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Microbiology and Molecular Biology Reviews, December 2006, p. 939-1031, Vol. 70, No. 4
1092-2172/06/$08.00+0 doi:10.1128/MMBR.00024-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria
Josef Deutscher,1*
Christof Francke,2 and
Pieter W. Postma3
Microbiologie et Génétique Moléculaire, INRA-CNRS-INA PG UMR 2585, Thiverval-Grignon, France,1
Department of Molecular Cell Physiology, Biocentrum, Vrije Universiteit, Amsterdam, and Wageningen Centre for Food Sciences at CMBI, Radboud University, Nijmegen, The Netherlands,2
Swammerdam Institute for Life Sciences, BioCentrum, University of Amsterdam, Amsterdam, The Netherlands3
The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.
From a few peaks rising above the fog we try to imagine what the hidden landscape underneath might look like.
—Pieter W. Postma, during a hiking trip in the Beaujolais region in October 1997.
Carbon Catabolite Repression
Given a certain extracellular environment, a bacterium needs only a subset of the enzymes encoded by the genome to propagate, and therefore, it regulates gene expression differentially. For instance, in case a particular substrate is absent, the genes encoding the enzymes required for uptake and subsequent metabolism of the substrate are often repressed. Availability of the substrate then leads to the relief of repression. The classical example of this mode of transcription regulation is the lac operon of Escherichia coli (367). More complex responses of cells can occur when they are exposed to multiple nutrients at the same time. More than a hundred years ago, it was observed that growth on glucose can lower the activity of certain enzymes in bacteria and yeast. This phenomenon became known as the glucose effect (see also reference 744). One of the first descriptions of the glucose effect stems from the work of Dienert and was published in 1900 in Paris, France. He observed that Saccharomyces cerevisiae cells, which had been adapted to galactose (i.e., they were able to utilize galactose), rapidly lost the adaptation when the cells were exposed to glucose or fructose (188). This observation was later confirmed, for example, by Söhngen and Coolhaas, in Wageningen, The Netherlands, who measured the influence of temperature on the glucose effect in yeast (820). One of the first quantitative analyses of the glucose effect was carried out by Stephenson and Gale in Cambridge, England, who measured the activity of E. coli galactozymase. Galactozymase was understood as being the entity of enzymes necessary for the degradation of galactose by a specific organism. Those authors found that glucose exerted a strong repressive effect on galactozymase activity (more than sevenfold), while the utilization of glucose was not affected by galactose (836). In the early 1940s, Jacques Monod observed that when cultivated in a synthetic medium containing sucrose and dextran, the gram-positive bacterium Bacillus subtilis first utilized sucrose and then stopped growing for a certain period (lag phase or phase of adaptation) before the cells resumed growth by utilizing dextran (572). Owing to the biphasic growth he had observed, Monod called this phenomenon diauxie. He extended his studies with B. subtilis and found that diauxie was a more general phenomenon. When this organism was provided with sucrose or glucose, for example, and a second carbohydrate such as maltose, mannitol, inositol, or sorbitol, it also showed diauxic growth; i.e., sucrose or glucose was utilized first before the bacterium started to transport and metabolize the "less favorable" carbon source. He finally extended his studies to the gram-negative bacterium E. coli, which exhibited diauxie when grown in the presence of glucose or fructose, for example, as the preferred carbon source and xylose, arabinose, rhamnose, maltose, lactose, or glucitol as the less favorable sugar (572). It appeared that in each organism, a specific hierarchy existed for the utilization of carbon sources, with glucose usually being on top of it. In subsequent years, it was found that preferred sugars such as glucose, fructose, or sucrose, as long as they are present in sufficient amounts in the growth medium, repress the synthesis of the enzymes necessary for the transport and metabolism of less favorable carbon sources. The phenomenon therefore became known as carbon catabolite repression (CCR) (137). When the preferred carbon source is exhausted, bacteria first need to synthesize the enzymes necessary for the transport and metabolism of the less favorable carbon source (lag phase) before they can resume growth. The glucose effect reported at the beginning of the last century can therefore be considered CCR.
The regulatory mechanisms underlying CCR have since been intensively studied, and it was found that the preferential use of one carbon source over the other involves either the prevention of transcription activation or the repression of transcription. Here, we define CCR, somewhat more broadly, as the inhibitory effect of a certain carbon source in the growth medium on gene expression and/or the activity of enzymes involved in the catabolism of other carbon sources. Gene expression and enzyme activity are regulated via protein-DNA, protein-protein, and protein-metabolite interactions, and these interactions are in turn modulated via protein modification. Interestingly, the bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), which catalyzes the uptake and concomitant phosphorylation of numerous carbohydrates, plays a major role in bacterial CCR. However, quite different mechanisms have evolved in gram-negative and gram-positive organisms, and different PTS proteins are implicated in the two types of bacteria (for earlier reviews on the subject, see references 90, 756, and 845). In some bacteria, CCR is governed by other preferences and is possibly mediated by additional mechanisms. For instance, 
-proteobacteria of the genera Sinorhizobium, Rhizobium, and Bradyrhizobium (83, 889) and 
-proteobacteria of the Pseudomonas family (135) prefer acetate or tricarboxylic acid (TCA) cycle intermediates such as succinate over carbon sources such as glucose, fructose, or lactose. The underlying mechanisms have not yet been unraveled, but in pseudomonads, they involve the catabolite repression control (Crc) protein (22, 331, 332, 573, 747, 982), and rhizobacteria, they involve inducer accumulation (83).
