Phosphoenolpyruvate: Carbohydrate Phosphotransferase Systems
Chapter
75
P. W. POSTMA, J. W. LENGELER, and G.R. JACOBSON
Bacteria can utilize a wide range of carbon sources, and their translocation across the cytoplasmic membrane is catalyzed by a variety of specific transport systems. Most bacteria can also adapt to their continuously changing surroundings in order to effectively compete with other organisms for limiting nutrients. To monitor their environment and to choose between the various carbon sources, cells contain sensing devices. In this chapter, we describe the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase systems (PTSs), which are both transport and sensing systems. The PTSs are involved in both the transport and the phosphorylation of a large number of carbohydrates (PTS carbohydrates), in the movement of cells toward these carbon sources (chemotaxis), and in the regulation of a number of metabolic pathways. The PTS was discovered in Escherichia coli more than 30 years ago (118). Since that time, we have learned many details about the properties and the functioning of the many PTS proteins in both gram-negative and gram-positive microorganisms.
In the first edition of this book, the literature on the PTS in enteric bacteria up until 1987 was covered. More than 400 new papers that deal with the PTS in both gram-negative and gram-positive organisms have been published since then. In particular, as a result of the cloning and sequencing of the corresponding genes, progress has been made in the characterization of the many carbohydrate-specific PTS proteins. The three-dimensional structures of a few of the soluble PTS proteins have also been determined.
In this chapter, we give an overview of the PTS in enteric bacteria, with particular emphasis on progress since 1987. References to the earlier literature are included if required to present a coherent picture. The reader is also referred to a number of other reviews that have dealt with specific or general aspects of the PTS of both gram-negative and gram-positive bacteria (131, 156, 202, 206, 222, 231, 243, 246).
The PTS catalyzes the following overall process:
Carbohydrate phosphorylation is coupled to carbohydrate translocation across the cytoplasmic membrane, the energy for these processes being provided by the glycolytic intermediate PEP.
The high free energy of hydrolysis (Δ G 0' ) of the phospho group of PEP (about –14.7 kcal/mol) (1 cal = 4.164 J) is utilized for both the translocation and the phosphorylation of its substrates and is largely retained in the phosphorylated derivatives of PTS proteins (303).
The proteins that make up the PTS in the enteric bacteria E. coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) catalyze the following reactions:
Enzyme I (EI) and histidine protein (HPr) are soluble cytoplasmic proteins that participate in the phosphorylation of all PTS carbohydrates and thus have been called the general PTS proteins. On the other hand, the EIIs are carbohydrate specific. They may consist of a single membrane-bound protein comprising three domains (A, B, and C) such as that for mannitol (EIIMtl), or they may consist of two or more proteins of which at least one is membrane bound (e.g., B and C) and one is soluble (IIA or EIII), such as the IICBGlc-IIAGlc pair for glucose (Fig. 1). In either case, the phospho group is transferred from PEP to the carbohydrate via obligatory phospho intermediates of EI, HPr, EIIA, and EIIB. The EIIC domain, the integral membrane portion of an EII, forms the translocation channel and presumably the specific substrate binding site. A third type of EII is found in the mannose PTS, in which both A and B domains are fused in a single soluble polypeptide, and two integral membrane proteins (IICMan and IIDMan) are involved in mannose translocation (Fig. 1). Figure 1 shows the phosphotransfer reactions of the PTS and its vectorial nature with respect to transport and phosphorylation for the mannitol, glucose, and mannose PTSs. We discuss these PTSs and other variations in more detail below.
EI has been purified to apparent homogeneity from E. coli (M r = 63,412) and S. typhimurium (M r = 63,489). Phosphorylation occurs at the N-3 position of a histidyl residue of EI (His-189 in E. coli) during autophosphorylation with PEP (reaction 1 of scheme 1) (3, 303) and requires, most likely, a dimeric form of the protein (117, 163, 303) and a divalent cation such as Mg2+ or Mn2+ (303). The stoichiometry of phospho group incorporation into EI from either E. coli or S. typhimurium is one per monomer (297, 303), and this reaction requires at least one free sulfhydryl group, which appears to be essential for dimerization (73, 74, 81).
The gene encoding EI (ptsI) has been cloned and sequenced from E. coli (40, 242) and S. typhimurium (141). The deduced amino acid sequences of the EIs from E. coli and S. typhimurium differ in only 16 residues, consistent with the fact that these proteins are functionally interchangeable in these organisms.
HPr is a small monomeric protein with a molecular weight of 9,109 in E. coli and S. typhimurium. Its size and heat stability have aided its purification. It is phosphorylated by P-EI at the N-1 position of a histidyl residue, His-15 (reaction 2 of scheme 1) (304). Primary sequence comparisons show that the enteric HPrs, encoded by the ptsH gene, are very similar (those from E. coli and S. typhimurium are identical) (23, 43).
An HPr-like domain is found in the fructose-specific, hybrid phosphotransfer protein (FPr) from S. typhimurium (63, 276) and E. coli (64, 219, 296). This protein also contains an active-site His consensus sequence in its HPr-like domain (63, 219). Moreover, FPr can substitute for HPr in E. coli and S. typhimurium mutants lacking HPr (64, 251, 296).
The first carbohydrate-specific PTS proteins to be studied were a complex of membrane proteins from E. coli that were involved in the phosphorylation of several hexoses (119) and were called II-A and II-B at that time. Subsequent studies showed that PTS-mediated phosphorylation required in a number of cases both soluble and membrane-bound, substrate-specific proteins (called factors III and EIIs, respectively). From the amino acid sequences of these carbohydrate-specific proteins, it became clear that in some cases there was no separate, soluble factor III but rather that a cytoplasmic structural domain of EII had a factor III-like function (252). A uniform nomenclature for the carbohydrate-specific PTS proteins, in which factor III or the factor III-like domain is called IIA, has been proposed (247). Other domains or proteins in the carbohydrate-specific complex are called IIB (a second hydrophilic domain), IIC (the membrane domain), and IID (a second membrane-bound protein found in a few PTSs, e.g., that for mannose). This nomenclature is used in this chapter. Table 1 summarizes the different EIIs that have been studied in enteric bacteria. We discuss some of the EIIs in more detail below.
Table 1EIIs in various enteric bacteria |
The best-characterized IIA protein is that specific for glucose in E. coli and S. typhimurium (IIAGlc, previously called IIIGlc). It works in conjunction with a membrane-bound IICBGlc protein. IIAGlc has been purified from both E. coli and S. typhimurium (157, 212, 262), and the crr gene, encoding IIAGlc, has been cloned and sequenced from both organisms (171, 242). The deduced monomer molecular weights are 18,230 and 18,226, respectively, and the deduced amino acid sequences of these proteins differ in only 3 of 169 positions. IIAGlc is extremely heat stable and has a strong tendency to form oligomers (263).
IIAGlc is phosphorylated by P-HPr at the N-3 position of His-90 (reaction 3, scheme 1) as an intermediate in PEP-dependent phosphotransfer to glucose via IICBGlc (Fig. 1) (44, 209). It is also required for the uptake and phosphorylation of other PTS carbohydrates such as sucrose (in cells harboring a naturally occurring plasmid that encodes a IIBCScr protein [132]), trehalose via IITre (14), and glucose via the cryptic maltose system MalX (214).
Soluble IIA proteins involved in uptake and phosphorylation of fructose (63, 276, 298), cellobiose (179, 180, 220), and glucitol (317) also have been partially characterized (Table 1). Their monomer molecular weights are in the range of 10,000 to 15,000, with the exception of the hybrid FPr, which contains an amino-terminal IIAFru domain in addition to a carboxy-terminal HPr-like domain and a middle domain (63), and the IIABMan protein involved in mannose transport in E. coli (56, 298).
In a number of cases, the IIA domain has been shown to be covalently linked to the other domains, IIB and IIC. This is the case for IIMtl, the EII specific for mannitol in E. coli (91, 122). Other examples include IINag, specific for N-acetylglucosamine (Nag) (192, 234), and IIBgl, specific for β-glucosides (16, 261).
Only a few EII complexes have been purified and studied. The 65,000-Da IIMtl of E. coli was purified to apparent homogeneity in a functional form (92) and catalyzed the PEP-dependent phosphorylation of mannitol in vitro in the presence of purified EI and HPr. Purified IIMtl was extremely sensitive to proteolysis by trypsin, which cleaved the protein nearly in half (91). The membrane-bound protein also could be cleaved by trypsin but only in everted membrane vesicles (90, 271). A 34-kDa amino-terminal fragment remained associated with the membrane, while a 29-kDa carboxy-terminal fragment was released from these vesicles by trypsin treatment (271). The mtlA gene, encoding E. coli IIMtl, was cloned and sequenced (122), and a hydropathy analysis of the primary sequence showed the amino-terminal half to be very hydrophobic and the carboxy-terminal half to be hydrophilic, supporting the direct topographical studies.
Purified IIMtl is phosphorylated by P-HPr (236, 298), and two phospho groups are incorporated per polypeptide chain (184). The sites of phosphorylation are His-554 in the IIA domain and, surprisingly, a cysteinyl residue (Cys-384) in the IIB domain. His-554 is probably phosphorylated at the N-3 position of its imidazole ring (182, 183).
Integral-membrane EIIs specific for glucose from E. coli (15, 159) and S. typhimurium (15, 52) have also been purified to apparent homogeneity from detergent-extracted membranes. They both have apparent subunit molecular weights of 45,000 and are very similar. Purified EIICBGlc catalyzes the PEP-dependent phosphorylation of glucose and its analog methyl α-d-glucopyranoside (αMG) in the presence of EI, HPr, and soluble IIAGlc. During this reaction, IICBGlc is phosphorylated by P-IIAGlc (8, 50). The site of phosphorylation of IICBGlc is also a cysteine residue in the IIB domain (Cys-421) (158).
The E. coli IIMan complex (encoded by the manXYZ operon) was highly purified in a functional form and shown to consist of three proteins: a soluble IIABMan (35 kDa) and two membrane proteins, IICMan (25 kDa) and IIDMan (27 kDa) (53, 56). PEP-dependent mannose phosphorylation by the IIMan complex requires all three proteins (56, 254, 310), and the purified complex appears to have a subunit stoichiometry of 2 IIABMan:1 IICMan:2 IIDMan (227).
Several of the soluble proteins and the hydrophilic IIA and IIB domains of the PTS have been structurally analyzed by physicochemical methods. The three-dimensional structures of a few have also been determined. By contrast, models of the membrane-bound EII domains have been derived only from the analysis of amino acid sequences and by gene fusion techniques. In this section, we review recent evidence on the structures of PTS proteins as they relate to each other and to their functions.
EI.
The amino acid sequences of EIs from E. coli and S. typhimurium have been deduced from DNA sequencing. Local similarity around the phosphorylated histidine (His-189 in E. coli EI) is found with pyruvate phosphate dikinase (199, 316) and PEP synthase (175). Similar to EI, both enzymes also autophosphorylate at an active-site histidine with PEP or ATP.
