The Bacterial Phosphotransferase System Chapter Abstracts
Chapter 1
The Bacterial Phosphotransferase System: Structure, Function, Regulation and Evolution
In Memoriam to Dr. Jonathan Reizer
Milton H. Saier, Jr.
First Paragraph: The PTS: An Overview
Thirty-seven years ago, Kundig, Ghosh and Roseman reported the discovery of a novel sugar-phosphorylating system in Escherichia coli (Kundig et al., 1964). The unique features of this phosphotransferase system (PTS) included the use of phosphoenolpyruvate (PEP) as the phosphoryl donor for sugar phosphorylation and the presence of three essential catalytic entities, termed Enzyme I, Enzyme II and HPr (heat-stable, histidine-phosphorylatable protein). The discovery of this system provided an explanation for pleiotropic carbohydrate-negative mutants of E. coli described as early as 1949 (Doudoroff et al., 1949).
Chapter 2
The Complete Phosphotransferase System in Escherichia coli
Jason H. Tchieu, Vic Norris, Jeremy S. Edwards, and Milton H. Saier, Jr.
Abstract
We here tabulate and describe all currently recognized proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and their homologues encoded within the genomes of sequenced E. coli strains. There are five recognized Enzyme I homologues and six recognized HPr homologues. A nitrogen-metabolic PTS phosphoryl transfer chain encoded within the rpoN and ptsP operons probably serves regulatory roles exclusively. A tri-domain PTS protein encoded within the dha (dihydroxyacetone catabolic) operon serves as a component of a soluble PTS Enzyme II, the other components being homologous to ATP-dependent DHA kinases. In addition to several putative PTS proteins, there are 21 (and possibly 22) recognized Enzyme II complexes. Of the 21 Enzyme II complexes, 7 belong to the fructose (Fru) family, 7 belong to the glucose (Glc) family, and 7 belong to the other PTS permease families. All of these proteins are briefly described, and phylogenetic data for the major families are presented.
Chapter 3
Three-Dimensional Structures of Protein-Protein Complexes in the E. coli PTS
Alan Peterkofsky, Guangshun Wang, Daniel S. Garrett, Byeong Ryong Lee, Yeong-Jae Seok, and G. Marius Clore
Abstract
The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport. The soluble proteins of the glucose transport pathway also function as regulators of diverse systems. The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy. The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIAGlc have been elucidated. An analysis of the binding interfaces of HPr with enzyme I, IIAGlc and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions. Similarly, a common surface on IIAGlc interacts with HPr, IIBGlc and glycerol kinase. Thus, there is a common motif for the protein-protein interactions characteristic of the PTS.
Chapter 4
Routes for Fructose Utilization by Escherichia coli
Hans L. Kornberg
Abstract
There are three main routes for the utilization of fructose by Escherichia coli. One (Route A) predominates in the growth of wild-type strains. It involves the functioning of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and a fructose operon, mapping at min. 48.7, containing genes for a membrane-spanning protein (fruA), a 1-phosphofructose kinase (fruK) and a diphosphoryl transfer protein (fruB), under negative regulation by a fruR gene mapping at min. 1.9. A second route (Route B) also involves the PTS and membrane-spanning proteins that recognize a variety of sugars possessing the 3,4,5-D-arabino-hexose configuration but with primary specificity for mannose (manXYZ), mannitol (mtlA) and glucitol (gutA) and which, if over-produced, can transport also fructose. A third route (Route C), functioning in mutants devoid of Routes A and B, does not involve the PTS: fructose diffuses into the cell via an isoform (PtsG-F) of the major glucose permease of the PTS and is then phosphorylated by ATP and a manno(fructo)kinase (Mak+) specified by a normally cryptic 1032 bp ORF (yajF) of hitherto unknown function (Mak-o), mapping at min. 8.8 and corresponding to a peptide of 344 amino acids. Conversion of the Mak-o to the Mak+ phenotype involves an A24D mutation in a putative regulatory region.