In this review, we will focus on mechanisms that control carbohydrate transport and metabolism in PTS-containing bacteria, and we will concentrate on the regulatory roles played by the components of the system. We start by discussing the individual components of the PTS and their properties. Subsequently, we will describe their role in catabolite repression and in other regulatory mechanisms governing carbon metabolism. We will pay particular attention to the mechanisms that have developed in the enteric bacteria E. coli and Salmonella enterica serovar Typhimurium (-Proteobacteria) and in low-G+C gram-positive bacteria (also known as Firmicutes) such as B. subtilis and Lactobacillus casei, but we will also refer to other proteobacteria and to gram-positive bacteria with high G+C content (also known as Actinobacteria). The two groups of gram-positive bacteria can be distinguished not only on the basis of the different G+C content of their DNA but also based on many other different characteristics. For example, actinobacteria have been shown to contain numerous proteins that have been detected only in members of this phylum so far (corynebacteria, streptomyces, mycobacteria, nocardiae, propionibacteria, bifidobacteria, leifsoniae, and rhodococci, to name just a few) (253). Important for this review is that most high-G+C gram-positive bacteria possess PTS components and transport sugars via this system but seem to be devoid of adenylate cyclase and HPr kinase/phosphorylase (41). Nevertheless, other PTS-related control mechanisms of carbon metabolism are operative.
The Phosphoenolpyruvate:Carbohydrate Phosphotransferase System
The PTS was discovered in E. coli by Kundig, Ghosh, and Roseman as a system that uses PEP to phosphorylate a number of hexoses, including N-acetylmannosamine, glucose, mannose, glucosamine, and N-acetylglucosamine (434). Subsequently, it was recognized that the PTS is in fact a transport system that catalyzes the uptake of numerous carbohydrates and their conversion into their respective phosphoesters during transport. After its discovery in E. coli, the PTS was found in many other bacterial species. The coupled transport and phosphorylation of carbohydrates were originally described as a two-step reaction catalyzed by two enzymes, enzyme I (EI) and enzyme II (EII), with the protein HPr as an intermediate phosphoryl donor:
However, this representation is a bit misleading in the sense that EI, HPr, and EII behave in a functionally identical manner. They accept a phosphoryl group from a donor and donate it to an acceptor (i.e., they transfer a group), thus cycling between the phosphorylated and unphosphorylated states (depicted in Fig. 1). It is precisely this aspect of the mechanism that is exploited by the cell in PTS-mediated regulation.
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FIG. 1. Carbohydrate transport and phosphorylation by the PTS and their coupling to glycolysis. Carbohydrates are transported and concomitantly phosphorylated by the PTS. The phosphorylated carbohydrate feeds into glycolysis, normally at the glucose-6-P or fructose-6-P level. Two phosphoenolpyruvate molecules are usually formed in glycolysis, one of which is used to drive the transport and initial phosphorylation of the carbohydrate. As a result, the phosphorylation state of the PTS proteins depends on both the concentration of extracellular carbohydrates and the ratio of internal phosphoenolpyruvate and pyruvate. Abbreviations for enzymes (in boldface type) are as follows: Pgi, phosphoglucose isomerase; Pfk, phosphofructokinase; Fba, fructose-1,6-bisphosphate aldolase; Tpi, triose-phosphate isomerase; Gap, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Pgm, phosphoglycerate mutase; Eno, enolase; Pyk, pyruvate kinase.
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The basic composition of the PTS is in fact similar in all species studied so far (for reviews, see references 678 and 730). It is comprised of two "general" cytoplasmic components, EI and HPr, which are common to all PTS carbohydrates. Carbohydrate specificity resides in EII, and hence, bacteria usually contain many different EIIs. Each EII complex consists of one or two hydrophobic integral membrane domains (domains C and D) and two hydrophilic domains (domains A and B), which together are responsible for the transport of the carbohydrate across the bacterial membrane as well as its phosphorylation. In a sense, the EII complexes constitute parallel transport pathways connected to a common PEP/EI/HPr phosphoryl transfer pathway. E. coli contains at least 15 different EII complexes, and the existence and properties of these enzymes have been established by genetic, biochemical, and physiological studies. A similar number of PTSs is present in B. subtilis (178, 706). EII complexes are formed either by distinct proteins or by a single multidomain protein. Likewise, fusion proteins that contain EI and/or HPr domains exist. A prominent example of the latter is FPr, which consists of HPr and an EIIA domain and mediates the phosphotransfer in the uptake of fructose by E. coli and Salmonella enterica serovar Typhimurium (266). In Rhodobacter capsulatus, a similar fusion protein also contains an EI domain (958).
Genome sequencing projects have uncovered many proteins that are similar to either one of the two general PTS proteins or to parts of carbohydrate-specific EII complexes (345, 867, 935). For example, in E. coli, the protein DhaM is composed of an EIIAMan-like regulatory domain followed by an HPr and a truncated EI domain (304). In other cases, the newly discovered proteins are formed by fusions between domains that originate from both PTS and non-PTS proteins, e.g., a Clostridium acetobutylicum response regulator (built from HPr and an NtrC-like protein) (721) and the Streptococcus thermophilus LacS protein (built from a melibiose carrier and EIIAGlc) (673). The functions of most of these chimeric proteins remain unknown, but for some proteins, like LacS and DhaM, the function has been elucidated. Indeed, one of the present challenges is finding the function of the newly discovered PTS-like proteins, most of which have not been detected by any biochemical or mutant study. A very intriguing group consists of the paralogs of EI and HPr. In E. coli, at least five paralogs of each general PTS protein were uncovered (345, 720). We will discuss some of these proteins below (see "SOME UNUSUAL PTS PATHWAYS AND PROTEINS").
The general PTS proteins EI and HPr.