A dimer of identical EI subunits has been proposed to autophosphorylate on each monomer in the presence of Mg2+ and PEP (73, 74). The phosphorylated dimer dissociates to the monomers which phosphorylate HPr. The rate-limiting step in the whole cycle is apparently subsequent reassociation of the dephosphorylated monomers. Microcalorimetry (141), fluorescence spectroscopy studies (174), and proteolysis studies (121) revealed two autonomous domains, probably connected through a linker. The isolated amino-terminal domain, which contains His-189, cannot be phosphorylated by PEP or P-EI but can be phosphorylated by P-HPr in a fully reversible reaction. The carboxy-terminal domain, in contrast, appears to be necessary for autophosphorylation and dimerization. It could thus play a key role in regulating EI activity (141), as has recently been suggested by monitoring the monomer-dimer equilibrium under various conditions by fluorescence anisotropy of a pyrene-labeled EI (25, 26).
HPr.
HPr from E. coli was the first PTS protein whose three-dimensional structure was determined by X-ray crystallography (at 2.0-Å [0.2-nm] resolution) and by two- and three-dimensional nuclear magnetic resonance (NMR) techniques (72, 98, 283, 285). According to these data, the overall folding topology is an open-face β-sandwich consisting of four antiparallel β-strands and three α-helices. These results are consistent with studies measuring the binding of monoclonal antibodies to mutant HPrs (266). Although HPrs differ considerably at the primary amino acid level, the three-dimensional structure of HPr is conserved throughout the gram-positive and gram-negative bacteria (97).
These structural studies have confirmed previous reports (106, 107, 222) that had established an essential role for His-15 (the phosphorylated residue) and Arg-17. The active center consists of His-15, which is hydrogen bonded to a sulfate anion, and Arg-17. His-15 is localized close to the surface. In the "open" conformation, the side chains of His-15 and Arg-17 are directed as far away from each other as possible. A "closed" conformation, in which the side chains are close together with a sulfate anion in the active center, likely resembles the phosphorylated form of HPr (78).
As indicated before, EIIs may comprise from one to four proteins, at least one of which is membrane bound (Fig. 2). Each EII contains three (IIA, IIB, IIC) and sometimes four (IID) functional domains (Table 1). The variations in the number of proteins which form an EII are most likely the result of fusion and splitting of these domains during evolution. They do not reflect essential mechanistic differences.
EIIs may be grouped into at least four families based on sequence alignments (Table 1) (135, 216, 244). The amino acid sequences of the EIIs belonging to one family share more than 25% identical amino acid residues over the entire molecule, while the similarities for members of different families are restricted to more local motifs. Furthermore, functional complementation between equivalent domains of the members of a family is often possible. Lack of interfamily complementation most likely reflects the existence of specific interactions between the different domains.
Most EIIs share several conserved features (Fig. 2) (127): (i) three autonomous structural domains (IIA, IIB, and IIC), which may be free or fused, comprising a total of about 630 residues; if fused, they are coupled by linkers (135, 264, 277) and may be arranged in different orders (e.g., BCA and CBA); (ii) a hydrophilic IIA domain (ca. 100 residues) with a conserved His residue which can be phosphorylated by P-HPr; (iii) a hydrophilic domain IIB (ca. 100 residues) which is phosphorylated by P-IIA at a conserved cysteine (or in some cases a histidine); and (iv) a IIC domain of about 350 residues which includes six to eight potential transmembrane helices, at least one large hydrophilic loop, and an essential glutamate residue.
The Glucose-Sucrose Family.
Members of the glucose-sucrose family, listed in Table 1, all phosphorylate their substrates (i.e., glucose, glucose-containing disaccharides, and amino sugars) at the C-6 position of a glucopyranoside ring. As for other EIIs, the hydrophobic domain IIC constitutes the translocation channel. In this family, domains IIC and IIB are always fused, either in the order IICB for the glucose group or in the order IIBC for the sucrose group. Provided that IIC is in the form of a bundle of α-helices (see below), the IICB and IIBC arrangements could be structurally equivalent. The IIA domains, however, can be free proteins (e.g., IIAGlc) or can be connected by a linker to the carboxy-terminal end of the previous domains (e.g., IICBANag and IIBCABgl). Several EIIs (e.g., IICBMal [214], IICBTre [14], and IIBCScr [132]) share IIAGlc with IICBGlc. This corroborates further the close relationship within this family and the hypothesis (252) that the fused forms are the equivalent of the split versions of different EIIs. Intermolecular complementation has been shown between IICBGlc and IIANag and between IIBCScr and IIANag or IIABgl (82, 223, 259, 260, 290, 292).
These observations strongly support a modular evolution of the EIIs in the glucose-sucrose family from autonomous protein domains. The interdomain DNA then corresponds to recombination sites which allow variable arrangements of gene fragments, and the protein sequences encoded by these DNAs correspond to linkers. In agreement with this hypothesis, nucleotide and amino acid sequence differences between closely related and fused EIIs, e.g., IINag from E. coli and K. pneumoniae, are more frequent within the linkers (294).
The Mannitol-Fructose Family.
IIMtl from E. coli, perhaps the best-understood EII, shows only local similarity to members of the glucose family. It is a large protein of the IICBA type. Various combinations of these domains have been cloned and expressed in functional form in E. coli (69, 144, 229, 286, 287, 308). Recently, two cryptic genes, cmtA and cmtB (for cryptic mannitol transport), which lack a promoter, were found in E. coli (268). If cloned behind a functional promoter, they express a IICBCmt and a IIACmt, respectively. The sequences of these proteins resemble those of IICBMtl and IIAMtl (about 51% identical residues), and the proteins are active in mannitol transport and phosphorylation.
Because of sequence similarities, IIFru complexes which generate fructose 1-phosphate during transport belong to the same class as IIMtl and IICmt, but they are of the IIBC type. The domain arrangements and sizes, however, are unusual. In enteric bacteria, a membrane-bound protein (gene fruA) of 553 residues (210) has the structure IIB'BC, in which IIB' represents an intramolecular duplication (residues 1 to 100) of IIB (residues 116 to 215). The two domains are fused through a linker to each other and to the amino-terminal end of IICFru (127, 316). The IIBFru domain contains a cysteine residue located in a consensus sequence which also surrounds the phosphorylated Cys-384 of the E. coli IIMtl. The IIB' domain, in contrast, lacks this consensus sequence. In the fru operon of enteric bacteria, gene fruF, which encodes a soluble protein FruF (376 residues), is found together with gene fruA for the IIB'BC protein. FruF, also called FPr or pseudo-HPr, contains three domains (63): (i) an amino-terminal IIAFru domain which resembles the IIAMtl and IIACmt domains (>38% identical residues), (ii) a central part of unknown function with a weak similarity to the receiver part of two-component systems (316), and (iii) a carboxy-terminal part which resembles HPrs from enteric bacteria (about 35% identical residues).
The Mannose-Sorbose Family.
Three highly similar EIIs (45 to 65% identical residues) for mannose (56) and l-sorbose (301, 302) in enteric bacteria and for fructose in Bacillus subtilis (152) contain four (IIABCD) instead of the usual three (IIABC) domains (Fig. 2). The mannose PTS is made up of a large soluble protein (ManX, 323 residues, gene manX) in which domains IIA and IIB are fused through a linker (Fig. 2) (51, 56, 310). Two proteins constitute the membrane-bound part of this family of EIIs. Neither of the membrane-bound proteins seems to be phosphorylated, but all proteins (three in the case of mannose and four in the case of sorbose and fructose) are required for transport and phosphorylation (56, 310). The mannose PTS is also required during infection of E. coli cells by bacteriophages lambda (48, 56, 254, 310) and N4 (109). The hydrophilic amino-terminal end of ΙΙDMan seems essential in phage recognition and thus may protrude into the periplasmic space (56). Neither IIABMan nor phosphorylation is required for phage infection, perhaps indicating the formation of a transmembrane channel by IICMan and IIDMan alone.
Structural details for IIAGlc from E. coli have become available through NMR spectroscopy and X-ray crystallography studies (188, 189, 190, 315). The protein consists of 13 β-strands forming an antiparallel β-barrel with three β-sheets. The binding interface between IIAGlc and HPr comprises predominantly hydrophobic residues, which form a shallow depression at the surface near the active-site His-90 of IIAGlc. His-75 is a second histidine residue found in the hydrophobic binding interface of IIAGlc. On the basis of mutagenesis studies, His-75 appears to have an important role in phosphotransfer from P-IIAGlc to IIBGlc (209, 223). Upon phosphorylation-dephosphorylation of His-90, only small structural rearrangements occur, and these rearrangements are limited to the active site (187). IIBGlc, in contrast, comprises four β-strands which form an antiparallel β-sheet, two larger α-helices at both ends, and a smaller helix composed of residues 52 through 58 (66). Cys-421 is at the carboxy-terminal end of a β-strand close to a β-turn and hence is probably at the protein surface.
A secondary structural model of IIaMtl was published recently (115, 284). The predicted structure is quite different from that reported for IIAGlc and consists of five helical regions and only two β-strands forming an antiparallel β-sheet. This structural difference between the IIA domains of the glucose and mannitol families may account for the lack of interfamily complementation in the studies mentioned earlier. The predicted and NMR secondary structures for IIAMan (150, 265, 274) and IIBCel (1) are also different from the structures proposed for IIAGlc and IIAMtl and for IIBGlc, respectively. Finally, studies on denaturation of the IIABMan protein have shown that the two domains unfold independently, indicating the absence of stabilizing interactions between these domains (151).
Secondary-structure models have been proposed for several IIC domains. The models rely primarily on hydropathy analyses, the location of strictly conserved and essential residues, and gene fusion techniques that use PhoA and LacZ fusions. For E. coli IICMtl, such studies predict six membrane-spanning structures (probably α-helices approximately 20 residues in length) connected by three short periplasmic loops (≤30 residues) and two large cytoplasmic loops (≥80 residues) (275). The model is consistent with secondary-structure predictions and biochemical studies (94). Of the ca. 120 residues of ΙΙCMtl located in putative transmembrane helices, only 20 are polar and/or capable of forming hydrogen bonds with the substrate, a property the model shares with other proposed EII models (131).
Studies with lacZ and phoA fusions indicated eight instead of six transmembrane helices in the IIC domain of IICBGlc (20). EIIs of the mannose class seem to form a stable membrane-associated complex in which the soluble protein(s) associates in a reversible form with the two membrane-bound proteins (51, 53, 55, 310). Fusion studies indicate that the carboxy-terminal end of IICMan (four to six helices) is close in space to the amino-terminal end of IIDMan (two helices), also suggesting a six- to eight-helix structure for IIMan (264). It remains to be shown whether the IIC domains from these different families deviate in structure or whether the models are not yet accurate.
As mentioned previously, P∼His is the protein-bound phospho intermediate for EI and HPr. The phosphoryl transfer potential of P-EI is nearly as high as that for PEP (303). Steady-state kinetic (300, 305) and isotope exchange (80) studies indicate that P-EI is an intermediate in reactions 1 and 2 in scheme 1. For EI from S. typhimurium, the Km for PEP for phosphorylation of HPr is 0.2 to 0.4 mM (250, 305), and that for HPr is about 5 μM (305). Similar Kms were obtained for EI from E. coli (223, 300). EI can catalyze a phospho exchange reaction between PEP and pyruvate (80, 250) and this reaction has been used as an assay for EI. The amino-terminal domain of EI contains the active-site His residue (His-189 in E. coli) and participates in phospho exchange with HPr, while the carboxy-terminal domain appears to be important for interaction with PEP and dimerization (141).