Chapter 5
Facilitation of Bacteriophage Lambda DNA Injection by Inner Membrane Proteins of the Bacterial Phosphoenol-pyruvate:Carbohydrate Phosphotransferase System (PTS)
Margarita Esquinas-Rychen and Bernhard Erni
Abstract
Infection of Escherichia coli by bacteriophage lambda depends on two membrane protein complexes: (i) maltoporin (LamB) in the outer membrane for adsorption and (ii) the IICMan-IIDMan complex of the mannose transporter in the inner membrane for DNA penetration. IICMan and IIDMan are components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) which together with the IIABMan subunit mediate transport and phosphorylation of sugars. To identify structural determinants important for penetration of lambda DNA, the homologous IIC-IID complexes of E. coli, K. pneumoniae and B. subtilis, and chimeric complexes between the IIC and IID were characterized. All three complexes support sugar transport in E. coli. Only IIC-IID of E. coli and B. subtilis also support bacteriophage lambda infection. The six chimeric complexes had lost transport activity, but three containing IIC of E. coli or B. subtilis continue to support bacteriophage lambda infection. Complexes containing IICMan and fusion proteins between truncated IIDMan and alkaline phosphatase or b-galactosidase support penetration of lambda DNA if less than 100 residues are missing from the C-terminus of IIDMan. Truncation of IICMan renders the complex unstable. Taken together, these results suggest, that IIC is the major specificity determinant for lambda infection but that the IIC subunit is stably expressed only in a complex with the IID subunit. Lambda DNA in transit across the periplasmic space, but not transforming plasmid DNA, is inaccessible to the non-specific nuclease NucA of Anabaena sp. targeted to the periplasmic space either in soluble form or as a fusion protein to the C-terminus of IIDMan.
Chapter 6
Regulation of PTS Gene Expression by the Homologous Transcriptional Regulators, Mlc and NagC, in Escherichia coli (or How Two Similar Repressors Can Behave Differently)
Jacqueline Plumbridge
Abstract
NagC and Mlc are paralogous transcriptional repressors in E.coli. Unexpectedly they possess almost identical amino acid sequences in their helix-turn-helix (H-T-H), DNA binding motif and they bind to very similar consensus operator targets. Binding to each others sites can be demonstrated in vitro but no cross regulation can be detected in vivo with physiological amounts of the two proteins. Although both proteins are involved in regulating the expression of PTS genes, the characteristics of their repression and induction are very different. NagC is a dual-function, activator-repressor which co-ordinates the metabolism of the amino sugars, N-acetylglucosamine (GlcNAc) and glucosamine, by repressing the divergent nagE-BA operons and by activating the glmUS operon. Repression (and activation) by NagC requires that NagC binds simultaneously to two operators, thus forming a DNA loop. This chelation effect allows use of lower affinity sites which would not individually bind the repressor. The specific inducer for NagC is GlcNAc-6-P, the product of GlcNAc transport by the PTS and a key compound in amino sugar metabolism. Mlc represses several genes implicated in the uptake of glucose; ptsG, ptsHI and manXYZ, and malT, the activator of the mal regulon. Glucose behaves like the inducer but growth on glucose only produces an overall increase in expression for ptsG and ptsHI. All Mlc repressed genes are also controlled by cAMP/CAP, so that glucose affects their transcription in two opposing ways: increasing expression by acting as the inducer for Mlc but decreasing expression by lowering the cAMP/CAP concentration. The Mlc protein is not directly responsive to glucose per se but to the activity status of the PTS. Displacement of Mlc from its binding sites
Chapter 7
Regulation of Galactoside Transport by the PTS
Masayuki Kuroda, Thomas H. Wilson, and Tomofusa Tsuchiya
Abstract
Inducer exclusion, regulation of activity of transporter, is mediated by phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). To elucidate the molecular mechanism of the inducer exclusion, numerous biochemical and genetic studies have been performed. It is now well known that non-phosphorylated IIAGlc inhibits the transport via direct binding to the transporter. Analysis of inducer exclusion resistant mutants of lactose transporter and melibiose transporter in Escherichia coli and Salmonella typhimurium revealed amino acid residues that are involved in the interaction with IIAGlc. It is concluded that there are multiple interaction sites for IIAGlc in these transporters.
Chapter 8
Regulation of E. coli Glycogen Phosphorylase Activity by HPr
Yeong-Jae Seok, Byoung-Mo Koo, Melissa Sondej, and Alan Peterkofsky
Abstract
Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases.