EI is encoded by the ptsI gene. Sequence comparisons reveal that the EIs (about 570 residues; 63 kDa) from various gram-positive and gram-negative bacteria exhibit significant identity (345, 678). EI is autophosphorylated in the presence of Mg2+ (940) at the N-3 position of the imidazole ring of a conserved histidine (His-189 in E. coli EI) (16, 940), which is located on the N terminus of the protein. The C terminus contains the PEP binding site and is necessary for dimerization (114, 254, 798). The two-domain structure of the protein was discovered by high-sensitivity differential scanning calorimetry (478). Limited proteolysis of E. coli EI in vitro results in a specific split and provides a stable peptide representing the N-terminal domain (455). The N-terminal domain of EI (EI-N) (115) as well as the C-terminal domain (EI-C) (234) were cloned and characterized. EI-C was shown to complement EI-N both in vivo and in vitro; i.e., EI-N was phosphorylated in the presence of EI-C, PEP, and Mg2+ (234). A truncated protein composed of the first 259 amino acids has been synthesized and purified, and both the crystal (475) and solution (258) structures were resolved. It appears that phosphorylation does not drastically change the conformation of the N-terminal domain per se. Recently, the crystal structure of the C-terminal domain of EI of the thermophile Thermoanaerobacter tengcongensis was also elucidated (619). EI exhibits about 30% sequence similarity with pyruvate phosphate dikinase (PPDK) (328, 605, 670), and the various domains in both proteins have similar structures (619). PPDK, which converts ATP, Pi, and pyruvate into AMP, pyrophosphate (PPi), and PEP; PEP synthase, an enzyme that catalyzes the conversion of pyruvate and ATP into PEP, AMP, and Pi and that is also phosphorylated on a histidyl residue (590); and EI have related functions. For PPDK, a structural model with three domains has been proposed: a C-terminal PEP/pyruvate binding domain, an N-terminal nucleotide binding domain, and a P
His domain linked to the other two domains via two flexible polypeptide segments (328). Although the nucleotide and PEP/pyruvate binding sites are approximately 45 Å apart, the PHis domain is thought to bridge the distance by swiveling between the two other domains to enable the phosphoryl transfer. Recent data obtained from a comparison of several PPDK crystal structures are consistent with the proposed model (587). Similar to PPDK, a large distance (>20 Å) between the PEP/pyruvate binding site and the phosphorylatable histidine was observed in a model of EI in which the N-terminal and C-terminal EI structures were fused (619). Swiveling of the N-terminal domain by changing the position of the domains with respect to each other but retaining the structure of the individual domains reduces the distance significantly (to 7.4 Å). In the case of EI, the N-terminal domain contains the HPr binding site rather than the nucleotide binding site of PPDK and PEP synthase. This model was confirmed by recent studies in which the structure of full-length EI of Staphylococcus carnosus was determined (516). The phosphohistidine and HPr-binding domain are clearly separated from the C-terminal dimerization and PEP binding domain. The structure also revealed the extensive interaction surface related to the dimer. Thermodynamic analyses of the influence of several effectors on the conformational stability of EI of Streptomyces coelicolor showed that at a low pH, EI is partly unfolded and that the presence of PEP causes structural changes (356). Two other recent studies on the structure of EI (639) and its C-terminal domain (638) suggest a relatively large structural variability for monomeric EI, which progressively diminishes when EI dimerizes and binds Mg2+ and PEP. The observed effects are explained by a swiveling mechanism, as described for PPDK. The structure thus provides additional evidence for the proposed conformational change necessary to facilitate phosphotransfer.
HPr (about 90 residues; 9 to 10 kDa) is encoded by the ptsH gene and has been purified from a variety of organisms (678). Similarity is most pronounced around the active-site histidyl residue, His-15 in the HPr of most enteric bacteria and firmicutes (183, 681, 941). HPr is phosphorylated at the N-1 position of the imidazole ring of His-15. The crystal and solution structures of HPrs from E. coli and B. subtilis as well as a few other species are known (for reviews, see references 33, 123, 329, and 936). The molecule forms an open-faced ß-sandwich consisting of a four-stranded antiparallel ß-sheet that is covered at one side by one short and two long -helices. The active-site His-15 is located in the N-terminal part of the first long -helix and is exposed to the solvent.
The solution structures of the complex between HPr and the N-terminal domain of EI (260) and between HPr and EIIAGlc of E. coli (929) have been determined. The absence of large changes of the chemical shift values for the backbone atoms upon complexation indicates that the interactions of HPr with EI and EIIAGlc do not require considerable conformational changes in any of the domains of the PTS binding partners. The binding interface of HPr and glycogen phosphorylase (see "REGULATION OF CARBON METABOLISM IN GRAM-NEGATIVE ENTERIC BACTERIA") was also mapped, and the interaction surface of the three structures was compared (930). EI and EIIAGlc interact with the same narrow region of HPr, whereas the region of interaction with glycogen phosphorylase is somewhat wider (929, 930). This is consistent with the higher binding affinity reported for HPr with glycogen phosphorylase than for HPr with EI or EIIAGlc (259, 799). A similar interaction surface was reported for E. coli HPr binding either EIIAMtl (139, 908) or EIIAMan (949) and B. subtilis HPr binding EIIAGlc (123). The key interacting residues are located in helices 1 and 2 and the loops preceding helix 1 and following helix 2. Studies with a large number of ptsH mutants, which show lowered affinity for binding to EI, suggest that the same residues are important for the interaction between EI and HPr (85).
In most low-G+C gram-positive bacteria and a few gram-negative organisms, HPr can also be phosphorylated by an ATP-dependent protein kinase on a seryl residue, e.g., Ser-46 in B. subtilis. We will discuss the importance of the second regulatory phosphorylation in detail below (see REGULATORY FUNCTIONS OF P-Ser-HPr). The regulatory phosphorylation is not part of the phosphoryl transfer to carbohydrates, but phosphorylation of the seryl residue slows the phosphoryl transfer from PEI to HPr at least 100-fold. E. coli HPr contains a Ser-46 residue but lacks an HPr kinase. Nevertheless, replacement of Ser-46 in E. coli HPr by an aspartyl residue lowers the affinity of PEI for HPr almost 1,000-fold (589). This effect is most likely caused by electrostatic repulsion, as the mutation caused by the replacement of Ser-46 with an aspartyl residue (Ser46Asp) leads to only weak structural changes (952).
Enzyme II complexes.