P-(His)-HPr, the product of the EI-catalyzed reaction, phosphorylates all IIA domains or proteins for which these reactions have been studied (scheme 1, reaction 3), even though many of these have quite different primary structures. Phosphorylation of His-15 at the N-1 position rather than the N-3 position in E. coli HPr may be favored, because the N-3 atom of this residue is apparently protonated at physiological pH values and is an H-bond donor in unphosphorylated HPr (282). Thus, the N-1 atom would be a better nucleophile for the phospho group in P-EI. Upon phosphorylation of HPr, this hydrogen bond involving the N-3 atom breaks, suggesting a conformational change in the protein (282), although structural comparisons suggest that any phosphorylation-induced conformational change in HPr may be rather small (97).
As mentioned earlier, three-dimensional structural studies of HPr suggest an essential role of Arg-17 in HPr function. Possibly P-(His-15)-HPr is stabilized by the interaction of Arg-17 with the phospho group (78). Mutation of Arg-17 of E. coli HPr to Gly, Lys, or several other amino acids reduces the catalytic efficiency 100- to 1,000-fold (6).
The IIGlc Complex.
A His residue is the phospho acceptor from P-HPr in IIAGlc and all other IIA proteins or domains studied to date. Kinetic studies have shown that for the IIAGlc protein from E. coli, the apparent Km in carbohydrate phosphorylation assays for E. coli P-HPr is 0.3 μM (223). As in EI and HPr, the phosphoryl transfer potential of P-IIAGlc is near that of PEP (Δ G 0? for hydrolysis is ca. –14 kcal/mol [303]). The extreme amino terminus of IIAGlc appears to be important for its interaction with and phosphorylation of IICBGlc. IIA
, a form of IIAGlc which migrates faster on nondenaturing gels than native IIAGlc because of proteolytic loss of the first seven amino-terminal residues, is phosphorylated by P-HPr at a rate similar to that seen for IIAGlc but donates its phospho group to IICBGlc at only 2 to 3% of the rate seen with P-IIAGlc (155, 157). Chemical modification of the amino-terminal Gly residue of IIAGlc (87) results in similar properties. A mutant IIAGlc in which Phe-5 is replaced by Trp is actually more efficient than the wild-type protein in in vitro phosphorylation of αMG (209).
Site-directed mutagenesis studies (209) and the three-dimensional structure determination of E. coli IIAGlc (315) have provided clues to structure-function relationships in the phosphotransfer activity of this protein. Replacement of the active-site His-90 in IIAGlc from E. coli with Gln results in an inactive protein that cannot be phosphorylated by P-HPr (209). A similar replacement in the nearby His-75 results in a mutant protein that can still be phosphorylated by P-HPr but cannot transfer the phospho group to IICBGlc (209). The His-75 residue may have some important role in phosphotransfer from P-IIAGlc to IICBGlc (209, 315), but the exact function of this His residue in phosphotransfer remains to be determined.
Purified IICBGlc catalyzes PEP-dependent phosphorylation of glucose in the presence of EI, HPr, and IIAGlc. It also catalyzes phospho exchange between P-IIAGlc and IIAGlc and between glucose 6-phosphate and glucose, indicative of reactions 4 and 5 in scheme 1, respectively (54, 224). The first evidence that IICBGlc is phosphorylated as a catalytically important intermediate was obtained by examination of the stereochemical course of the phosphotransfer reactions carried out by the E. coli glucose PTS, using chiral PEP (8). Subsequently, covalent labeling of purified IICBGlc from S. typhimurium with 32PEP was also demonstrated, and 0.6 to 0.8 phospho group was incorporated per IICBGlc polypeptide (50). P-IICBGlc can transfer its phospho group to glucose to form glucose 6-phosphate in the absence of IIAGlc, but the rate of phosphotransfer is greatly stimulated by adding unphosphorylated IIAGlc (50), suggesting that IIAGlc may allosterically activate the phosphotransfer reaction from P-IICBGlc to glucose. As mentioned earlier, the residue in IICBGlc that is phosphorylated by P-(His-90)-IIAGlc is Cys-421 in the IIB domain (158). It was shown previously that changing Cys-421 to Ser results in an inactive protein that cannot be phosphorylated by P-IIAGlc (178). The IICGlc and IIBGlc domains have been separately expressed and purified (21). In combination with IIAGlc, they are active in sugar phosphorylation but at only 2% of the level found with native IICBGlc (21).
Like many PTS EIIs, IICBGlc from E. coli or S. typhimurium has a high affinity for its substrates in the carbohydrate phosphorylation reaction. The apparent Km for glucose has been reported to be from 3 to 10 μM (162, 273), and that for αMG has been reported to be from 6 to 28 μM (68, 273), while the Km for P-IIAGlc is between 2 and 5 μM (68, 157, 223).
The IIMtl Complex.
As discussed previously, the IIA, IIB, and IIC domains of E. coli IIMtl are combined in a single polypeptide. IIMtl catalyzes PEP-dependent phosphorylation and transport of mannitol in the presence of EI and HPr. Purified IIMtl also catalyzes two phospho exchange reactions, one between P-HPr and HPr (278) and the other between mannitol 1-phosphate and mannitol (91, 92). These reactions are presumably simply indicative of the reversibility of reactions 3 and 5 in scheme 1. Like IICBGlc, IIMtl can be phosphorylated by 32PEP (236, 298). E. coli IIMtl gives ping-pong kinetics for mannitol phosphorylation, with mannitol and P-HPr as the varied substrates (68, 235), consistent with a catalytically important P-IIMtl intermediate. The apparent Km for mannitol has been reported to be from 2 to 11 μM (68, 91, 125, 235), and that for P-HPr has been reported to be from 1 to 7 μM for IIMtl (68, 235).
The interaction of IIAMtl with HPr has recently been studied by using NMR techniques (284). The spectra show that two blocks of residues in HPr (residues 13 to 21 and 48 to 56) are most strongly affected by binding of IIAMtl to HPr. Not surprisingly, His-15, the active-site His in HPr, is included in the first group of residues. However, some of the other residues whose spectra are affected are not facing the surface of HPr, suggesting that the binding of IIAMtl induces structural rearrangements in HPr. Also not surprisingly, no interaction of P-IIAMtl with P-HPr could be detected in these studies.
Approximately two phospho groups per polypeptide chain are incorporated into IIMtl (184), and two different phospho peptides are isolated from fully phosphorylated IIMtl (183). These studies as well as others show that the phosphorylated residues are His-554 and Cys-384 (69, 182, 286, 287, 288, 308). Two catalytically important phosphorylation sites in IIMtl were also inferred from the stereochemical course of mannitol phosphorylation with chiral PEP, EI, HPr, and IIMtl (166).
Recent studies have shown that His-554 in the IIAMtl domain is the phospho acceptor from P-HPr, that P~Cys-384 (in the IIBMtl domain) is the phospho donor to mannitol, and that phosphotransfer between these residues occurs in intact IIMtl. When His-554 is changed to Ala or Asp (288, 306), the resulting proteins are inactive in PEP-dependent mannitol phosphorylation but still catalyze mannitol:mannitol 1-phosphate phospho exchange. The H554D mutant protein cannot be phosphorylated at all by PEP in the presence of EI and HPr (306), a situation consistent with the assignment of His-554 as the phospho acceptor from P-HPr. When Cys-384 is changed to Ser (288) or to Asp or His (306), the resultant proteins are inactive in PEP-dependent phosphorylation, but at least the C384D and C384H mutants (306) can be phosphorylated by PEP (at His-554).
In IICMtl, an additional His residue (His-195) is involved in high-affinity substrate binding and phosphorylation, and a functional His-195 must be on the same subunit as a functional Cys-384 for phospho transfer to mannitol (307). A second residue in this domain, Glu-257, is also essential for IIMtl activity. An E257A mutant protein is defective in all activities of IIMtl, apparently because it cannot bind the substrate (89, 93). A Glu residue in similar sequence contexts is found in most IIC domains (127, 246) and therefore may be critical for the activities of all these proteins.
The IIMan Complex.
IIMan differs from most other PTS EIIs because the IIA and IIB domains are found in a single hydrophilic protein and because two proteins, IIC and IID, make up the integral membrane part of the complex. The 35-kDa IIABMan protein has been purified (56), and its two domains can be separated by mild trypsinolysis. Each domain has also been separately expressed by subcloning the appropriate parts of the manX (ptsL) gene (55). The amino-terminal IIA domain (M r = 13 kDa) was phosphorylated by PEP in the presence of EI and HPr at the N-3 position of His-10. The carboxy-terminal IIB domain (M r = 20 kDa) was phosphorylated by PEP only in the presence of EI, HPr, and catalytic amounts of IIAMan. In the IIBMan domain, the phosphorylated residue is His-175 (at the N-1 position). No phosphorylation of the IIC or IID domains was detected in these experiments (55). Phosphotransfer was demonstrated between His-10 and His-175 of the same as well as of different subunits of IIABMan. Another histidine residue, His-186, is not phosphorylated but plays an important role, since in a H186N mutant, in vitro phosphorylation is almost completely abolished (274). When the IIAMan and IIBMan domains are expressed in E. coli as individual polypeptides, their activities are similar to that of intact IIABMan (55).
These results show that His-10 of IIABMan is the phospho acceptor from P-HPr and that this reaction is followed by phosphotransfer from His-10 to His-175 of the same protein. In the presence of the IICMan and IIDMan proteins, phospho exchange activity between glucose 6-phosphate and 2-deoxyglucose can be catalyzed by IIBMan but not by IIAMan (55). Thus, P∼His-175 in the IIB domain is likely to be the phospho donor to the carbohydrate (reaction 5, scheme I). Replacement of His-10 or His-175 by cysteine (or Asn/Gln) abolishes phosphotransfer and shows that this protein is different from IIGlc and IIMtl, in which the second phosphorylation site is a cysteine (274). Apparently, in this family of EIIs, P∼His is the phospho donor to the carbohydrate, while in the other EII families thus far examined, this role is played by P∼Cys.
Considerable evidence suggests that both the PEP-dependent and the phospho exchange activities of IIMtl are optimally catalyzed by an oligomeric form of this protein, probably a dimer (13, 88, 108, 137, 145, 181, 228, 236, 237, 272). This raises the question of whether phosphotransfer between the two phosphorylation sites of IIMtl occurs within a single polypeptide in the oligomer or between these two sites in different subunits. Mutant proteins of IIMtl at positions 554 and 384, each of which is inactive alone in PEP-dependent mannitol phosphorylation, can catalyze this reaction when they are present together in detergent solution (288) or in the membrane (306). These experiments show that intersubunit phosphotransfer between His-554 and Cys-384 can occur. However, at low concentrations of the purified protein, a dissociated form of IIMtl can also catalyze PEP-dependent phosphorylation, although with a lower specific activity (145). Thus, it is possible that intrasubunit phosphotransfer can also occur in IIMtl. Recently, it was shown that a chimeric protein consisting of IICGlc and IIBAMtl, while inactive in glucose phosphorylation, can work in combination with purified IICMtl in mannitol phosphorylation (13). Moreover, purified IICMtl stimulates the phosphorylation activity of purified native IICBAMtl by up to twofold. These results suggest that intrasubunit phosphotransfer between the A and B domains can occur. Furthermore, stimulation of IICBAMtl by IICMtl was taken as evidence that the individual subunits in the native dimer may be coupled functionally and may work in this structure at only half the maximal rate (13).
