in high concentration (around 1 mM and in 30- to 50-fold molar excess over the intrinsically membrane-bound proteins of the system) (74). This maltose binding protein (MalE protein or MBP) consists of two nearly symmetrical lobes between which the binding site is formed (197, 257). Substrate-loaded and substrate-free forms of MBP, as well as of other substrate-binding proteins, differ dramatically in their conformations, so that substrate bound to the protein no longer has access to bulk solvent. In the substrate-free form, the lobes are open and the substrate-binding site becomes accessible to the bulk solvent. Several physical, spectroscopic, and thermodynamic properties of MBP change when ligand is bound to it (100, 101, 109, 175, 244, 247, 265). Structural data on the open (244) and closed substrate-loaded (243, 244, 257) forms of the protein are available (Fig. 3A). The two lobes not only perform a bending movement relative to each other but also perform a twisting movement (Fig. 3B). In addition, the structures of a MBP with a dominant negative mutation (248) as well as of MBPs harboring a foreign epitope (226) or deletions and insertions (245) have been solved. Both the substrate-loaded and the substrate-free forms of MBP have access to the membrane components MalF and MalG (17, 158), even though it is unclear whether the interacting substrate-free form is of the open or the closed configuration.

View larger version (166K): [in this window] [in a new window]   View larger version (104K): [in this window] [in a new window]   FIG. 3.   Crystal structures of the open and closed forms of MBP. (A) Structural representation of MBP in the open (top) and closed (bottom) forms. The yellow structure represents maltose bound within the binding site. Courtesy of Sherry Mowbray (247); reprinted with permission of the publisher. (B) Perspective view of the superimposed backbone structure of unliganded (mauve) and maltose-bound (blue) MBP (looking into the binding cleft from the side) with bound maltose (ball-and-stick model). The direction and magnitude of the conformational change when going from the unliganded to the ligand-bound form are shown. Reprinted from reference 244 with permission of the publisher.

MBP has been subjected to a detailed mutational analysis, and regions affecting transport (78, 264) or chemotaxis (77) have been defined. Also, intensive studies on the renaturation process of denaturated MBP (37, 96) and on the biosynthesis and secretion of the protein (37, 73, 90, 261, 272) have been published. A recent report even indicates that periplasmic binding proteins, MBP among them, exhibit properties of molecular chaperonins in the periplasm (212).

The interaction of MBP with MalF and MalG has been studied by the genetic approach of mutant and suppressor analysis (274). This study, in combination with the knowledge of the crystal structure of MBP, has led to the conclusion that one lobe of MBP interacts with MalF and the other lobe interacts with MalG (128). The starting point for this genetic analysis was the isolation of mutants (actually, two point mutations were always required in either malF or malG) that transported maltose in the absence of MBP. Surprisingly, some of these MBP-independent mutations became maltose negative in the presence of wild-type MBP, a situation that allowed isolation of mutants with suppressor mutations in malE (274). Most of these suppressor mutations in MBP were dominant negative in combination with a wild-type MalFGK₂ complex, indicating a higher affinity towards the membrane components when in the non-ATP hydrolysis-triggering mode (128, 248). Similarly, after the introduction of two cysteines (G69C and S337C) by site-directed mutagenesis into each domain of MBP, the formation of an interdomain disulfide cross-link that holds the protein in a closed conformation could be observed. This mutant MBP confers a dominant negative phenotype for growth on maltose, for maltose transport, and for maltose chemotaxis (303). The observation that wild-type MBP interferes with the transport activity of the MalFGK₂ complex of the MBP-independent mutant permitted further studies on the interaction of MBP with the MalFGK₂ complex. Transport studies with vesicles revealed that the wild-type binding protein inhibited transport only at high concentrations while it actually stimulated transport at low concentration (59). This analysis, together with the properties of dominant negative mutations in MBP, has led to the proposal that the MalFGK₂ complex must be able to attain at least two different conformations in its interaction with MBP, only one of which is able to trigger ATP hydrolysis by the MalK subunit (21, 248).

Even though the major recognition entity of the maltose transport system is the MBP, it is clear that not all substrates that are bound by the binding protein are transported. This is particularly obvious with cyclodextrins and the p-nitrophenyl derivatives of maltooligosaccharides that are bound very well by MBP but are not transported by the intact system (87, 150, 269). This is due to the modes of substrate recognition by MBP. Substrates that can be attached to MBP at the reducing end are transported; those which are bound within the dextrinyl chain (such as cyclodextrins) are not (107, 108, 173).