The carbohydrate specificity of the PTS resides in the EIIs, which consist of an integral membrane domain facing both the periplasmic space and the cytoplasm and cytoplasmic domains or proteins. All EIIs are built basically in a similar modular manner (764) and can consist of up to four separate proteins (for reviews, see references 464, 678, 730, and 813). The PTSs were classified into four (super)families with distinct evolutionary origins on the basis of the phylogenies of the EIIs (759) as follows: (i) the glucose-fructose-lactose superfamily, comprised of the glucose family (e.g., E. coli EIIAGlc/EIICBGlc or B. subtilis EIICBAGlc [CB, CBA, etc., indicate the domain order in multidomain EIIs]), the fructose-mannitol family (e.g., E. coli EIICBAMtl), and the lactose family (e.g., L. casei EIIALac/EIICBLac); (ii) the ascorbate-galactitol superfamily, comprised of the ascorbate family (e.g., E. coli SgaA/SgaB/SgaT) (358, 989) and the galactitol family (e.g., E. coli EIIAGat/EIIBGat/EIICGat) (610); (iii) the mannose family (e.g., E. coli EIIABMan/EIICMan/EIIDMan or B. subtilis EIIALev/EIIBLev/EIICLev/EIIDLev [a mannose- and fructose-specific PTS]); and (iv) the dihydroxyacetone family (e.g., E. coli EIIA-HPr-EIDha [DhaM] [304] or EIIADha in firmicutes).
The presence of a common preserved domain in the latter two families (411) might indicate a similar evolutionary origin of the two families and thus the existence of a third superfamily. The sequence-based classification of the various PTSs is supported by X-ray crystallography and nuclear magnetic resonance (NMR) studies, which clearly show that the structures of the EIIA (474, 617, 818, 858, 906, 955) and EIIB (1, 2, 98, 459, 623, 778, 906) domains/proteins belonging to the various classes are quite different. Information on the structure of the integral membrane domain EIIC (and EIID) is limited. The membrane topologies of these proteins have been studied using Cys replacement mutagenesis and chemical modification by thiol reagents for EIIBCABgl (960) and EIICBAMtl (918).
Extensive work on the intricate properties of the EII complexes and the detailed mechanisms by which they transport and phosphorylate carbohydrates has been performed in the laboratories of G. T. Robillard, B. Erni, and G. R. Jacobsen. The results that focus predominantly on EIICBAMtl, EIICMan/EIIDMan, and EIICBGlc have been reviewed extensively (369, 730, 813), and we will therefore discuss only a few important aspects. Some more recent papers included work on the properties of a cyclized protein formed by the soluble EIIAGlc and the membrane-bound EIICBGlc (812), the dimerization of EIICBAMtl (903, 904), sugar recognition by the EIICMan/EIIDMan and EIICBGlc of E. coli (256), the membrane topologies of EIIBCABgl (960) and EIICBAMtl (918), and transient-state kinetics of enzyme EIICBGlc (544).
The glucose-specific EII complex of enteric bacteria consists of two distinct proteins, the cytoplasmic protein EIIAGlc (gene crr) and the membrane-associated protein EIICBGlc (gene ptsG), in which the EIIB domain is hydrophilic and in contact with the cytoplasm and the EIIC domain is buried within the membrane. The EIIA and EIIB domains are defined as the domains that receive the phosphoryl group from PHPr and PEIIA, respectively. Whereas the phosphorylated residue in the EIIA domain/protein is a histidyl residue, that in the EIIB domain/protein can be either a histidyl residue (mannose family) or a cysteyl residue (all other families). The latter was first demonstrated in E. coli EIIMtl (634), and the specific phospho-cysteyl residue was thereafter identified in a number of cases by analytical methods (547, 633). The phosphoryl group of the EIIB domain is transferred to the carbohydrate after translocation of the carbohydrate by the EIIC domain across the membrane and delivery at the inside face of the cytoplasmic membrane. In the case of the mannitol-specific E. coli EIIMtl or the glucose-specific B. subtilis EIIGlc complex, the EII is a single polypeptide that contains the two hydrophilic domains EIIA and EIIB as well as the transmembrane domain EIIC. Again, in other EII complexes, such as the mannose-specific E. coli EIIMan, phosphoryl groups are carried by two histidyl residues present in the A and B domains of the cytoplasmic EIIABMan, whereas translocation of the carbohydrate requires the two transmembrane subunits EIIC and EIID.
Organization of the PTS.
Phosphoryl transfer between the PTS proteins proceeds by an in-line associative mechanism (48). The transfer is accommodated by the formation of heterodimeric transition complexes, and considering the kinetics of the transfer, the heterodimeric association should be transient. The solution structures of different transition complexes, including EI and HPr (260), HPr and EIIAGlc (124, 929), HPr and EIIAMtl (139), HPr and EIIAMan (949), EIIAGlc and EIIBGlc (98, 270), and, finally, PEIIAChb and Cys10Ser mutant EIIBChb (398), could be determined. The formation of the heterodimeric complexes has a significant effect on the flux control properties of the individual components of the PTS (738, 896, 901).
Based on the observation that E. coli EI and HPr are attached to membrane fragments of E. coli (757), it was speculated that the PTS components associate into a metabolon (611), which would improve PTS function. However, it is not clear how phosphotransfer from one component to the next could take place in a fully associated system without linkers to provide room for the domains to move, and furthermore, as it was calculated that diffusion should not limit glucose influx (239, 240), there should be no functional driving force to fully associate into an EI-HPr-EII complex. Formation of larger complexes by EI linked to yellow fluorescent protein has been observed in cells grown on PTS as well as non-PTS carbohydrates when they reached stationary phase and high cell densities (637). The observed distribution of the fluorescence over the cells was punctate and sometimes even bipolar (after induction of yellow fluorescent protein-EI with IPTG [isopropyl-ß-D-thiogalactopyranoside]). The aggregation appeared to be reversible, as it disappeared after the addition of a carbohydrate, which led to renewed growth of the cells and dispersion of the fluorescence. Thus, phosphotransfer activity within the PTS, as invoked by the addition of a carbohydrate, in fact seems to promote the overall dissociation of the PTS components rather than their association into a metabolon.