IICBGlc was first suggested to be active as a dimer (50), but later experiments suggested that it could catalyze at least phospho exchange between glucose 6-phosphate and glucose as a monomer (159). It has been shown, however, that IICBANag and IIBCABgl can complement IICBGlc in an E. coli strain that is lacking IIAGlc (290). Furthermore, a truncated IINag from Klebsiella pneumoniae that lacks its IIA domain can be complemented in vivo by IIAGlc (292). These results suggest that most likely, a functional heterodimer is (at least transiently) formed in the membrane between IINag or IIBgl and IICBGlc.
Finally, it has been shown that IIABMan forms a very stable dimer (56). It was subsequently shown that P-IIABMan phosphorylated at both sites can phosphorylate either the isolated IIAMan or the isolated IIBMan domain (55). More recently, intermolecular phosphotransfer between IIABMan subunits has been demonstrated by utilizing mutant proteins (274).
In the past, it was often assumed that transport and phosphorylation of carbohydrates by the PTS are concomitant events. However, in principle, transport of a PTS substrate through the membrane either could be directly coupled to phosphorylation of the substrate or could be the consequence of phosphorylation of the EII followed by phosphorylation of the carbohydrate in a separate step on the inside surface of the membrane. Current evidence favors the latter mechanism, and in this section, we review recent studies designed to understand the translocation mechanism.
IIMtl.
Binding, transport, and phosphorylation of mannitol by IIMtl have been studied in detail. Purified IIMtl contains one high-affinity binding site per dimer (Kd = 0.1 μM) and a lower-affinity site (Kd = ca. 9 μM) (184). Both high- and lower-affinity binding sites for mannitol with Kds of about 40 nM (144, 307) and 0.3 to 1 μM (13, 18, 69) have been found in the membrane-bound protein. The IIC domain is sufficient for mannitol binding (13, 69, 144), but deletion of as little as the last putative transmembrane helix destroys detectable binding activity (18). It is not known, however, which residues specifically form the binding site, although some mutations that affect binding and/or transport have been isolated (93, 148, 307).
The kinetics of mannitol binding, translocation, and phosphorylation by IIMtl have been studied both in inside-out and right-side-out vesicle systems (143, 144, 146) and in proteoliposomes reconstituted with purified IIMtl (46). Unphosphorylated IIMtl can apparently catalyze facilitated diffusion of mannitol but at a very slow rate, and this process can be carried out by the IIC domain alone (46, 144). Phosphorylation of IIMtl increases the translocation rate by several orders of magnitude (46, 146), but translocation in this case is not strictly coupled to the transfer of the phospho group to mannitol to form mannitol 1-phosphate. Approximately half of the mannitol molecules translocated by the phosphorylated protein in inside-out vesicles are not phosphorylated before they dissociate from the protein. These mannitol molecules, however, can apparently rebind to IIMtl at a cytoplasm-facing binding site and can subsequently be phosphorylated by the protein (146).
From these results, it has been concluded that the loaded, periplasm-facing mannitol binding site of IIMtl converts to a cytoplasm-facing orientation only slowly in unphosphorylated IIMtl. Phosphorylation of the protein greatly increases the rate, i.e., lowers the activation energy, of translocation. Therefore, phosphorylation of mannitol by P-IIMtl is not strictly coupled, mechanistically, to translocation (i.e., the protein can carry out facilitated diffusion). A kinetic model for mannitol translocation and phosphorylation incorporating these features was recently proposed (232). This model may also explain the observation that under certain conditions, EIIs can catalyze the intracellular phosphorylation of PTS carbohydrates (reviewed in reference 206).
IIGlc.
From studies of a hybrid protein in which the E. coli IICGlc domain is fused to the IIBNag domain, it was concluded that the binding-translocation site of IICBGlc must reside in the IICGlc domain (82). This binding site has a high affinity for glucose (Kd = 1.5 μM) (240). The coupling between transport and phosphorylation has been studied in two classes of mutants. Using S. typhimurium ptsHI deletion strains that lack EI and HPr and are unable to grow on glucose, mutations in the ptsG gene which allowed IIGlc to transport glucose in the absence of phosphorylation (facilitated diffusion) were selected (201, 239). Similar mutants were isolated by using the plasmid-encoded E. coli ptsG gene (240). These mutant proteins, like the ones isolated from S. typhimurium, had apparent Kms for glucose oxidation (0.5 to 2.5 mM) that were about 100-fold higher than that for the wild-type protein. In the presence of EI, HPr, and IIAGlc, these mutant EIIs were still able to catalyze PEP-dependent phosphorylation of αMG. Four different ptsG mutations with this phenotype were found (240). According to a topology model of IIGlc (20), three of the four mutations are located in helix 6 and 7, whereas one mutation (I296N) lies within a sequence (GITE) that is highly conserved in many EIIs (135, 246) and that includes the essential Glu residue in IIMtl mentioned earlier.
It is interesting that all mutations in IIGlc which allow efficient facilitated diffusion of the substrate also significantly increase the Km for transport. It had been shown previously that facilitated diffusion of galactose, a poor substrate of IIGlc and IIMan, can be carried out by IIGlc with low affinity in E. coli (113) and by IIMan in S. typhimurium (200). Similarly, trehalose can be taken up with low affinity by facilitated diffusion via IIMan in S. typhimurium (204). Possibly, low-affinity substrates of wild-type or mutant EIIs cannot tightly "lock" the enzyme into the state in which facilitated diffusion is slow (e.g., the "occluded" state that has been observed for unphosphorylated IIMtl [143]), and facilitated diffusion is therefore more rapid in these cases.
A second class of mutants has been isolated in which the mutant IIGlc had a severely impaired ability to transport αMG while retaining significant PEP-dependent phosphorylation activity for both glucose and αMG (19). The mutations cluster in three hydrophilic areas of the protein: the amino-terminal region, which is postulated to be cytoplasmic, and two other regions that are presumed to be periplasmic loops (19). The periplasmic mutations could thus either define part of an extracellular binding site or be involved in a conformational change related to translocation of the substrate.
Glucose, generated intracellularly in E. coli from maltose, can be phosphorylated via IIGlc (178). Thus, even the wild-type protein can carry out phosphorylation "from within," without transport, at least under these physiological conditions. In this respect, then, IIGlc also resembles IIMtl, although for the latter protein, nonvectorial phosphorylation has to date been convincingly demonstrated only in vesicular systems, as discussed in the previous section.
On the basis of the preceding discussion, the following sequence of events leading to the transport and phosphorylation of a PTS carbohydrate can be proposed.
(i) The periplasmic substrate binds with high affinity to its specific EII. If the EII is not in its phosphorylated form or is not complexed with its IIA protein (e.g., IICBGlc and IIAGlc), the substrate is translocated by facilitated diffusion to a cytoplasm-facing orientation only slowly if at all.
(ii) Phosphorylation at the IIB site allows rapid translocation of the substrate to a cytoplasm-facing orientation (i.e., facilitated diffusion occurs via a conformational change in the protein).
(iii) Phosphorylation of the bound substrate by the P-IIB domain then occurs and is followed by dissociation of the phosphorylated carbohydrate into the cytoplasm. In the case of IIMtl, at least, the substrate can also dissociate without phosphorylation with about equal probability but can rebind at the cytoplasm-facing site and then be phosphorylated.
Thus, under normal physiological conditions, transport appears to be coupled to phosphorylation (nearly every molecule that is transported is phosphorylated unless there is a fast competing reaction involving the unphosphorylated substrate), but mechanistically, the two processes are separate.
A number of important questions remain to be answered. For example, as previously reviewed by us (205) and others (156, 231), there are conflicting reports concerning whether PTS substrates generated on the inside of intact cells can be phosphorylated in situ by various EIIs without the substrate first leaving the cell, as would be predicted by the above model. It should be recognized that much (but certainly not all) of the evidence for phosphorylation without transport has been obtained with membrane vesicle systems or with mutant EIIs as described above. It is entirely possible that soluble factors or proteins in the cytoplasm could inhibit phosphorylation "from within" in vivo and that this process could also be influenced by the physiological conditions and the specific EII being studied. Perhaps the most important unanswered question regarding the mechanisms of the EIIs, however, is exactly how the carbohydrate substrate is translocated from "outside" to "inside" through the protein. A complete answer to this question awaits, among other things, a three-dimensional structure determination of a well-studied EII such as IIMtl or IIGlc.
To ensure that uptake and phosphorylation of PTS substrates do not greatly exceed the cells’ capacities to metabolize them under a given set of environmental conditions, the activities of many EIIs must also somehow be regulated. Indeed, uptake of PTS substrates by whole cells is rarely linear over time, and in many cases, initial uptake rates are difficult to determine. This suggests that as a consequence of PTS carbohydrate uptake, the activities of the EIIs are regulated by some type of feedback. A number of different mechanisms have been postulated to account for this, including energy-dependent efflux of PTS substrates, regulation by the membrane potential, competition of the EIIs for P-HPr, and regulation by intracellular phospho compounds. Because each of these possibilities has been extensively reviewed elsewhere (156, 205, 231) and their contributions to regulation of EII activity in vivo remain unclear, we will consider these only briefly and put our emphasis on more recent work.
If direct regulation of EII activity is physiologically relevant, then the flux of substrates through the EIIs might be expected to be a rate-limiting reaction of the PTS. By changing in a controlled manner the amount of IIGlc in E. coli, it was shown that the activity of IIGlc exerts considerable control over flux through the glucose PTS (238). Although in these experiments IIGlc had a low control coefficient over growth and glucose oxidation at high glucose concentrations (238), other experiments have shown that under glucose-limited conditions, the activity of the glucose PTS does limit growth (83). The EIIs could therefore be targets for direct regulation of PTS activities in vivo, at least in the case of IIGlc.
Much work has shown that the steady-state level of accumulation of αMG by E. coli cells can be rapidly lowered by exogenous addition of an energy source (for references to the older literature, see references 205 and 206). This is apparently due to the efflux of free αMG from the cells upon addition of the energy source, after hydrolysis of αMG 6-phosphate by an intracellular phosphatase. The abilities of at least some EIIs to carry out facilitated diffusion could help explain this phenomenon. If P-EIIs can catalyze rapid facilitated diffusion, as appears to be the case at least for IIMtl, then PTS substrates that bind to P-EII on the inside of the cell could also be translocated to the outside before they are phosphorylated by the P-IIB domain (i.e., efflux of the free carbohydrate occurs) (146). However, the exact nature of the energy requirement for this process and whether IIGlc is directly involved in the efflux remain to be determined.
Influx of substrates via EIIs is also controlled by the energy state of the cell. For example, uncouplers of oxidative phosphorylation and anaerobiosis stimulate αMG accumulation in energized whole cells and membrane vesicles of E. coli, while donors to the electron transport chain, such as d-lactate, inhibit accumulation (213). Uncouplers of oxidative phosphorylation also stimulate the initial rate of αMG uptake in S. typhimurium but have very little effect on the final steady-state level of accumulation (273). The mechanism of this membrane potential-mediated inhibition of IIGlc activity is unknown. Although it was proposed that a high membrane potential might inhibit IIGlc activity by promoting oxidation of thiols in the protein, leading to an increase of the Km for αMG (230), subsequent experiments showed that oxidized IIGlc, as well as IIMtl, was essentially inactive (67).