MBP also functions as the substrate recognition site for maltose chemotaxis (114). The sites in MBP that are essential for the interaction with the maltose transport machinery and the chemotaxis machinery are distinct but partially overlapping (77, 97, 98, 302, 303). The  helix 7 of MBP appears to be exclusively involved in the interaction with the membrane components of the transport system. Mutations in this  helix belonging to the C-lobe of MBP affect transport without affecting the binding of substrate or the interaction with the chemotactic machinery (264).

The application of the phoA fusion technique allowed determination of the two-dimensional topology of both MalF (24, 82, 94) and MalG (25, 52). Accordingly, MalF consists of eight membrane-spanning -helical segments (MSS) with both termini of the polypeptide chain protruding into the cytoplasm. MalG, as judged by the same criteria, consists of six MSS, and again both termini extend into the cytoplasm (Fig. 4). Near their C termini, both proteins carry a sequence between MSS 6 and 7 (MalF) and MSS 4 and 5 (MalG), respectively, that is very similar to the consensus sequence EAA-X₃-G-X₉-I-X-LP conserved in all intrinsically membrane-bound subunits of binding protein-dependent ABC systems (227, 228). Substitutions at the same positions in MalF and MalG cause different phenotypes, and mutations in malG or malF that slightly affect or do not affect transport by themselves cause a completely defective phenotype when present together. This indicates that MalF and MalG are acting together asymmetrically in the translocation step. Suppressor mutations to transport negative mutations in malF or malG were found in malK, indicating that the EAA-harboring domain interacts with MalK (167).

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Two-dimensional structure of MalF and MalG. A topological model of MalF and MalG based on the analysis of malF::phoA and malG::phoA fusions is shown. MBP-independent mutants always require two mutations, one in the p (proximal) region and one in the d (distal) region. The G338R mutation in MalF alone causes MBP-dependent lactose transport. The EAAXXLG consensus motif is located in the cytoplasmic loop between MSS 6 and 7 of MalF and MSS 4 and 5 of MalG. Reprinted from reference 41 with permission of the publisher.

Studies with mutations in malF and their intragenic suppressors represent the first attempts to determine the positions of the different MSS relative to each other. Thus, mutations in MSS 7 were suppressed by mutations in MSS 6 or 8, perhaps an indication that these helices are next neighbors. In addition, mutations in MSS 6 leading to altered substrate specificity occur only on one side of the helical wheel, pointing to a participation of this surface in the substrate-specific transport channel (80).

The MalF subunit of the maltose transport system of E. coli and other gram-negative enteric bacteria (44) represents something of an exception among the membrane-bound components of binding protein-dependent ABC transporters, since these MalF proteins contain a large periplasmic loop between MSS 3 and 4. This loop may not mediate the interaction with the binding protein, since all pairs of mutations in malF leading to an MBP-independent phenotype (and which can be suppressed by mutations in MBP for the dominant negative phenotype caused by wild-type MBP) are positioned outside the loop. It may be involved in the docking of MBP to the membrane components. Consistent with this view is the observation that in none of the MBP-independent mutants is docking of MBP prevented. Also, no periplasmic loop is found in the MalF homologs of binding protein-dependent maltose transport systems in gram-positive bacteria (196, 225, 276) or even in the Archaea (299), whose members contain lipid-anchored high-affinity binding proteins for maltose on the outside of the cytoplasmic membrane. In the latter case, docking is no problem because close vicinity to the membrane components is based on the lipid anchorage.

Even though it represents the major substrate recognition site, MBP cannot be the only substrate binding site of the transport system. MBP-independent mutants with mutations in MalF or MalG that retain specificity for maltose have been isolated, indicating the presence of a latent substrate binding site in the MalFGK₂ membrane complex (273). The V_max of these mutants for maltose transport can sometimes be similar to the wild-type value, while the apparent K_m of uptake is always increased by a factor of about 10³. Transport in these mutants is still active, i.e., against the concentration gradient, and dependent on the MalK-mediated hydrolysis of ATP. Characteristically, these MBP-independent mutants with mutations in MalF always carry two mutations, one in MSS 5 near the periplasmic surface, in the p (proximal) region, and the other one deep within the cytoplasmic membrane, in either MSS 6, 7, or 8, in the d (distal) region (41). It is interesting that substrate specificity mutations have been isolated in MSS 6 and that nearest-neighbor analysis predicts that MSS 6, 7, and 8 are close to each other (80) and at the same time form the d region. The attractive concept that mutations in the p region affect binding-protein recognition and mutations in the d region affect the coupling to the ATPase activity mediated by the associated MalK subunit apparently does not hold true, however. Both mutations are required for the uncoupled MalK-ATPase phenotype.