Notwithstanding, several PTS proteins were experimentally proved to associate to functional homodimers. The first PTS protein that was found to functionally dimerize was EI (432, 560; also reviewed in reference 114). Later, it was shown that the N-terminal domain does not dimerize (115) but that the C-terminal domain is responsible for dimerization (86). In fact, the association properties of the isolated C-terminal domain are similar to that of entire EI, although the binding constants are orders of magnitude larger (638). It was proposed that the monomer/dimer transition of EI could potentially regulate uptake flux (940), and as a result, the transition has been extensively studied. The idea was supported by the fact that PEP phosphorylates only dimeric EI, and the rate of association/dissociation is very slow, especially compared to sugar uptake (114, 539). The monomer/dimer transition has been studied by various analytical techniques, and many physiological conditions have been tested. It was observed that PEI has a 10-fold increased dimerization constant with respect to unphosphorylated EI and that the dimerization of EI is promoted by PEP and Mg2+ and inhibited by pyruvate (190-192). It was concluded that the stimulatory effect of Mg2+ and PEP on EI association results from a change in conformation. Association of both normal EI and the nonphosphorylatable mutants H189E, H189A, and H189Q was studied by carrying out sedimentation equilibrium experiments (639). The association constant appears to be very sensitive to pH, temperature, and ionic strength and may vary by as much as 700-fold. It was concluded that the ligands Mg2+ and PEP are the major determinants for dimerization and that their binding leads to conformational changes. A model that accommodates the known facts about the dimerization was presented. A potential regulatory function of EI dimerization with regard to the role of PEP is discussed below.
Several membrane-spanning EIIs have also been shown to dimerize, such as EIICBAMtl (730) and EIICBGlc (213, 214, 548). Proteome analysis of stable oligomeric protein complexes confirmed the existence of EIICBAMtl and EIICBGlc oligomers in the membrane of E. coli (826). In addition, oligomers of EIINag and EIITre were detected, which complies with a generalized picture in which the PTS permeases function as dimers (or oligomers). In contrast, it is generally assumed that HPr and EIIA do not form functional homooligomers. However, several experimental observations suggest the opposite. Biochemical evidence strongly suggested that EIIALac forms homotrimers (176, 319). This suggestion was supported by the crystal structure of EIIALac from Lactococcus lactis, which also revealed a homotrimer (818). In addition, it was reported that four molecules of EIIAGlc associate with one EIICBGlc homodimer (213). Moreover, multimeric EIIAGlc was observed during gel filtration (786) and in crystals (224). Dimeric EIIAChb and PEIIAChb were observed when ultracentrifugation was carried out (398). EIIABMan was found in the dimeric form both in the cytoplasm and linked to the membrane (216, 826). Also, EIIANtr, a protein homologous to EIIAs that are functional in sugar transport, crystallizes as a dimer (73), although it seems to be a monomer in solution (927). Crh, an HPr-like protein of B. subtilis, was shown to form homodimers in crystals (644), and F29W HPr from Geobacillus stearothermophilus (previously Bacillus stearothermophilus) forms similar domain-swapped dimers in crystallization experiments (827). On the other hand, several measurements performed with the protein in solution suggested that F29W HPr forms monomers. Recently, in vitro experiments provided kinetic evidence that all components of the E. coli glucose PTS form functional dimers (R. Bader, J. G. Blom, K. J. Hellingwerf, M. A. Pelletier, H. V. Westerhoff, and C. Francke, unpublished results).
Although the supposed primary function of the PTS is the transfer of the phosphoryl group from PEP to carbohydrates, all reactions up to and including the EIIs are reversible. The equilibrium constant, Keq, for the reaction PEP
PEIPHPr is about 80 for the E. coli enzymes (539), whereas the Keq for the phosphoryl group transfer between HPr and EIIAGlc from E. coli has a value of between 1 and 1.5 (540). Thus, the overall Keq for the phosphoryl group transfer from PEP to EIIAGlc is in the order of 80 to 120. Furthermore, the Keq for the reaction PEIIAGlc + EIICBGlcEIIAGlc + PEIICBGlc was determined to be 12 (539), very close to the estimated value of 7 (737). The number signifies that the phosphotransfer potential of the thiophospho bond in PCys-EIICBGlc is very close to that of the phosphoamidate bond in phosphohistidine of the other PTS proteins. Only the final step, PEIIBGlc + glucoseEIIBGlc + glucose-6-phosphate (glucose-6-P), is virtually irreversible, with a measured forward rate of 3.2 x 106 M–1 s–1 (539). These values indicate that under physiological conditions, the phosphoryl transfer reactions catalyzed by the PTS between PEP and PEIIBGlc can run in both directions, from one high-energy Penzyme intermediate to the next or back to the previous reactions. This differs from most eukaryotic signal transduction systems in which the seryl, tyrosyl, and threonyl residues that are often phosphorylated represent low-energy phosphoenzyme intermediates (837). The reversibility of the phosphoryl group transfer in the PTS has regulatory consequences because it allows the metabolic network to control the phosphorylation state of the PTS proteins in a number of ways, and we will discuss how numerous cellular processes are regulated by the phosphorylation states of certain PTS proteins below (see Table 1 for a summary).
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REGULATION OF CARBON METABOLISM IN GRAM-NEGATIVE ENTERIC BACTERIA
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In the following sections, we will discuss the numerous regulatory functions carried out by EIIAGlc in enteric bacteria. This protein not only is involved in the regulation of adenylate cyclase, and therefore in CCR, but also interacts with several non-PTS permeases and glycerol kinase to inhibit their activity. The latter phenomenon is described by the term inducer exclusion. We will present new insights into the complex regulation of the intracellular cyclic AMP (cAMP) level and the binding of EIIAGlc to adenylate cyclase and to non-PTS permeases.