Various IIA proteins or domains present in the same organism have different Kms for phosphorylation by P-HPr (206). Therefore, preferential utilization of one PTS carbohydrate over another in cells expressing the EIIs for both substrates could occur and has, in fact, been observed (for a review of the older literature, see references 205 and 206). However, it is not clear that in all cases this is due exclusively to competition for P-HPr. Undoubtedly, discrimination between PTS substrates by a single EII, which transports more than one carbohydrate with different affinities (Table 1), is also a mechanism for preferential PTS carbohydrate uptake. This has been reviewed elsewhere (205) and will not be further considered here.
Another potential feedback control mechanism is modulation of EII activity by carbohydrate-phosphates and other intracellular metabolites. Intracellular carbohydrate-phosphate products of the PTS inhibit, directly or indirectly, various EIIs (see references in reference 206). On the other hand, since we now know that carbohydrate phosphorylation is probably not directly coupled to transport, it is still possible that the phosphorylated carbohydrate products of the EIIs could feedback inhibit transport at lower concentrations than that at which they inhibit phosphorylation. This could be through direct competition for a substrate binding site (i.e., product inhibition) or through an as yet uncharacterized allosteric binding site.
Numerous mutants which lack one or more of the PTS proteins have been isolated and analyzed. Subsequent mapping of the different mutations in strains of E. coli and S. typhimurium revealed that the structural genes ptsH and ptsI, encoding the general PTS proteins HPr and EI, respectively, are clustered in a pts operon together with a gene, crr, for IIAGlc (formerly EIII or IIIGlc). In contrast, the genes for the substrate-specific EIIs, with few exceptions, are clustered in specific operons or regulons together with the genes for the corresponding catabolic enzymes (205). Most specific systems are inducible with the major substrate as the external inducer. The pts operon, however, is expressed in a constitutive way, controlled only by global regulatory systems.
Mapping of the pts genes in E. coli and S. typhimurium gave the identical gene order cysK...ptsHI crr...cysAM. Cloning and sequencing of the genes and identification of the corresponding gene products revealed three major open reading frames (ORFs) (23, 40, 171, 242). They correspond to the genes ptsH (HPr), ptsI (EI), and crr (IIAGlc), which are highly similar in both species (≥97% identical amino acid residues).
In enteric bacteria, expression of the pts operon increases about threefold during growth on PTS substrates and requires an intact cyclic AMP (cAMP)-cAMP receptor protein (CRP) system (40, 153). Anaerobic growth conditions also favor higher expression (133). Two regulatory mechanisms are involved in the expression of the pts operon, a glucose-mediated and a cAMP-CRP-mediated activation. Two promoters, P0 and P1, are located upstream of ptsH and are separated by about 100 bp. Expression from these promoters generates one large transcript that covers the entire pts operon and a short transcript that covers ptsH and terminates within ptsI (42, 62). An anti-ptsH RNA that originates within ptsI has been implicated in transcription termination of the short transcript (138). A third promoter, P2, is located within ptsI. A short transcript that covers crr initiates at P2. The short ptsH- and short crr-specific mRNAs account for 80% of the total ptsH and crr mRNAs. This transcription pattern explains the relative abundance of HPr and IIAGlc compared to that of EI (about 5:1) (153, 299). The arrangement also explains why many strains which contain polar mutations or deletions in the ptsH and ptsI genes still express the crr gene at near-normal levels (29, 262, 263).
While the expression of crr from P2 is not affected drastically by the cAMP-CRP complex or the glucose-mediated activation, transcription from P0 and P1 is complex. The activity of the P0 and P1 promoters was analyzed first in vivo by means of fusions between P0 and P1 and indicator genes located on low-copy plasmids, by the use of mutated copies of both promoters, by primer extension studies with mRNAs isolated from cells, and by the use of strains lacking cAMP (Δ cya*) or CRP (Δ crp*) (40, 42). These studies were complemented by in vitro studies with DNaseI footprinting and gel retardation assays (62, 241). According to these studies, both the P0 and the P1 promoter exhibit a switching mechanism which causes transcription to initiate at different start sites, designated a and b. In the presence of the cAMP-CRP complex, initiation is from P1a and P0a, while in the absence of cAMP or CRP, transcription starts only from P1a. When glucose is added to wild-type cells, transcription partly switches from P1a to P1b and increases from P0a. This suggests that there is some barrier to P1b initiation that can be relieved by glucose. The process requires uptake of glucose through EIIGlc, but it is independent of the cAMP-CRP complex (41, 42). It rather complements CRP-dependent activation, because under conditions of weak catabolite repression, expression of the pts operon will be predominantly from P1a and P0a, while during uptake of glucose (strong catabolite repression), expression will be from P1b and especially from P0a. Whether Px, another transcription initiation site in front of ptsH with specificity for a σ 32-dependent RNA polymerase, has any physiological role, e.g., in this complex regulation, is unclear (241).
As summarized schematically in Fig. 3, operons and regulons which encode substrate-specific EIIs and the corresponding catabolic enzymes are scattered over the chromosome. In general, similar systems are located at identical sites on the chromosomes of both species and are closely related. A more detailed description of the PTS-dependent catabolic pathways with the corresponding enzymes (see chapter 20), and of the exact mapping of the corresponding genes (see chapters 109 and 110), is given elsewhere in this book.
In the enteric bacteria, many PTSs (e.g., those for sucrose, sorbose, and galactitol) are found in one strain but not in another strain of the same species. Others (e.g., those for sucrose) are encoded on plasmids or on transposable elements. Still others (e.g. those for cellobiose and β-glucosides) are encoded in a cryptic form; i.e., they can be expressed only after mutation. A total of 500,243 contiguous base pairs, or slightly over 10% of the cellular chromosome from E. coli K-12, has been published recently (12, 22, 198). Within these new DNA sequences were seven ORFs which resembled EI (f711), IIFru (f485, o359, o106, o113), IIBgl (f161), and IINag (f455) and had not been recognized previously because they did not express functional PTS proteins. The frv operon, including f485, and the glv operon, including f161 and f455, encode putative PTSs involved in fructose and β-glucoside transport and phosphorylation, respectively (218).
Mutations affecting the activity and expression of the glucose PTS in E. coli and S. typhimurium, i.e., IIAGlc-IICBGlc, have been mapped in three loci called umgC, ptsG, and crr. An umgC mutant expresses IICBGlc constitutively. The E. coli umgC gene maps close to its structural gene ptsG (103). By contrast, the gene crr, for the corresponding IIAGlc, maps in the pts operon. This separation of the structural gene for an EIICB from the gene of the corresponding EIIA is rare. We have speculated before (205) that one reason for this separation is that IIAGlc is the essential regulatory molecule in catabolite repression and inducer exclusion.
In E. coli K-12, the glucose PTS is the major glucose transport system (35, 102, 126), while in S. typhimurium, the mannose PTS is also a major transport system for glucose (273). The ptsG gene from E. coli, encoding IICBGlc, has been cloned and sequenced (15, 54). Promoter or regulator genes adjacent to ptsG which control this system have not been identified. The system is inducible by glucose in wild-type strains of E. coli K-12 (128) but apparently is constitutive in S. typhimurium (225).
A number of other PTSs use IIAGlc for transport and phosphorylation of their substrates. These PTSs include ones specific for trehalose, "maltose," and sucrose. This may explain why the crr gene maps separately from ptsG. In E. coli, a trehalose PTS (tre genes) is induced by growth on trehalose (14, 149). The internal inducer is trehalose 6-phosphate (110).
Maltose is generally a non-PTS substrate in enteric bacteria. The various enzymes involved in its complex metabolism are encoded by genes which form the mal regulon. Among these genes, malX codes for a protein which resembles IICBGlc (35% identical residues). It is apparently controlled by a repressor (malI gene) (214). The natural substrate(s) and function of the malX product have yet to be identified.
Finally, genes involved in sucrose utilization through a PTS-dependent pathway are found on large conjugative plasmids (e.g., pUR400) from certain strains of E. coli and Salmonella spp. (75, 256, 257, 312). In these systems, sucrose is taken up through a sucrose PTS of the IIBCScr type (scrA gene) which requires IIAGlc for activity (132). All scr genes, including scrK for a fructokinase, scrY for a sucrose- and glucan-specific porin, and scrB for a sucrose-6-phosphate hydrolase, are encoded in a regulon (256). This scr regulon is regulated by a repressor, ScrR (scrR gene), that recognizes intracellular fructose and, to a lesser degree, fructose 1-phosphate as the molecular inducers (95). ScrR is a member of the GalR-LacI repressor family. A characteristic operator motif is present once in the scrK promoter for the first scr operon and twice, together with a CRP-binding site, in the scrY promoter for the second scr operon (31, 95).
Nag and its analogs (including the antibiotic streptozotocin) are taken up through the Nag PTS (IICBANag) (104, 126, 309). The structural gene encoding IINag, nagE, is part of a nag regulon which has been sequenced completely from E. coli (191, 195, 234). Two nag operons are clustered on the chromosome and transcribed from two divergent promoters which share a central CRP binding site. The first operon expresses nagE at a semiconstitutive level, while the second contains, in addition to structural genes for catabolic enzymes, the gene nagC, encoding a repressor protein. NagC binds Nag 6-phosphate (and perhaps d-glucosamine 6-phosphate) as the inducer (195, 196, 197, 293, 295).
Many strains of enteric bacteria cannot utilize β-glucosides because the corresponding genes are cryptic, i.e., intact but not expressed in wild-type cells (71, 255). However, mutations which restore growth on β-glucosides arise spontaneously. Three cryptic metabolic pathways for β-glucosides which involve a PTS have been identified in E. coli (71, 114, 179). The EIIs involved in two of these pathways, encoded by genes asc (for arbutin, salicin, and cellobiose) and bgl (for β-glucosides), are highly similar and have the domain structures IIBCAsc and IIBCABgl, respectively. Most mutations which activate the cryptic bgl operon are IS1 and IS5 insertions upstream of the bgl promoter (226, 258, 261). The operon is inactive in wild-type strains because of local supercoiling of the chromosomal DNA upstream of the bgl genes. The insertion sequences probably act by altering this supercoiling. The first gene of the operon, bglG, encodes an antiterminator protein which is modulated by the activity of IIBgl, the product of the second gene, bglF. Phosphorylation of the intracellular regulator molecule BglG by EI, HPr, and IIBgl renders it inactive in antitermination. In E. coli, the bgl operon is expressed from a constitutive promoter (147, 259). Because the first gene, bglG, is flanked on both sides by terminators, transcription termination occurs in the absence of β-glucosides in the medium. During uptake of substrate, the phospho groups are transferred preferentially from P-IIBgl to its substrates. This causes a drain through backward flow of the phospho group from P-BlgG and a dephosphorylation of the antiterminator. Free BglG binds to specific RNA sequences located upstream of both terminators (79), prevents termination, and thus causes induction of the bgl operon. The model is supported by mutations in ptsI, ptsH, bglF (IIBgl), and bglG, all of which cause constitutive expression of the bgl operon (4, 5, 208, 259). This is a very elegant regulatory system in which the membrane-bound transporter also represents the sensor for the presence of inducing substrates in the medium. Its cytoplasmic domains IIA (IIB) interact directly with the response regulator BglG, and the entire system represents a "two-component"-like signal transduction system.