The screening of mutations in malF and malG for the ability to transport other sugars revealed that one mutation, malF515, which changes Leu334 to Trp (L334W) on the periplasmic side of MSS 5, permitted the transport of lactose in addition to maltose. MalK as well as MalG is required for lactose transport, as is MBP. The requirement for MBP is particularly intriguing because MBP clearly does not bind lactose. Therefore, these results are consistent with the idea that the unliganded form of MBP interacts with the membrane complex (160). The position for such a "specificity" mutation is surprising since it is on the periplasmic side of MalF, which is not expected to form the substrate recognition site. This may indicate that substrate specificity for lactose is already present in MalF or MalG but needs the proper opening of the gate for the substrate to enter. In a different construct, large portions of the MalF protein have been deleted (beginning from the C terminus up to MSS 1). These deletion mutants are able to transport lactose in a MalG-, MalK-, and MBP-dependent manner without an additional mutation (159). This may indicate that the substrate recognition site of the membrane complex for lactose resides in MalG.

The MalK subunit contains a classical consensus sequence found in all ATP-hydrolyzing proteins (284). It consists of two subsites, the A and B domains. These two motifs, as well as the sequence surrounding these sites, are conserved in the equivalent subunits of all binding protein-dependent transport systems (123). In addition, they are found in many proteins of prokaryotic and eukaryotic origin whose function is in the transport of molecules, including polysaccharides, peptides, and proteins (83, 122). MalK and equivalent proteins of other binding protein-dependent transport systems have been characterized not only as ATP-binding proteins but also as enzymes that hydrolyze ATP. When MalK is embedded in the membrane as a complex with MalF and MalG, maximal rates of ATP hydrolysis are achieved only in the presence of substrate-loaded MBP (54, 57-59, 184). The ATPase activity of the detergent-solubilized MalFGK₂ complex of the MBP-independent mutant F500 has also been measured in solution (53). The active complex consists of two MalK subunits connected by one molecule each of MalF and MalG (55). When this complex is reconstituted in liposomes, ATP-dependent active transport of maltose into the liposomes can be measured, demonstrating the function of the MalK dimer as an energy module driving the active transport of maltose. In agreement with the dimeric structure of MalK in the translocation complex is the observation that the ATP binding sites of both subunits are essential (56) and that the kinetics of ATP hydrolysis show cooperativity (53). Using the technique of Hu (132) to probe proteins for their ability to form dimers by fusing them to a truncated  repressor (lacking the dimerization domain but retaining the operator binding), we were able to show that wild-type MalK has the ability to form dimers in vivo (68).

Little is known about the mechanism of energy coupling of ATP hydrolysis to the accumulation of maltose. Since neither the substrate nor any of the proteins involved have been seen to become phosphorylated during transport, it has become commonplace to interpret energy coupling as the transfer of the energy gained through ATP hydrolysis to protein conformational energy in MalF and MalG, followed by its subsequent binding protein-triggered release, resulting in the unidirectional translocation of the substrate through the membrane. Mutants with mutations in malF or malG that no longer require the triggering by substrate-loaded MBP have been isolated. It is noteworthy that the MalFGK₂ complex of these mutants no longer requires the presence of substrate-loaded MBP to stimulate the ATPase activity of MalK (57). ATPase activity has become uncoupled in these mutants, similar to the uncoupled ATPase activity of the purified wild-type MalK subunit (166). This has led to the notion that the transport system is a signal transduction pathway that begins in the periplasm with the recognition of maltose by the binding protein and ends with the control of MalK-ATPase activity in the cytoplasm (41, 57). The helical domain of the ATPase in MalK is likely to be involved in the signal transduction between MalF and MalG (167).

No quantitative studies on the synthesis of MalK in comparison to its partners in the membrane, MalF and MalG, are available. Even though the stoichiometric composition of the biochemically active complex has been determined as MalFGK₂ (55), it is unclear whether MalK is synthesized in excess or, depending on the regulatory conditions, in varying amounts. The latter possibility is not unlikely since malF and malG are located in a different operon from malK and since substantial amounts of MalK have been found, in contrast to MalF and MalG (7, 251). As discussed below, MalK plays an important role in the MalT-dependent control of mal gene expression. It is only natural to see MalK not only as an energy module to drive transport but also as a control unit of transport activity. Obviously, the availability of ATP should influence transport activity. However, since the K_m of MalK for ATP is in the micromolar range (58) and the physiological concentration of ATP is in the millimolar range, the cells would presumably have to become quite exhausted of ATP before they stopped transporting maltose.