EIIAGlc Is the Central Processing Unit of Carbon Metabolism in Enteric Bacteria
The integration of hundreds of enzymatic reactions into a metabolic network and the complex response of such a network to environmental changes require global regulation at the level of enzyme activity and gene transcription (89, 592, 917). A bacterium that is confronted with changes in the supply of nutrients can adapt its metabolic potential through the induction of specific catabolic operons (most often induced by the substrate) (90, 94). More drastic changes may require global adaptation and therefore the induction of complete regulons (89, 592, 917). Transcriptome data indicate that when E. coli cells that have been growing on a rich carbon source such as glucose are exposed to a carbon source allowing only slow growth, the number of induced genes increases progressively (485). Low growth rates result in the expression of many transport systems and catabolic genes for nutrients that might be absent but that would stimulate growth if present. Results obtained by high-throughput nutrient screening of glucose-grown cells and cells grown under carbon limitation confirmed the effects observed by transcriptome analysis: cells grown under carbon limitation do utilize a large variety of carbon sources, whereas cells growing on glucose do not (the result of CCR) (360). The large-scale transcriptional responses in E. coli are mediated via a limited number of global regulators (527). In enteric bacteria, global regulation of carbon metabolism involves several 
and transcription factors such as Crp (cyclic AMP receptor protein) (reviewed in references 76, 95, 96, 416, and 636), Mlc (666), FruR (145, 267, 268, 762), CsrA (carbon storage regulator) (451, 742, 743, 752, 861), and 54 (25, 150, 843). The activities of some of these regulators are directly affected by the PTS.
The importance of the PTS in the regulation of carbon metabolism in enteric bacteria became apparent when ptsHI mutants containing defective EI and/or HPr were isolated. These mutants failed to grow not only on PTS carbohydrates but also on a large number of non-PTS carbon sources like lactose, melibiose, glycerol, and maltose (for reviews, see references 677, 678, and 763). Analysis of suppressor mutations, which restored growth of ptsHI mutants on the non-PTS carbon sources (765, 767), revealed that these mutations were in the crr gene (carbohydrate repression resistant), which was later shown to be the structural gene for EIIAGlc (542, 543, 786). While ptsHI-crr triple mutants grew on the non-PTS carbon sources, they did not resume growth on PTS carbohydrates. These results indicated that not only is EIIAGlc involved in glucose transport and phosphorylation via the glucose PTS, it also regulates the transport and/or metabolism of several non-PTS carbon sources.
The changes of structure or charge induced by phosphorylation or dephosphorylation determine the regulatory role of EIIAGlc. PEIIAGlc is required for the activation of adenylate cyclase. S. enterica serovar Typhimurium and E. coli crr mutants exhibit a low residual adenylate cyclase activity, generally 5 to 20% of the activity found in the parental strain (227, 469, 595). Unphosphorylated EIIAGlc can bind to and inhibit numerous non-PTS proteins, including the permeases for lactose (LacY) and melibiose (MelB), the ATP-hydrolyzing component of the maltose transport system (MalK), and glycerol kinase (GlpK). As a result, induction of the genes involved in the uptake and metabolism of these substrates is prevented, and hence, the inhibitory process was called inducer exclusion.
Both control mechanisms, inducer exclusion and modulation of adenylate cyclase activity, have a physiological function and allow the cell to choose between different carbon sources as long as the uptake of glucose and other PTS carbohydrates leads to the dephosphorylation of EIIAGlc. The connections between carbohydrate uptake, the phosphorylation state of EIIAGlc, adenylate cyclase activity, and the intracellular concentration of metabolites such as PEP and pyruvate relate metabolic flux directly to the regulation of solute uptake and gene expression. In Fig. 2, a summary of the mechanisms underlying CCR in enteric bacteria is given. Although EIIAGlc-mediated regulation has been found only in enteric bacteria so far, this signal transduction system has served and serves as a model for other regulatory processes that involve reversible phosphorylation, such as PTS-regulated transcription activation or antitermination.
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FIG. 2. Mechanisms underlying CCR and inducer exclusion in enteric bacteria. The import of glucose and other PTS carbohydrates leads to net dephosphorylation of the PTS proteins (including EIIAGlc and the B domain of EIIBCGlc) and thereby to inducer exclusion and recruitment of the transcription regulator Mlc to the membrane. The upper left part of the figure shows that unphosphorylated EIIAGlc blocks the import of lactose, maltose, and melibiose and the phosphorylation of glycerol by binding to the respective transporter or kinase. The upper right part of the figure shows recruitment of Mlc by unphosphorylated EIIBCGlc, which prevents the regulator from binding to its target sites on the DNA. In the absence of glucose and in the presence of phosphoenolpyruvate, the PTS proteins are found mainly in the phosphorylated state. The central part of the figure shows that phosphorylated EIIAGlc activates adenylate cyclase (AC) but probably only in the presence of an unknown adenylate cyclase activation factor (ACAF). Adenylate cyclase binds phosphorylated as well as unphosphorylated EIIAGlc (see reference 632). The bottom part of the figure shows the effect of the activated transcription factors (free Mlc and Crp:cAMP) on the transcription of the genes encoding Crp, adenylate cyclase, Mlc, and EIIBCGlc, respectively. The inset shows the Crp:cAMP concentration dependence of crp transcription (deduced from data reported in references 310 and 311). The arrow indicates the physiological concentration of activated Crp in exponentially growing cells in the absence of PTS carbohydrates.
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Transcription Regulation by Crp/cAMP and Role of PEIIAGlc
In enteric bacteria, Crp is one of the few truly global regulators, as it controls the expression of a vast number of genes/operons (34, 291, 527, 990). Crp is activated by binding cAMP, which is synthesized from ATP by adenylate cyclase (gene cyaA) (for recent papers on the structure of the Crp/cAMP regulator, see references 316, 471, 671, and 888). Different genes and operons require different levels of Crp/cAMP for full expression because the Crp/cAMP complex can adopt several conformationally active states and because the affinity for the recognition sites on the DNA varies (149, 316, 417, 484, 853, 888).