The third cryptic PTS for β-glucosides found in E. coli is the cellobiose PTS (179, 180). The genes celABC encode the domains IIACel, IIBCel, and IICCel, respectively. They constitute a IICel which genetically belongs to the lactose PTS family, for which no other representatives have been identified thus far in enteric bacteria. Interestingly, a repressor CelD and a phospho-β-glucosidase CelF, also found in the cel operon (Fig. 3), resemble the melibiose-specific repressor and α-galactosidase from E. coli.
In enteric bacteria, the three naturally occurring hexitols (mannitol, glucitol [or sorbitol] and galactitol [or dulcitol]) are PTS carbohydrates (124). The similarity in their metabolic pathways and in the apparent gene order within the corresponding operons originally prompted speculations about a common ancestor (134). The three operons have been sequenced, and the data reveal interesting and puzzling differences. The genes involved in mannitol catabolism are clustered in the mtlADR regulon (267). The gene mtlA codes for a large EII of the type IICBAMtl (637 residues), and mtlD codes for a mannitol-1-phosphate dehydrogenase. Both are regulated by a repressor encoded by the gene mtlR (60, 99, 122, 134). A cryptic mannitol transport system (genes cmtAB) of the type IICBCmt-IIACmt which resembles IIMtl was recently discovered (268). The genes lack a functional promoter, but when cloned behind lacZp, they expresses a IICmt which transports mannitol.
The E. coli gutABDMR(Q) operon consists of the structural genes gutA (IICBGut), gutB (IIAGut), and gutD (glucitol-6-phosphate dehydrogenase). Although the proteins encoded by gutA (506 residues) and gutB (123 residues) are likely the equivalent of IICBAMtl, no similarity can be found at the DNA or amino acid level (317, 318). The gut locus contains two (gutM and gutR) and possibly three (gutQ) regulatory genes which seem to act antagonistically (319). Upon induction, the repressor GutR is inactivated, and this causes an increased synthesis of the activator GutM from the main promoter, gutAp, a process which is dependent on the cAMP-CRP complex. The model confirms previous genetic data (34, 134, 154) and also explains the delayed induction (ca. 25 min) of the gut operon and the preferred utilization of mannitol when cells are grown on a mixture of mannitol and glucitol (123).
The gat operon for galactitol metabolism contains six structural genes (gatYZABCD) and one regulatory gene (gatR). Three of these genes code for the galactitol PTS and its three proteins, IIAGat (gatA), IIBGat (gatB), and IICGat (gatC). The IIAGat and IIBGat proteins are not related to any known PTS, but IICGat shows extended similarity to the E. coli IICFru domain (176a).
d-Fructose can be taken up in enteric bacteria by several PTSs (Table 1). Only one of these, however, is linked at the genetic level to the corresponding catabolic genes. It is induced by fructose and will be called the fructose PTS (fru genes; Fig. 3). Its product is fructose 1-phosphate. However, the system can also take up and phosphorylate mannose to mannose 6-phosphate (112). Because its repressor, FruR, has a special role in the control of gluconeogenesis and in the suppression of ptsH mutants, the system is discussed in a later section.
Two PTSs that contain two soluble and two membrane-bound domains or proteins (about 850 residues), one specific for d-mannose (Man) and one specific for l-sorbose (Sor), are found in enteric bacteria. Both accept fructose as a substrate. Genetically, however, they form a PTS family distinct from the fructose PTS. The mannose PTS has an unusually broad substrate specificity (Table 1) but is the only efficient mannose transport system; hence its name. The uptake and phosphorylation of mannose, glucose, and fructose by IIMan yield the corresponding sugar 6-phosphate. While IIMan in S. typhimurium is a major uptake system for glucose, it is a minor system for this substrate in E. coli, as stated before. The manXYZ (formerly ptsLPM) locus encodes a IIABMan, a IICMan, and a IIDMan protein, respectively (53, 253, 310). It is controlled through the NagC repressor (197, 293) and, under anaerobic conditions, through a regulatory factor encoded by gene dgsA (233). Similar to IINag, the mannose PTS appears to be a scavenger system for amino sugars generated during cell wall synthesis and turnover. To avoid futile cycles involving the amino sugar biosynthetic enzyme glucosamine-6-phosphate synthetase (gene glmS), the catabolic enzyme glucosamine-6-phosphate deaminase (gene nagB), and mannose-6-phosphate isomerase (gene manA), the systems have to be regulated strictly (196, 295).
Finally, uptake of sorbose through the l-sorbose PTS (genes sorFBAM) yields l-sorbose 1-phosphate, which is degraded through a reductase (SorE) to glucitol 6-phosphate and through a dehydrogenase (SorD) to fructose 6-phosphate (269, 301, 302, 313; U. F. Wehmeier, B. M. Wöhrl, and J. W. Lengeler, Mol. Gen. Genet., in press). Uptake of fructose through this system yields fructose 1-phosphate. The sor operon, similar to nag, gut, and gat, is another example of autoregulation for PTS-related systems. A regulatory protein, SorC, is encoded within the sorCDFBAME operon. In the absence of sorbose, SorC acts as a repressor (SorCR). It is converted during induction into an activator (SorCA) which activates the main promoter sorCp (314). Similar to other autogenously regulated systems, the sor operon shows a delayed induction (up to 20 min) (270).
Mutants defective in EI and/or HPr are unable to utilize PTS carbohydrates as the sole source of carbon for growth. In addition, such ptsHI mutants are unable to grow on a number of non-PTS carbon sources, including lactose, maltose, melibiose, glycerol, Krebs cycle intermediates, rhamnose, xylose, and others (Table 2). In this section, we discuss how the presence of a PTS carbohydrate in a wild-type cell or the absence of the general proteins of the PTS, EI and HPr, affects growth on quite a range of non-PTS carbon sources and the attendant mechanisms underlying PTS-mediated regulation.
Table 2Phenotypes of ptsI, ptsH, and crr mutants of enteric bacteria |
Two different observations are crucial in understanding the process of PTS-mediated regulation. First, Pastan and Perlman (185) reported that growth of an E. coli ptsI or ptsH mutant on the non-PTS carbohydrate lactose can be restored by the addition of cAMP to the growth medium (Table 2). Second, mutations in the S. typhimurium crr (carbohydrate repression resistance) gene, encoding IIAGlc, that restore growth of ptsHI mutants on several non-PTS carbon sources simultaneously were isolated (Table 2) (248, 249). To simplify the discussion of PTS-mediated regulation, we begin by showing in Fig. 4 the essential elements. The central regulatory molecule is IIAGlc, which can exist in two states: phosphorylated IIAGlc (P-IIAGlc) and nonphosphorylated IIAGlc. The phosphorylation state of IIAGlc is determined by the balance between phosphorylation via P-HPr and dephosphorylation via IICBGlc in the presence of its substrate. IIAGlc and P-IIAGlc interact with and regulate different proteins. IIAGlc binds to and inhibits proteins essential in the metabolism of several carbohydrates, e.g., lactose, melibiose, maltose, and glycerol. The direct result is inhibition of uptake and of subsequent metabolism of these carbon sources. This process is called "inducer exclusion." P-IIAGlc is involved in the activation of adenylate cyclase. Since cAMP is required for the expression of many catabolic genes, regulating the cAMP level allows control of enzyme synthesis. When cells are growing on a non-PTS carbohydrate, the PTS proteins most likely will be predominantly in their phosphorylated forms. Addition of a PTS carbohydrate will lower the phosphorylation state of the PTS proteins, including that of IIAGlc. Under those conditions, the entry of the non-PTS carbon source is inhibited (high concentration of the inhibitor, IIAGlc), and expression of the catabolic genes is prevented (low concentration of the activator, P-IIAGlc). We discuss both processes in more detail below.
Transport and metabolism of class I non-PTS carbohydrates, e.g., lactose, melibiose, raffinose, maltose, and glycerol, are inhibited by PTS carbohydrates (Table 2). Inhibition can be brought about by glucose or any other PTS carbohydrate as long as the carbohydrate’s corresponding EII is present at a sufficiently high level. The model depicted in Fig. 4 offers an explanation. Each PTS carbohydrate can dephosphorylate P-IIAGlc, either directly via EIICBGlc in the case of glucose or indirectly, because transport and phosphorylation of all other substrates via their respective EII complexes will result in dephosphorylation of P-HPr. Since the phosphorylation of IIAGlc by P-HPr is a reversible process, P-IIAGlc will be dephosphorylated as a consequence.
The role of IIAGlc in inducer exclusion is well established. Binding of purified IIAGlc to the lactose carrier, glycerol kinase, and to the MalK component of the maltose transport system has been demonstrated (for references, see references 205 and 206). In liposomes reconstituted with the purified lactose permease, thiomethyl-β-d-galactoside transport was inhibited by the addition of IIAGlc (173). In proteoliposomes made from membranes that overexpressed the maltose transport system, inclusion of IIAGlc into the liposomes inhibited maltose transport to a maximum of 60% (37).
Two features of IIAGlc-mediated inducer exclusion are similar, regardless of the target protein. (i) Only nonphosphorylated IIAGlc binds to the target proteins; phosphorylation of IIAGlc prevents its binding. (ii) Binding of IIAGlc occurs only when a substrate of the target protein, i.e., a β-galactoside, glycerol, or maltose, is present (see below). These observations suggest that a conformational change in the target proteins is required before IIAGlc can bind.
What do we know about the interaction between IIAGlc and its target proteins? The three-dimensional structure of the complex of E. coli IIAGlc with glycerol kinase has been determined (84). Tetrameric glycerol kinase binds four IIAGlc molecules. The contact between both molecules is limited to a rather small area and involves a very hydrophobic environment in which residues 472 to 481 of glycerol kinase project directly into the active site of IIAGlc. Since phosphorylation of IIAGlc does not result in large structural changes (187), most likely the introduction of a negatively charged phospho group on His-90 is sufficient to prevent the binding of IIAGlc to proteins of class I uptake systems. Phosphorylation of His-90 would lead to direct electrostatic and steric repulsion between the covalently bound phospho group and glycerol kinase and would disrupt the interaction. Because the IIAGlc binding site is far removed from the active site of glycerol kinase, its activity is presumably allosterically inhibited by IIAGlc.
Mutations have been isolated in the genes encoding the other target proteins. These mutations restore the growth of ptsHI mutants on the specific class I compound used for selection (Table 3). Most likely, such mutations result in proteins that cannot bind IIAGlc. A number of these mutations in the lacY (203, 311), malK (37, 116), and melB (120) genes have been sequenced recently and may eventually help in establishing which residues or domains are recognized by IIAGlc in these proteins, whose structures are not yet known. However, the region of glycerol kinase known to bind to IIAGlc shows no sequence similarity with the regions of LacY, MalK, and MelB in which these mutations have been isolated. Possibly, IIAGlc recognizes only a few amino acid residues on a hydrophobic interaction surface as well as a coordinating ligand for a Zn(II) ion (194).