We would like to postulate an additional level of control for the function of MalK in transport, which works by regulating the affinity of MalK for its partners, MalF and MalG. We observed that transport of glycerol-3-phosphate by the binding protein-dependent Ugp transport system is inhibited by internal P_i (29). The Ugp system exhibits a surprising degree of sequence similarity to the maltose system in all subunits in spite of the differences in substrate specificity and regulation (178). In an attempt to find whether UgpC, the MalK analog of the Ugp system, was the target of P_i inhibition, we exchanged MalK with UgpC (115) and tested a possible inhibition of maltose transport by P_i. Although P_i did not inhibit the UgpC-mediated uptake of maltose, it also failed to inhibit the uptake of glycerol-3-phosphate via the Ugp system when UgpC was overproduced. Apparently, the reduced affinity of UgpC for its cognate membrane partners in the presence of P_i can be compensated for by increasing the UgpC concentration. It suggests the possibility that the degree of association of the ATP-hydrolyzing subunit with the membrane components may control transport activity. An equivalent finding in the histidine transport system in Salmonella typhimurium has been interpreted in the same way. High concentrations of internal histidine were able to inhibit transport of histidine in trans (152). These observations may indicate that the transport activity of ABC transporters is controlled from the inside via the ATP-hydrolyzing subunit by binding the accumulated substrate.

The MalK subunit is usually pictured as being peripherally membrane associated through binding to the intrinsic membrane proteins MalF and MalG. This membrane association of MalK may in fact be more intimate (296). Studies with the functionally related binding protein-dependent histidine transport system of Salmonella typhimurium have shown that HisP, the MalK analog of this system, might actually be accessible from the periplasm (6), even though the interaction of the cognate periplasmic binding protein, HisJ, takes place with the intrinsic membrane proteins of the system and not with HisP (192). Similar findings of accessibility of MalK on the periplasmic side of the membrane have been reported for MalK in S. typhimurium (236). Models of the transport mechanism mediated by binding protein-dependent ABC systems picture the input of energy by ATP hydrolysis as conservation of a strained protein structural conformation in MalF and MalG that is released by triggering with substrate-loaded binding protein (21, 59, 250). The analogy of the repetitive movement of SecA through the membrane that has been postulated as crucial for Sec-dependent protein secretion (140, 293) to a possible repetitive movement of a subdomain of MalK through the membrane (236) is less convincing. The transported molecule would be far smaller than the moving machinery, and the moving part of MalK should then exhibit substrate specificity for the transported molecule, which has never been observed. In contrast, UgpC, the ATP-hydrolyzing subunit of the glycerol phosphate transport system, can complement malK mutants to transport maltose (115), indicating that the ATPase subunit does not carry substrate specificity for the transported molecule.

Efficient uptake of maltose and, particularly, longer maltodextrins at low concentrations requires the presence of -receptor (maltoporin), the specific diffusion pore for maltodextrins and other carbohydrates, in the outer membrane (62, 63, 92, 141, 263, 265). It is interesting that lamB, the gene for the -receptor, is, together with malK, located in a different operon from the remaining genes of the transport system (malE, malF, and malG). Since MalK is involved in the regulation of the system, and since the -receptor is also required for the diffusion of carbohydrates other than maltodextrins (63, 141), a differential regulation of these two genes (in comparison to the malEFG operon) should be considered. In addition, translational control of lamB (72, 110) may be evoked when considering the need of carbohydrate uptake under starvation conditions (62, 174).

A number of studies have analyzed the function of LamB (maltoporin) in the diffusion of maltodextrins (16, 88, 92, 136, 154, 171). All these studies demonstrate the presence of a maltodextrin binding site that is essential for the facilitated diffusion process of maltodextrins. A major contribution to understanding the molecular basis of transport through porin channels has come from determination of the three-dimensional structure of LamB, which was done in the presence of a series of maltooligosaccharides. These studies showed that each subunit of the trimeric protein contains a wide channel formed by an 18-stranded (230), antiparallel -barrel. Three inwardly folded loops contribute to a constriction about halfway through the channel (231) (Fig. 5). The crystal structures of maltoporin complexed with maltose, maltotriose, or maltohexaose reveal an extended binding site within the channel. The maltooligosaccharides are in apolar van der Waals contact with the "greasy slide," a hydrophobic path that is composed of aromatic residues and is located at the channel lining (79, 287). Interestingly, the LamB protein contains two of four sequences (235) that are conserved in amylases and form the maltodextrin binding sites there (282). One of them, FYQRHD, at positions 106 to 111 of LamB, is actually part of the sugar binding site as determined by X-ray crystallography (79). A similar structure for the maltoporin from S. typhimurium, including the prominent greasy slide for maltodextrins, has been reported (162).