The concentration of Crp/cAMP is tightly regulated by the PTS. Soon after the discovery of cAMP in E. coli, it was observed, for example, that the addition of glucose to cells growing on lactate lowers the concentration of cAMP (508). Later results confirmed this finding (212, 384), and experiments with toluenized cells established that adenylate cyclase activity is inhibited by glucose (318, 648-652). Other PTS carbohydrates were also shown to inhibit cAMP synthesis, provided that the cognate EII complex was intact and induced (317, 767). Studies with ptsHI and crr mutants demonstrated that adenylate cyclase activity is low in both types of mutants (227, 469, 595). Based on these and other studies (see references 678 and 763), a regulatory mechanism for the activation of adenylate cyclase by PEIIAGlc was proposed. In this scheme, transport and phosphorylation of glucose or other PTS carbohydrates decrease the extent of phosphorylation of the PTS proteins, including EIIAGlc, and thus lower the activity of adenylate cyclase, whereas growth on non-PTS carbohydrates, particularly poor carbon sources like lactate and succinate, results in fully phosphorylated PTS proteins and the activation of adenylate cyclase. However, this model does not explain how growth on non-PTS carbon sources such as glucose-6-phosphate or gluconate can lead to intracellular cAMP levels similar to, or sometimes lower than, those observed in glucose-grown cells (212, 337). Moreover, the inhibitory effect of glucose-6-phosphate is found only in intact but not in toluenized cells (318), and we will discuss this important point later.
Regulation of cyaA and crp transcription.
Most of our current knowledge regarding Crp/cAMP-mediated regulation is derived from the study of cyaA, crp, and/or ptsHI-crr mutants (76, 416, 636, 677, 678, 763, 890). The effect of different carbon sources on growth, gene expression levels, cAMP concentrations, and Crp levels has been tested with these mutants. The addition of exogenous cAMP was used to distinguish between the effects caused by concentration changes of either cAMP or Crp. One class of mutants, designated crp* or crp(In), produced Crp, which is active without cAMP. Unfortunately, an interpretation of the mutant data is not easy, as the factors involved in cAMP-mediated regulation are interdependent, as depicted in Fig. 2.
Inactivation of the cyaA gene disables bacteria like E. coli and S. enterica serovar Typhimurium from growing on a large number of carbon sources (76), while cyaA overexpression appears to be detrimental (699). Inactivation of crp also affects the cAMP concentration by leading to a drastic increase in the rate of cAMP synthesis (75, 170, 241, 363, 679). The increase in the cAMP concentration depends on an intact crr gene. In crp crr double mutants, cAMP overproduction is markedly reduced (from about 60-fold of the wild-type level in the crp strain to only 4-fold of the wild-type level in the double mutant) (143, 170).
The phenomena described above are related to feedback regulation on the level of transcription. Expression of cyaA is negatively regulated by Crp/cAMP in both E. coli (10, 387, 395, 576) and S. enterica serovar Typhimurium (221), whereas expression of crp is both positively (310) and negatively (9, 140, 311) affected by Crp/cAMP. As a result, strong crp expression occurs at low and high Crp/cAMP concentrations (due to reduced inhibition and increased stimulation, respectively), whereas low crp expression is observed at intermediate Crp/cAMP concentrations (310). The concentration of Crp/cAMP in exponentially growing cells is an order of magnitude higher than that allowing minimal crp transcription (310, 365). Consequently, a decrease in the cAMP concentration caused by the addition of glucose to these cells should lower the transcription of crp and hence the concentration of Crp. Glucose-grown E. coli cells indeed contain less Crp, and this was shown not to be due to faster degradation of the crp mRNA (364, 365). The glucose effect on crp expression is absent in mutants lacking either adenylate cyclase or Crp, consistent with the postulated role of Crp/cAMP. The growth stage of E. coli cultures also has an influence on the amount of Crp (365). These variations might explain why some authors reported complete relief from glucose repression after the addition of cAMP (647), whereas others observe only partial relief (932).
Expression of the EIIAGlc-encoding crr gene is almost completely independent of Crp/cAMP (857). In contrast, expression of the EIICBGlc-encoding ptsG gene is almost fully repressed in cyaA or crp mutants (727). Owing to the low level of the glucose transporter EIICBGlc in these mutants, the addition of glucose to cyaA or crp mutants does not lead to the same extent of dephosphorylation of PEIIAGlc as in the wild-type strain. The regulation of crp transcription by factors other than Crp/cAMP adds further complexity to catabolite repression data. crp transcription is negatively regulated by both the DNA binding protein Fis (280), the transcription of which is in turn regulated by Crp/cAMP (591), and SpoT (378), the protein that mediates the stringent response and that catalyzes the synthesis of ppGpp (guanosine tetraphosphate).
Regulation of adenylate cyclase activity.
Whether PEIIAGlc exerts its regulatory effect on adenylate cyclase directly or indirectly has been an open question for a long time. Early experiments with permeabilized cells containing an intact PTS showed that adenylate cyclase activity is stimulated by potassium and phosphate and inhibited by -methylglucoside (-MG) and pyruvate (476, 477, 701), which is in line with a stimulatory effect by PEIIAGlc. However, in vitro experiments with purified adenylate cyclase were not conclusive (963). The effect of EIIAGlc phosphorylation on the activation of adenylate cyclase was studied in crr mutants producing EIIAGlc in which the phosphorylatable His-90 (197) and the catalytically important His-75 had been changed. In comparison to wild-type EIIAGlc, His75Gln EIIAGlc synthesized in a crr mutant caused an approximately fourfold activation of adenylate cyclase. In contrast, neither His90Gln nor His90Glu EIIAGlc activated adenylate cyclase (700, 852). The latter two mutant proteins are not phosphorylated by PHis-HPr, while His75Gln EIIAGlc is. Takahashi and coworkers also found that His75Glu EIIAGlc, which is barely phosphorylated in vitro (700), can slightly activate adenylate cyclase (852). In wild-type EIIAGlc, His-75 and His-90 are in very close proximity, and the replacement of His-75 with a negatively charged Glu possibly mimics phosphorylated His-90. Although these results indicate that phosphorylation of His-90 of EIIAGlc is important for the activation of adenylate cyclase, they do not prove a direct interaction. Recent results with adenylate cyclase tethered to the membrane showed that although both PEIIAGlc and unphosphorylated EIIAGlc bind with similar affinity to the protein, it is activated by PEIIAGlc only when an additional, not-yet-identified factor from cell extracts is present (632) (Fig. 2).