Table 3Mechanisms of suppression of PTS-mediated regulation |
Mutations in the crr gene that lead to the absence, or lowered synthesis, of IIAGlc have been isolated. These types of mutations reveal very little about the nature of the interaction between IIAGlc and its target proteins. Other crr mutations result in IIAGlc molecules that are defective in regulation but still active in glucose transport and phosphorylation (207, 209, 320). Most mutated residues are in the region of IIAGlc known to interact with glycerol kinase. We stress that the crr mutations studied to date that result in regulation-defective IIAGlc molecules affect all class I systems, suggesting strongly that the same region of IIAGlc interacts with the different target proteins.
Can we understand the response of cells to PTS carbohydrates and the resulting PTS-mediated regulation? A number of factors are important for inducer exclusion: (i) the number of IIAGlc molecules and the number of target protein molecules; (ii) the phosphorylation state of IIAGlc (and other PTS proteins); and (iii) the presence of a substrate of the target protein.
In cells in which the respective class I catabolic operons are maximally induced, transport and metabolism of non-PTS substrates are often not inhibited by 2-deoxyglucose (a substrate of the mannose PTS) or αMG. This can easily be seen in cells in which the operon encoding a class I uptake system is expressed constitutively. Such mutations in the repressor or operator have been isolated and restore growth on one particular carbon source (in contrast to the general crr mutation) (Table 3). An increase in the amount of IIAGlc above the chromosomal level (164, 169, 281) or a decrease in the amount of a target protein by partial induction (170, 245, 281) results in cells that are more sensitive to inducer exclusion. Since IIAGlc and its target protein form a stoichiometric complex, the number of protein molecules and the dissociation constants are important. An E. coli or S. typhimurium cell contains 1 × 104 to 2 × 104 IIAGlc molecules (25 to 50 μM [263]). Because the Kd of the lactose permease-IIAGlc complex is between 5 and 16 μM (173) and the number of lactose carriers in a lactose-grown E. coli cell is estimated to be 8 × 103 per cell (0.2 nmol/mg of membrane protein), most, if not all, lactose permease molecules can be complexed, resulting in almost complete inhibition of lactose transport. Similarly, from the amount of glycerol kinase (15 to 20 μM [77, 281]) and the Ki of IIAGlc for inhibition of glycerol kinase (10 and 4 μM at pHs 7 and 6.5, respectively [177] and somewhat higher at pH 7.5 [38]), it can be calculated that sufficient IIAGlc is available to inhibit most glycerol kinase molecules. In fact, complete inhibition of glycerol uptake in E. coli by PTS carbohydrates is found when approximately four IIAGlc molecules are present per glycerol kinase tetramer (281). It was shown recently that the Ki of IIAGlc for glycerol kinase (16.6 μM at pH 7) decreases to 1.1 μM in the presence of 0.01 mM Zn2+ and to 0.28 μM in the presence of 0.1 mM Zn2+, allowing complete inhibition by IIAGlc at the intracellular concentration. The Zn effect is quite specific. The Zn2+ ion is coordinated by the two active-site histidines (His-75 and His-90) of IIAGlc, Glu-418 of glycerol kinase, and an H2O molecule (58).
Still other specific suppressor mutations that, as in the cases described above, restore growth on a single carbon source in ptsHI mutants have been found (Table 3). As mentioned above, promoter-up mutations have been found, e.g., in the glp operon that allows E. coli ptsI mutants to grow on glycerol, presumably by increasing the level of glycerol kinase above that which can be inhibited by IIAGlc (11). Another type of suppressor mutation resulted in a glycerol kinase that had become insensitive to feedback inhibition by fructose 1,6-bisphosphate (10). Such mutant glycerol kinase proteins are still sensitive to inhibition by IIAGlc, however (38, 142).
As mentioned earlier, the phosphorylation state of IIAGlc is essential in PTS-mediated regulation and is determined by the rate of phosphorylation via P-HPr and the rate of dephosphorylation via IICBGlc (in the case of glucose) or HPr (in the case of all other PTS carbohydrates). Addition of αMG to wild-type cells of S. typhimurium results in dephosphorylation of P-IIAGlc from roughly 80% IIAGlc being phosphorylated to less than 10% (172). No other data about the phosphorylation state of IIAGlc in intact cells are available.
Another important observation is that IIAGlc binds to its target protein only when a substrate is present. In this way, IIAGlc, present at a constant and limited level in the cell, is not being wasted on nonproductive binding. This interpretation is supported by the observation that induction of a second target protein, e.g., glycerol kinase, in a cell already containing a certain level of the maltose transport system results in less inhibition of the maltose system by 2-deoxyglucose only if glycerol is present, i.e., if glycerol kinase can bind IIAGlc and can lower the free concentration of IIAGlc (169).
It was pointed out in a previous section that some EIIs, like IINag and IIBgl from E. coli and IIGlc from B. subtilis, contain IIAGlc-like domains (Table 1, Fig. 2) and that these IIAGlc-like domains can replace IIAGlc in glucose transport and phosphorylation. These IIAGlc-like domains can also function in PTS-mediated regulation, although not as efficiently as IIAGlc. In S. typhimurium crr nagE double mutants lacking both IIAGlc and IINag, inhibition of glycerol and maltose uptake by PTS carbohydrates is absent. A plasmid encoding the membrane-bound EIINag restores inducer exclusion (207). Similarly, introduction of the soluble IIAGlc domain of B. subtilis IIGlc into an E. coli ptsHI-crr deletion strain results in inhibition of lactose fermentation, while the synthesis of β-galactosidase in an E. coli crr strain containing the B. subtilis IIAGlc domain is sensitive to glucose repression (223). Thus, the B. subtilis equivalent of the enteric IIAGlc restores PTS-mediated regulation in a crr mutant.
cAMP plays a central role in gene expression in enteric bacteria. Together with CRP (also called CAP), it is involved in the (generally) positive global regulation of a large number of catabolic genes (see chapter 84). As mentioned above, the addition of cAMP restores the growth of ptsHI mutants of E. coli on certain non-PTS compounds (185). This observation has been extended to all class I and class II compounds (Table 2) (also see reference 206). In addition, mutations in the crp gene, which encodes CRP, that restore the growth defects of ptsHI mutants on class I and II carbon sources have been isolated. These crp* mutations result in a CRP that has become independent of cAMP.
What is the connection between pts mutations or the presence of a PTS carbohydrate and the rate of cAMP synthesis? Peterkofsky and coworkers (76, 193) demonstrated that adenylate cyclase activity in toluenized E. coli cells is strongly inhibited by PTS carbohydrates. This inhibition is dependent on the presence of the EII specific for the inhibitory carbohydrate. As shown in Fig. 4, the phosphorylated form of IIAGlc is proposed to be an activator of adenylate cyclase. In contrast to the interaction between nonphosphorylated IIAGlc and the different uptake systems, a demonstration of the direct stimulation in vitro of adenylate cyclase by P-IIAGlc (with or without additional EI and HPr) has not yet been successful (212). In fact, the addition of EI or EI together with HPr and IIAGlc to bacterial extracts inhibits adenylate cyclase activity. In contrast, addition of HPr stimulates the activity. The phosphorylated PTS proteins do not stimulate adenylate cyclase unless phosphate is present. Addition of αMG lowers the adenylate cyclase activity at most by twofold under these conditions (212). Unexpectedly, adenylate cyclase activity is higher in extracts containing only PEP than in extracts containing the PTS proteins and PEP, i.e., the phosphorylated form of the proteins. In a later paper (140), it was shown that in the presence of phosphate, nonphosphorylated IIAGlc inhibits adenylate cyclase. It had previously been suggested, on the basis of mutant studies, that IIAGlc might be an inhibitor of adenylate cyclase in addition to P-IIAGlc being an activator (170).
Mutants that lack CRP produce large amounts of cAMP. This increased production is dependent on IIAGlc, since it is not seen in crr mutants (32, 39). Several mutations in the E. coli cya gene that result in a modified regulation of cAMP synthesis and abolish the stimulation of cAMP synthesis in crp strains have been isolated (32). Possibly, interaction between IIAGlc and adenylate cyclase is altered. In other cya mutants, cAMP synthesis is high even in the absence of IIAGlc (33). In such mutants, a truncated adenylate cyclase that lacks the carboxy-terminal regulatory domain is found. It has been suggested that this regulatory domain is inhibitory to the activity of the amino-terminal catalytic domain and that P-IIAGlc possibly modifies this interaction (see below). It seems unlikely that most of the regulation of adenylate cyclase activity could be at the level of expression, since in a number of older studies (cited in reference 205) and in a more recent study with S. typhimurium (57), it was shown that inhibition of the expression of cya by the cAMP-CRP complex is from 2-fold to about 10-fold. This effect is not sufficient to explain the large changes observed in cAMP synthesis, which, under various conditions, can vary over at least a 100-fold range (105).
There are several possible explanations for the failure to reconstitute the regulation of adenylate cyclase in vitro. It might be difficult to mimic intracellular conditions, despite the facts that adenylate cyclase is overproduced by using a cloned cya gene and that the PTS proteins are added at concentrations approaching intracellular concentrations. Alternatively, other unknown (protein) factors could be involved. On the basis of the results obtained with crp and crr strains described above, it could be proposed that CRP binds to the regulatory domain of adenylate cyclase and decreases its activity. P-IIAGlc might prevent binding of CRP and thus activate adenylate cyclase, as does deletion of the carboxy-terminal domain.
It is important to point out that crr mutants and ptsHI deletion mutants have a residual level of cAMP synthesis. The fact that different genes and/or operons require different concentrations of cAMP for full expression (see chapter 82), may explain why crr mutants can grow on certain carbon sources (class I) but not on others such as succinate, citrate, or xylose (class II; Table 2). Expression of genes required for class I carbon source catabolism in general requires less cAMP than expression of those of class II carbon sources. It has been shown, however, that growth on different carbon sources leads to relatively small differences in the intracellular cAMP concentration (49). This may explain the different phenotypes of some ptsHI-crr deletion mutants of E. coli and S. typhimurium that are observed. As mentioned previously, the crr mutation was originally isolated in S. typhimurium as a suppressor of the ptsHI phenotype, i.e., by restoration of growth on maltose, glycerol, and melibiose (249). Similar mutations with a similar phenotype were also isolated in E. coli (24, 168). Recently, a set of isogenic E. coli mutants containing ptsI-crr and ptsHI-crr deletions was constructed (139). Those mutants were unable to grow on class I compounds, in contrast to the S. typhimurium and E. coli mutants described above. Growth of the E. coli ptsHI-crr mutant on class I compounds was stimulated by cAMP. In these particular E. coli strains, the cAMP level was only 3% of that of the parent, whereas the adenylate cyclase activity in S. typhimurium crr and ptsHI-crr strains and in some other E. coli mutants may be 10 to 20% of that in the parent strain (59, 170). Thus, the genetic background of a particular strain might explain the observed differences.
Finally, it should be stressed not only that glucose and other PTS carbohydrates lower the intracellular cAMP concentration and thus control the expression of cAMP-dependent genes and operons but also that the level of CRP is lowered by glucose (86). This decrease is due to lowered crp transcription and requires the cAMP-CRP complex (85). Thus, both the intracellular cAMP concentration and the level of CRP are important factors in glucose-mediated regulation.