View larger version (125K): [in this window] [in a new window]   FIG. 5.   Crystal structure of LamB. A schematic drawing of the -receptor (maltoporin) monomer is shown. The cell exterior is at the top, and the periplasmic space at the bottom. The area of the subunit in trimer contacts is facing the viewer. The 18 antiparallel  strands of the barrel are represented by arrows. Strands are connected to their nearest neighbors by loops or regular turns. Loops L1 (blue), L3 (red), and L6 (green) fold inward toward the barrel. L3 is the major determinant of the constriction site. The yellow bond symbolizes the disulfide bridge Cys22-Cys38 within loop 1. Loop 2, facing the viewer, latches onto an adjacent subunit in the trimer. Loops L4 to L6 and L9 form a large protrusion. The horizontal lines delineate the boundaries of the hydrophobic core of the membrane as inferred from the hydrophobic area found on the molecular surface. Reprinted from reference 231 with permission of the publisher.

Calculations show that the diffusion of maltose through LamB and the following uptake by the ABC transporter are closely matched at maltose concentrations in the micromolar range. The V_max for the disaccharide maltose in a fully induced strain has been measured to be 20 nmol per min per 10⁹ cells (265). The diffusion of maltose through the outer membrane, mediated by about 10,000 trimeric LamB porin molecules per cell, is apparently not yet limiting at micromolar maltose concentrations, the K_m of the transport system (27). We calculated the diffusion of maltose for different external concentrations through these 30,000 pores with an approximate diameter of 1 nm, using a diffusion constant of 10⁵ cm²/sec and an outer membrane thickness of 6 nm. We found that a rate of diffusion of 20 nmol per min per 10⁹ cells, the V_max of the fully induced ABC transporter, is reached at 0.1 µM. Considering that not all maltose molecules that reach the external face of the lambda receptor will actually enter the pore, this concentration is most probably a lower limit, and in reality the concentration might be closer to 1 µM. Therefore, one can conclude that the overall uptake of maltose at its K_m is close to the maximal rate of diffusion through the outer membrane. The validity of this conclusion has been experimentally tested with mutants containing reduced amounts of lambda receptor (27). In other words, a transport system that requires a V_max of about 20 nmol per min per 10⁹ cells and is equipped with at least equal amounts of a specific diffusion pore to those in the fully induced lambda receptor cannot exhibit an apparent K_m considerably lower than 1 µM, in spite of a periplasmic binding protein with a considerably lower K_D of binding. In contrast, transport systems with an inherently low V_max, such as binding protein-dependent amino acid transport systems, may very well approach in their K_m of transport the K_D of the corresponding binding protein.

Incoming maltose and maltodextrins of up to seven glucose moieties are metabolized to glucose and glucose-1-phosphate by the combined action of three cytoplasmic enzymes, amylomaltase (MalQ), maltodextrin phosphorylase (MalP), and maltodextrin glucosidase (MalZ) (Fig. 6). Since maltodextrins larger than six glucose moieties are not very well transported by the ABC transporter, they are reduced in size by a periplasmic amylase, the MalS protein.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Maltose degradation by the maltose enzymes. The enzymes amylomaltase (malQ), maltodextrin phosphorylase (malP), and maltodextrin glucosidase (malZ) are indicated by their genes. After transport of maltose by the binding protein-dependent ABC transporter, a maltosyl, maltotriosyl, or maltotetraosyl (and so on) residue is transferred from maltotriose, maltotetraose, or maltopentaose (and so on) onto the incoming maltose releasing glucose in the process. Maltopentaose and longer maltodextrins are recognized by the maltodextrin phosphorylase, forming glucose-1-phosphate and a maltodextrin that is smaller by one glucosyl residue. Maltodextrin glucosidase recognizes maltotriose and longer maltodextrins (up to maltoheptaose), releasing glucose consecutively from the reducing end of the maltodextrin. This scheme demonstrates that maltose degradation by the maltose enzymes requires the endogenous presence of a maltodextrin primer with the minimal size of maltotriose. The activity of the last maltose enzyme, the periplasmic -amylase, is not considered in this scheme. Adapted from reference 69 with permission of the American Society for Microbiology.

Amylomaltase (165, 194, 294, 295), encoded by malQ, is a dextrinyl transferase that can transfer maltosyl and longer dextrinyl residues onto glucose, maltose, and longer maltodextrins. The smallest substrate which amylomaltase recognizes is maltotriose (182). Acting on maltotriose, it releases glucose from the reducing end, forms a maltosyl-enzyme complex, and transfers the maltosyl residue onto the nonreducing end of an acceptor, be it glucose, maltose, or any larger maltodextrin. The paradoxical consequence of this scheme is that maltose itself should not be a primary substrate, but pure maltose should be a substrate only in the presence of small amounts of maltodextrins acting as a primer in the reaction. Amylomaltase, in contrast to the MalZ enzyme discussed below, is able to release not only glucose but also longer dextrins from the reducing end of its maltodextrin substrates (182). Since MalQ is not a hydrolase but a transferase, the sum of glycosidic linkages of all maltodextrins involved in the reaction must remain constant. Only when glucose is removed from this equilibrium of dextrins, for instance by glucokinase-mediated phosphorylation to glucose-6-phosphate, does the degradation of maltose continue while forming longer maltodextrins.