From studies with truncated adenylate cyclase, it can be concluded that the catalytic activity resides in the N-terminal domain, with the C-terminal domain required for EIIAGlc-mediated regulation (632, 699, 746). Starting from an E. coli ptsHI-crr deletion strain containing, in addition, an Asp414Asn mutation in cyaA, which prevents the large increase in adenylate cyclase activity in a crp mutant (143), suppressor mutants affected in the cyaA gene could be isolated. Most suppressor mutations were located in the region around Asp-414 and resulted in a truncated adenylate cyclase that was about half the size of the wild-type enzyme (144). The truncated molecules produce about 10 times more cAMP in a crr strain than the wild-type enzyme. These results suggest that the N-terminal domain of adenylate cyclase is active in the absence of PEIIAGlc and that the C-terminal domain acts as an inhibitor that may be removed/inactivated by PEIIAGlc.
Mutation of crp leads to a drastic increase of extracellular cAMP. This increase is 50-fold diminished when the C-terminal 48 amino acids of adenylate cyclase are cut off. In contrast, the increase is diminished by only fourfold when the CRP/cAMP-mediated transcriptional regulation of the cya gene is prevented by replacing the cya promoter with the constitutive bla promoter (363). Therefore, it was concluded that the regulation of cAMP production occurs mainly posttranslationally (at the level of enzyme activity) and not at the level of transcription. However, because the regulation of cyaA transcription is affected by the Crp/cAMP concentration, which in turn is affected by CyaA activity, that conclusion does not seem to be justified. This can be illustrated by the changes in adenylate cyclase activity caused by a crr null mutation. Adenylate cyclase activity measured in crr mutants amounts to only 5% to 20% of the activity measured in wild-type strains (227, 469, 595), and the reintroduction of plasmid-borne crr increases it four- to fivefold, thus approaching levels of wild-type activity (477, 700). If EIIAGlc was the only factor interacting with and stimulating adenylate cyclase, crr mutants and strains producing adenylate cyclase missing the last 48 amino acids should exhibit the same phenotype. However, it is evident that the positive effect on adenylate cyclase caused by the expression of plasmid-borne crr in a crr mutant is much smaller than the negative effect (50-fold) caused by the deletion of the 48 C-terminal amino acids of adenylate cyclase in a crp mutant. This difference probably owes to the absence of the Crp-dependent effect of cAMP on transcription in a crp mutant.
Regulation by Crp/cAMP.
Despite the enormous amount of experimental data, the control of cAMP and Crp levels in enteric bacteria is still not completely understood. Regulation occurs not only at the level of transcription but also at the level of enzyme activity. These two processes affect each other, making it difficult to quantify their individual contributions. Nevertheless, the general properties of the regulatory mechanism are clear. The immediate response of cells to the addition of a rapidly metabolizable carbohydrate involves regulation only at the level of enzyme activity. The addition of glucose or another PTS sugar causes the dephosphorylation of PEIIAGlc. This deactivates adenylate cyclase and hence lowers the concentration of cAMP and Crp/cAMP inside the cell. On a longer time scale, regulation on the transcriptional level becomes important. At normal physiological levels of cAMP and Crp, the crp gene is negatively regulated by Crp/cAMP. Thus, a reduced activity of adenylate cyclase leads to lower expression of crp. As a result, the concentration of Crp/cAMP drops even further. On the other hand, a lower Crp/cAMP concentration leads to an increased expression of cyaA, and more adenylate cyclase will accumulate, thereby counteracting the decrease in activity and raising the Crp/cAMP concentration. In principle, the elevated pool of adenylate cyclase provides a higher potential for cAMP production once the glucose is depleted, although the Crp concentration will be lower.
Some results cannot be explained by the proposed mechanism. For instance, in S. enterica serovar Typhimurium crp* (595) and E. coli crp* cya (851) mutant strains, the addition of glucose lowers the concentration of Crp* and hence repression. However, because Crp* is constitutively active, i.e., it does not need cAMP for activity, its concentration should not be affected by the presence or absence of glucose. The occurrence of a component that is able to interact with Crp in the presence of glucose could explain the observed decrease in the Crp concentration. In fact, such a mechanism regulates the activity of the repressor Mlc (588): when glucose is present, EIICBGlc is dephosphorylated and binds the transcription factor Mlc, thus relieving repression. It is tempting to speculate that unphosphorylated EIICBGlc might also bind Crp, but as yet, there is no experimental proof to support this assumption.
Factors other than those mentioned above have been reported to influence the activity of adenylate cyclase, but their physiological relevance remains uncertain. A decrease in adenylate cyclase activity, for instance, was observed upon lowering the membrane electrochemical potential (649). Mutation of fruB (formerly fruF) can have a negative (272) or positive (225) effect on adenylate cyclase activity. Also, when GTP or the elongation factor Tu, a GTP-binding protein (702), was added to purified adenylate cyclase, it stimulated its activity almost twofold (829).
Growth rate effects on cAMP concentration.
Although early studies reported hardly any effect of the growth rate on the cAMP concentration under glucose limitation (529, 957), more recent studies report a clear relationship, with elevated cAMP concentrations occurring at low growth rates (613, 614). During growth on glucose in continuous culture, expression of lacZ appears to be independent of ppGpp but directly related to the cAMP concentration and inversely related to the growth rate (438); i.e., growth rate and cAMP concentration are also inversely related. The effect of the growth rate on the cAMP concentration can be explained in terms of the stimulation of adenylate cyclase activity by PEIIAGlc. The phosphorylation state of the PTS components in these experiments should depend on the growth rate because the cells were grown in a chemostat under carbohydrate-limiting conditions, and the growth rate was varied by changing the rate of carbon source supply. As a result, at high growth rates, less PEIIAGlc and hence less
EIIAGlc
His-HPr AND P
EIIBs.
EIIBs are necessary for the phosphorylation of a histidine in PRD1.
His-HPr-Mediated Phosphorylation
His-HPr-Mediated Phosphorylation
GlpK dephosphorylation leads to CCR via inducer exclusion.
His-HPr?
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