Some carbohydrates are utilized in enteric bacteria in preference to other carbon sources (165). When a PTS carbohydrate causes diauxic growth, the available evidence suggests that net dephosphorylation of IIAGlc, caused by the utilization of PTS carbohydrates, is the major regulatory factor. The lowered phosphorylation state of IIAGlc leads in turn to a lower expression of the catabolic operons (less cAMP because of less P-IIAGlc) and (in some cases) inhibition of non-PTS uptake systems already synthesized. In Table 3 we have summarized the different mechanisms by which the effect of ptsHI mutations or the presence of PTS carbohydrates on growth can be suppressed. Some of the mechanisms (cAMP, crp*, elimination of an EII) affect all class I and class II carbon sources, and others (inducer exclusion) affect only one class. Finally, some mutations (promoter-up mutations or mutations in target genes) specifically affect only one carbon source.
It is important to point out that not only in ptsHI and crr mutants but also in wild-type strains growing on PTS carbohydrates, adenylate cyclase activity and cAMP synthesis are not decreased to zero (in contrast to the situation in cya mutants). This residual cAMP allows enteric bacteria to grow on certain carbon sources but not on others. Most important, this basal cAMP level allows wild-type cells to grow on PTS carbohydrates, many of which require cAMP. Otherwise, growth on a PTS carbohydrate would be self-inhibitory. Finally, preference among PTS carbohydrates is possible via competition of the various EIIs for P-HPr, as discussed earlier.
In the previous section, we described the interaction between IIAGlc, several non-PTS uptake systems, and adenylate cyclase, which results in inducer exclusion and regulation of cAMP synthesis. A number of other processes in which proteins of the PTS are involved in the regulation of various metabolic reactions or are phosphorylated by other non-PTS proteins have been described. In this section, we discuss (i) regulation of gluconeogenesis and related processes by the repressor of the fru operon; (ii) interaction between EI, acetate kinase, and possibly other kinases; and (iii) a possible link of the PTS with nitrogen regulation.
The fruFKA operon encodes the enzymes involved in the fructose PTS: FPr, fructose-1-phosphate kinase, and IIFru. FPr was discussed previously and can functionally replace HPr. Expression of the fru operon is controlled by the fruR gene, encoding the Fru repressor. fruR mutants express the fru operon constitutively (64, 111, 215). fruR mutations can be isolated as suppressor mutations that restore the growth of ptsH mutants on PTS carbohydrates, because the constitutively synthesized FPr can complement the absence of HPr. Surprisingly, fruR mutants are unable to grow on lactate and pyruvate. fruR mutants of S. typhimurium lack PEP synthase (64), which catalyzes the conversion of pyruvate and ATP into PEP, AMP, and Pi.
In other studies, it was shown that in addition to PEP synthase, other enzymes involved in gluconeogenesis, such as PEP carboxykinase, fructose-1,6-bisphosphatase, isocitrate lyase, and malate synthase, are affected by the fruR mutation (27, 28). The activities of glycolytic enzymes such as phosphofructokinase are elevated, however. It could therefore be argued that FruR acts as a global regulator.
The ppsA gene, encoding PEP synthase, has been cloned (65) and sequenced (175). By use of fruF-galK (63) and ppsA-galK (65) fusions, it was shown that transcription of both the fru operon and the ppsA gene is regulated by the FruR protein. FruR acts as a repressor in the case of fru and as an activator for ppsA. Since transcription of the ppsA-galK fusion is not affected by a fruF::Tn10 insertion mutation (65), none of the Fru proteins except FruR is required for ppsA activation.
The fruR genes of both E. coli and S. typhimurium have been sequenced (96, 289). The FruR protein contains a typical helix-turn-helix motif in the amino-terminal part that is common to regulatory proteins. Thus, the same effector protein regulates transcription of the fru operon negatively and that of the ppsA gene in a positive way. From in vitro DNA-binding studies, it has been concluded that binding of FruR to both the regulatory regions of the fru operon (negative regulation) (95, 211) and the upstream regions of the ppsA, icd, and aceB genes (positive regulation) (211) is reversed by fructose 1-phosphate, the putative intracellular inducer. The purified FruR protein was shown to bind to a 16-bp DNA sequence located 170 bp upstream of the ace operon (30). FruR possibly activates transcription as CRP does by inducing a bend in DNA and facilitating binding of RNA polymerase.
Interestingly, PEP synthase is a P~His enzyme, as are the PTS enzymes. Sequence analysis of the ppsA gene (175) shows that PEP synthase exhibits similarity to EI and to pyruvate, orthophosphate dikinases (PPDK) from plants and microorganisms (199, 217). The active-site region, identified in PPDK and EI, is also conserved in PEP synthase. PEP synthase, PPDK, and EI all carry out similar reactions, the transfer of a phospho group via a P~His intermediate between PEP and pyruvate.
Acetate kinase catalyzes the conversion of acetate and ATP into acetyl phosphate and Pi. During the reaction, a phospho group becomes linked to the enzyme via an acyl phosphate, a high-energy bond. In the presence of EI and HPr, IIAGlc could be phosphorylated by ATP via acetate kinase (61). P-EI can directly donate its phospho group to acetate kinase, and the phospho group of P-IIAGlc can be transferred to acetate kinase in the presence of EI and HPr. Potentially, this reaction could form a link between the PTS and the enzymes connected to the Krebs cycle (61). It remains to be demonstrated, however, that this alternative pathway for phosphorylating PTS proteins in the absence of PEP is operative in an intact cell since this sequence of reactions has been shown only in an in vitro system.
A protein kinase that can phosphorylate EI on the active-site His residue has been purified (36). The EI-kinase required NAD(P)+. The specific activity of the partially purified fraction is very low, however, compared to the flux through the PTS.
Analysis of an ORF (ORF162) found downstream of the K. pneumoniae rpoN gene, which encodes a minor sigma factor, σ 54, showed sequence similarity with the IIAMtl domains of E. coli and Staphylococcus carnosus and the IIAFru domains of S. typhimurium and Rhodobacter capsulatus (221), especially around and including the active-site histidine. σ 54 is required for the transcription of a number of genes important for nitrogen assimilation under conditions of nitrogen limitation (see chapter 86). Phosphorylation of the K. pneumoniae ORF162 protein by the PTS was demonstrated (9). Recently, the complete rpoN operon of E. coli was sequenced (101). In addition to an ORF162-like gene, an ORF encoding an HPr-like protein was found. Since ORF162 could be a regulator of σ 54-dependent transcription (100, 161), modulation of its phosphorylation state via the PTS could form a link between carbon and nitrogen metabolism.
Enteric bacteria carry out both positive chemotaxis (i.e., they swim toward attractants) and negative chemotaxis (they swim away from repellents). Elaborate signal transduction networks of chemosensors and response regulators underlie this behavior. One chemotaxis pathway involves methyl-accepting chemotaxis proteins (MCPs) as the membrane-bound sensors. Two proteins, CheA and CheY, which are the equivalent of a transmitter and a receiver in a two-component system, form the central unit of this pathway, which is described in detail in chapter 73 of this book.
The PTS is a second signal transduction system through which bacteria respond by positive chemotaxis to the presence of PTS carbohydrates (for a review, see reference 136). In PTS-dependent chemotaxis, stimulation requires uptake and phosphorylation of a substrate through an EII. No MCP is involved in this process, since mutants lacking the MCPs retain normal PTS-dependent chemotaxis (176, 186). Mutants lacking CheB and CheR, two proteins which are involved in the methylation-dependent adaptation of cells to MCP-dependent stimuli, also retained normal PTS-dependent chemotaxis.
For all EIIs analyzed thus far, transport, phosphorylation, and chemoreception are three closely related activities. Cells lacking a specific EII do not show chemotaxis toward its corresponding substrate(s), and the apparent affinities in wild-type cells for the three activities are very similar. No mutations in EIIs which eliminate chemotaxis but retain transport and phosphorylation have been found (see references in reference 279). In contrast, mutants lacking EI and HPr do not show a chemotactic response to any PTS carbohydrate, even if the corresponding EIIs are present (2, 124, 128, 160). In addition, mutant forms of IIMtl, which could not be phosphorylated but still could bind the substrate, could not trigger a chemotactic response (306). Extensive metabolism of the stimulating PTS carbohydrate is not necessary to elicit chemotaxis, however, since nonmetabolizable analogs are good attractants, and mutants defective in subsequent metabolism of PTS substrates also show a normal chemotactic response (2, 128). Furthermore, glucose 6-phosphate and fructose 6-phosphate do not cause chemotaxis in cells induced for an uptake system for hexose phosphates (Uhp). This result excludes the carbohydrate phosphates as the stimulating agents (186).
Essential roles for IIAGlc, adenylate cyclase, and cGMP had previously been postulated in linking the PTS to the general chemotaxis machinery. More recent studies show, however, that synthesis of the flagella, the Che proteins, and the EIIs is dependent on the cAMP-CRP complex, while PTS-dependent chemotaxis is normal in the absence of IIAGlc, adenylate cyclase, cAMP, or CRP. Moreover, no evidence of a role for cGMP can be found (280, 291).
Most sensory systems integrate various signals and adapt to long-lasting stimuli. Because all PTSs use the general proteins EI and HPr, these proteins would be logical candidates for the integration of PTS-dependent signals. All ptsH and ptsI mutants tested thus far have a pleiotropically negative phenotype in their chemotaxis towards PTS carbohydrates, and factors which modulate the PTS phosphorylation activity, as tested in vivo by means of transport assays, affect the chemotactic response in a similar way (186). A proposed model assumes that the signal in PTS-dependent chemotaxis is a change in the phosphorylation level of EI or HPr, or both (128). As is discussed in detail in chapter 73, positive stimuli through the MCP-dependent signal transduction pathway inhibit autophosphorylation of the protein kinase CheA (at a His residue). The receiver CheY, which is phosphorylated by P-CheA on an aspartate residue, cannot be activated under these conditions, and the cell runs. If it is assumed that both signal pathways act in an analogous manner, a net dephosphorylation of the general PTS proteins should decrease the level of phosphorylation of P-CheA (and P-CheY). Proof for a direct interaction of both systems is lacking, but circumstantial evidence for the missing link does exist. In a strain which lacks all MCPs and the general chemotaxis proteins (CheA, CheB, CheR, CheW, CheY, and CheZ), reintroduction of CheA, CheY, and CheW but not of CheZ is required to restore PTS-dependent chemotaxis (279). CheA, CheY, and CheW are most likely needed to restore tumbling and changes in the direction of swimming of the cells. CheZ, however, a P-CheY phosphatase, may be dispensable, because no P-CheY is generated during a positive stimulation. Mutants which lack HPr but synthesize FPr constitutively show normal growth on the various PTS carbohydrates and a normal PTS transport but lack chemotactic activity to PTS substrates, including fructose itself (70). PTS-dependent chemotaxis is restored in these mutants when the HPr-like domain of FPr is overexpressed (129). Furthermore, P11E mutants of HPr show only a small decrease (about twofold) in transport activity, but no chemotactic response. However, chemotaxis is restored to normal levels when the mutated HPr is overexpressed (70). These results suggest that the phosphorylation levels required for efficient transport are lower than those for chemotaxis and that HPr may play a role in signalling during PTS-dependent chemotaxis.
Portions of this chapter are based on a recent review by us that appeared in Microbiological Reviews (206). We thank C. A. Alpert for help with the figures.
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