It is important to stress that maltose, strictly speaking, is not a substrate of amylomaltase but only an acceptor in the transfer reaction catalyzed by the enzyme. Thus, it follows that for maltose degradation, the cell has to be able to internally produce small amounts of maltodextrins as primers with the minimum size of maltotriose. Amylomaltase is essential for maltose degradation, and malQ mutants are unable to grow on maltose. Not only are malQ mutants Mal, but also their growth is inhibited by maltose (127). Under these conditions, they accumulate large amounts of free maltose inside the cell (265). In the presence of an alternative carbon source, mutations arise that are found preferentially in malT or in malK. Why no mutations in other genes arise whose products are essential in transport remains a mystery (241). Even in the absence of external maltose, malQ mutants contain significant concentrations of free glucose, maltose, maltotriose, and larger maltodextrins, provided that the strain contains the glycogen-synthesizing enzymes (69, 81). Therefore, within the cell, glycogen must continually be degraded to maltodextrins, which cannot be repolymerized (under production of glucose) in the absence of amylomaltase.

The enzymatic reaction of amylomaltase can be exploited for the convenient synthesis of uniformly labeled maltodextrins (maltotriose and larger) of high specific activity starting from uniformly labeled maltose. When amylomaltase is incubated, even after extensive dialysis with radiochemically pure [¹⁴C]maltose as the only substrate, [¹⁴C]glucose and ¹⁴C-maltodextrins are formed immediately. They can be separated and purified by chromatographic procedures and exhibit the same specific radioactivity (with respect to the glucosyl moiety) as the initial maltose. The primers used for this reaction are present in minute amounts, bound to the enzyme (180).

Maltodextrin phosphorylase, encoded by malP, forms glucose-1-phosphate by sequential phosphorolysis of the nonreducing end glucose moieties of larger dextrins. As discussed above, amylomaltase does not split the glycosidic bond of maltose, but the net products of the action of MalP on maltose are glucose and maltodextrins. Since glucose will be removed in vivo by glucokinase to form glucose-6-phosphate, maltodextrins would accumulate. Indeed, malP malQ⁺ mutants can grow on maltose. Under these conditions, they become very large, are filled with long, linear dextrins (238, 239), and stain blue with iodine (1). Surprisingly, they do not transform these dextrins into glycogen, even though the glgB-encoded branching enzyme is present. Maltodextrins do not accumulate in the malP⁺ wild-type strain. Maltodextrin phosphorylase (8, 181, 242) recognizes maltopentaose and longer linear maltodextrins and forms -glucose-1-phosphate by phosphorolysis from the nonreducing end of the maltodextrin. The enzyme consists of a dimer with 796 amino acids per polypeptide. The three-dimensional structure of the enzyme has been solved recently (177, 290).

Obviously, it is important that maltodextrin phosphorylase does not attack maltotetraose and maltotriose, since dextrins of a minimum size are required for full activity of amylomaltase. Notably, glycogen phosphorylase, the glgP-encoded enzyme, supposedly involved in the degradation of glycogen and linear dextrins (300), cannot replace maltodextrin phosphorylase in the utilization of maltose and maltodextrins as a carbon source.

The reaction catalyzed by maltodextrin phosphorylase is, of course, reversible. The rate in the phosphorolysis direction will be stimulated by increasing concentrations of cytoplasmic P_i. In turn, the concentration of internal P_i will vary depending on the availability of external P_i and phosphorus-containing organic compounds, as well as the state of induction of the phoB-dependent pho regulon (298). There are other connections between the mal and the pho regulon. Aside from the sequence similarity between the proteins of the maltose- and phoB-dependent Ugp transport systems for glycerol-3-phosphate (178) and the functional exchangeability between their ATP binding subunits, MalK and UgpC (115), there is a common response (i.e., repression) of the two systems to dominant mutations in envZ, resulting in overphosphorylation of OmpR, the response regulator of the two-component regulatory EnvZ-OmpR system involved in osmoregulation (33).

In addition, the utilization of a fermentable carbon source such as glucose, trehalose, or maltose at P_i concentrations below 1 mM requires the derepression of the pho regulon. phoB mutants do not turn deep red on MacConkey indicator plates (0.3 mM P_i) unless 5 mM P_i is added. Also, whereas strains that turn red on these plates derepress the pho regulon, nonfermenters that appear pale remain repressed for the pho regulon. Apparently, the utilization of a fermentable carbon source requires higher cellular P_i concentrations than does that of a noncarbohydrate carbon source present in MacConkey plates (111).

MalZ was discovered in malF or malG mutants that transport maltose independently of MBP. In contrast to the wild type, these mutants also transport p-nitrophenyl -maltoside (NPG2) and are able to hydrolyze this compound in the cytoplasm (211). Since amylomaltase is unable to hydrolyze NPG2 and since the observed NPG2-hydrolyzing activity was maltose inducible and MalT dependent, it was clear from the start that the novel enzyme must be a member of the maltose regulon. The cloning and sequencing of the malZ gene and the isolation and biochemical characterization of the encoded protein revealed an enzyme that hydrolyzed maltoheptaose and smaller maltodextrins to glucose and maltose. The smallest substrate is maltotriose; maltose is not a substrate. In contrast to other glucosidases, the MalZ enzyme preferentially removes glucose (and to some extent maltose) consecutively from the reducing end of the maltodextrin chain (267). The deduced amino acid sequence of MalZ reveals homology to cyclodextrinyl transferases (190). We found that the enzyme does not hydrolyze -cyclodextrin or pullulan. However, -cyclodextrin (and, to a much smaller extent, -cyclodextrin) is an excellent substrate, forming maltose and glucose. The significance of this finding is unclear (185). -Cyclodextrin is not a carbon source for E. coli. The compound cannot diffuse through the outer membrane or be taken up by the maltose transport system. malZ mutants grow normally on maltose and maltodextrins. Only in combination with a pgm mutation do malZ mutants have a Mal phenotype.

malQ mutants cannot grow on maltose or maltotriose. This is somewhat surprising since MalZ should release glucose (which could be used as a carbon source) from maltotriose. Most probably, the simultaneous accumulation of maltose resulting from the MalZ-dependent hydrolysis of maltotriose in the malQ mutant is toxic. By selecting malQ mutants to grow on maltose, a mutation in malZ (malZ292) was obtained. Surprisingly, the purified mutant MalZ enzyme is not able to hydrolyze purified maltose and shows the same maltodextrin-hydrolyzing activity as the wild-type enzyme. However, by using [¹⁴C]glucose in the presence of unlabeled maltotriose or maltotetraose, the mutant enzyme, unlike the wild-type enzyme, is able to transfer maltodextrins onto the labeled glucose. The mutant MalZ enzyme has acquired a quality which is similar to that of amylomaltase. However, amylomaltase is strictly a transferase, which is not true for the mutant MalZ enzyme. The latter also remains a hydrolase, allowing the net hydrolysis of maltose to glucose in the presence of maltodextrins (185). As in the case of amylomaltase, the utilization of maltose by the mutant MalZ enzyme in vivo requires the endogenous formation of maltodextrins that can be used as primers. The first step in the catalysis performed by amylomaltase and the MalZ enzyme is identical, namely, release of glucose. However, whereas in the case of amylomaltase the maltodextrinyl residue can be transferred only onto the C-4 carbon hydroxyl group of the nonreducing glucose residue of the acceptor molecule, in the case of MalZ it can also be transferred to water.

The malZ gene has been found and characterized as a typical mal gene that is dependent on MalT (235, 267). Recently, we found that malZ expression can also occur in the absence of MalT, in particular under conditions of high medium osmolarity. Using the method of primer extension with reverse transcriptase, we found that besides the MalT-dependent transcript described first (235), malZ is also transcribed into a second mRNA, originating upstream of the start site of the main promoter (145). The significance of this phenomenon is unclear.

The final products of the combined action of amylomaltase, maltodextrin phosphorylase, and maltodextrin glucosidase are glucose and -glucose-1-phosphate. Therefore, to funnel these end products of the specific maltose enzymes into general metabolism, the cells rely on glucokinase (encoded by glk) for the phosphorylation of glucose to glucose-6-phosphate and on phosphoglucomutase (encoded by pgm) for the transformation of -glucose-1-phosphate to glucose-6-phosphate, which enters into glycolysis. Mutants unable to phosphorylate glucose due to the lack of glucokinase, enzyme II^Glc (encoded by ptsG) and enzyme II^Man (ptsM) of the PTS-mediated phosphorylation, are unable to grow on maltose (30). Similarly, they are unable to grow on trehalose, which is degraded by an enzyme that produces internal glucose (219). Therefore, the utilization of internally produced glucose appears to be essential for growth. It is unclear whether the inability to grow on these sugars in the absence of glucose phosphorylation is due to a possible toxic effect of accumulating internal glucose (requiring extrusion) or the insufficient flow of carbon and energy via the phosphoglucomutase pathway alone. The drain of glucose-1-phosphate for the biosynthesis of polysaccharides might be another limiting factor.

pgm mutants lacking phosphoglucomutase activity are able to grow on maltose, even though seemingly only one half of the maltose molecule can be used as a carbon and energy source. These strains exhibit a maltose blue phenotype (1, 220) caused by the massive production of dextrins due to the accumulation of glucose-1-phosphate and the reversal of the maltodextrin phosphorylase reaction. The problem created by the accumulation of maltodextrins is eased by the action of maltodextrin glucosidase, the MalZ enzyme, which produces glucose from the maltodextrins, followed by glucokinase-dependent phosphorylation to glucose-6-phosphate. Indeed, pgm malZ double mutants are unable to grow on maltose. Similarly, pgm mutants can grow on galactose (1) but only when all the enzymes of the maltose system, including MalZ, are present (69). pgm mutants, even null mutations created by insertion elements (153), still show residual Pgm-like activity when the enzyme activity is assayed by coupling the formation of glucose-6-phosphate from glucose-1-phosphate to the NADP⁺-dependent oxidation by glucose-6-phosphate dehydrogenase. This activity is caused by a sugar-phosphate transferase which is able not only to hydrolyze sugar phosphates but also to transfer the phosphate moiety to another sugar molecule (258). Thus, in the presence of free glucose, glucose-1-phosphate can be transformed to glucose-6-phosphate, mimicking phosphoglucomutase activity.

The malS gene (235) was discovered as a maltose-inducible and MalT-dependent gene by the lacZ fusion technique (91). MalS is a periplasmic -amylase with weak homology to the cytoplasmic MalZ enzyme but not to amylomaltase. MalS cleaves maltodextrins except maltose. Its preferred product released from larger dextrins is maltohexaose. The enzyme needs Ca²⁺ for activity and DsbA (157) for proper folding in the periplasm. The four cysteine residues of MalS form intramolecular disulfide bonds. The disulfide bond at Cys40-Cys58 is located in an N-terminal extension of about 160 amino acids which has no homology to other amylases but to the proposed peptide-binding domain of GroEL, the Hsp60 of E. coli. The N-terminal extension is linked to the C terminal amylase domain via disulfide bond Cys104-Cys520. Reduction of the disulfide bonds by dithiothreitol treatment led to aggregation, suggesting that the N terminus of MalS may represent an internal chaperone domain (256). malS mutants have no recognizable maltose phenotype. The function of the enzyme is most probably the degradation of longer dextrins that enter the periplasm to shorter dextrins that can be transported by the binding protein-dependent maltose/maltodextrin transport system (92). Even though MBP, the recognition site of the transport system, binds all maltodextrins from maltose to amylose, only dextrins up to the size of maltohexaose can be transported across the membrane (84, 86).

Figure 6 summarizes the pathway by which, according to our view, maltose is degraded into glucose and -glucose-1-phosphate by the maltose-specific enzymes.

Maltose uptake and utilization in other bacteria, notably in gram-positive bacteria, usually proceeds differently. In Bacillus subtilis, maltose is probably taken up by a proton motive force-dependent transport system and split internally into glucose and glucose-1-phosphate by a maltose-specific phosphorylase (266). The observation that Lactobacillus sanfrancisco takes up maltose and excretes half of it as glucose points to the same mechanism (172). In addition, maltose may also be taken up in lactobacilli by a PEP-dependent sugar PTS system and is internally hydrolyzed to glucose and glucose-6-phosphate (270). The isolation of a maltose-6-phosphate hydrolase and the vicinity of its gene, malH, to malB, encoding a putative IIB-like enzyme of the PTS, points to the same maltose degradative pathway in Fusobacterium mortiferum (23). This is quite in analogy to the uptake and degradation of trehalose in E. coli (142, 219).

NONCLASSICAL REGULATORY PHENOMENA

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MalK, the ATP-Hydrolyzing Subunit, as a Sensor for mal Expression

It has been known for a long time that mutations in the transport system, later identified as malK mutations, lead to elevated expression of the remaining mal genes (31, 125). This can be conveniently observed by using malK-lacZ fusions with a nonfunctional MalK, which exhibit high and constitutive -galactosidase activity. An intact MalT activator is required for this constitutivity. The same malK-lacZ fusion in a malK⁺ merodiploid genetic background exhibits low -galactosidase activity that can be induced by maltose, reflecting the induction observed in the wild type (31). These findings indicate that MalK acts as a repressor. Indeed, when plasmid-borne malK is overexpressed in the original malK-lacZ fusion strain, -galactosidase activity is abolished. This effect can also be observed in a wild-type strain where the overexpression of malK renders the strain unable to grow on maltose, even though the strain can grow normally on glycerol (210). The mutational analysis of MalK has shown that the regulatory function resides in the C-terminal domain of the protein and that its functions in transport and regulation are independent of each other (147, 151, 237). Surprisingly, a malT(Con) mutation leading to expression of the mal genes in the absence of inducer can largely counteract the repressing effects of overpr