Chapter 9
Use of Staphylococcus aureus 6-P-ß-Galactosidase and GFP as Fusion Partners for Lactose-Specific IIC Domain from Staphylococcus aureus
Claudia M. Kowolik and Wolfgang Hengstenberg
Abstract
The hydrophilic part of membrane proteins plays an important role in the formation of 3D crystals. The construction of fusion proteins using well crystallizing proteins as fusion partners is a possibility to increase the hydrophilic part of membrane proteins lacking large hydrophilic domains. These fusion proteins might be easier to crystallize.
Two bifunctional fusion proteins containing the membrane-bound, lactose-specific enzyme IIC domain of the lactose transporter (IICBlac) from S. aureus as N-terminal fusion partner were constructed by gene fusion. The C-terminal fusion partners were S. aureus 6-P-ß-Galactosidase and GFP, respectively. Both proteins were overexpressed in E. coli, purified to homogeneity and kinetically characterized: In the presence of the components of the lactose phosphotransferase system of S. aureus, the hybrid proteins phosphorylated their substrates, indicating that the fusion partners are sufficiently flexibly linked to allow the interaction of the IIClac domain with the IIBlac domain of the lactose transporter. The activity of the 6-P-ß-Galactosidase as well as the fluorescence of GFP were preserved in the fusion proteins. The Vmax values determined for the IIC domain in the fusion proteins were dramatically reduced compared with the values determined for the separate IIClac domain and the complete lactose transporter (IICBlac). The Km values were only slightly increased indicating that the Vmax values are much more influenced by the fusion than the substrate affinities. The substrate affinity and the Vmax value determined for the GFP-fused IIClac domain are higher than for the 6-P-ß-Galactosidase-fused IIClac. These results suggest that the fusion with GFP enables a better interaction with the IIBlac domain than the fusion with 6-P-ß-Galactosidase. Moreover, the GFP-fused IIClac domain proved to be more stable than the 6-P-ß-Galactosidase fusion protein.
Chapter 10
Hierarchical Control versus Autoregulation of Carbohydrate Utilization in Bacteria
M. G. W. Gunnewijk, P. T. C. van den Bogaard, L.M. Veenhoff, E.H. Heuberger, W. M. de Vos, M. Kleerebezem, O. P. Kuipers and B. Poolman
Abstract
The involvement of phosphoenolpyruvate:sugar phosphotransferase (PTS) proteins, like HPr and IIAGlc, in the regulation of carbohydrate utilization has been well established in Gram-negative and Gram-positive bacteria. The majority of the studies of PTS-mediated regulation have been concerned with the hierarchical control of carbohydrate utilization, which results in the preferential utilization of a particular carbohydrate from a mixture of substrates. The underlying mechanisms of PTS-mediated hierarchical control involve the inhibition of expression of other catabolic enzymes and transporters and/or the allosteric regulation of their activity, which prevents the transcriptional inducer to be formed or taken up into the cell. More recently, it has become clear that PTS components allow also the cell to tune the uptake rate(s) to the carbohydrate availability in the medium and the metabolic capacity of the cell. The different phosphorylated species of HPr play a central role in this autoregulatory control circuit, both at the gene and at the protein level. Our knowledge of hierarchical control and autoregulation of carbohydrate utilization in bacteria is discussed.
Chapter 11
Corynebacterium diphtheriae: a PTS View to the Genome
Stephan Parche, Andreas W. Thomae, Maximilian Schlicht, and Fritz Titgemeyer
Abstract
We have surveyed the publicly available genome sequence of Corynebacterium diphtheriae (www.sanger.ac.uk) to identify components of the phosphotransferase system (PTS), which plays a central role in carbon metabolism in many bacteria. Three gene loci were found to contain putative pts genes. These comprise: (i) the genes of the general phosphotransferases enzyme I (ptsI) and HPr (ptsH), a fructose-specific enzyme IIABC permease (fruA), and a fructose 1-phosphate kinase (fruK); (ii) a gene that encodes an enzyme IIAB of the fructose/mannitol family, and a novel HPr-like gene, ptsF, that encodes an HPr domain fused to a domain of unknown function; (iii) and a gene for a glucose-specific enzyme IIBCA (ptsG). A search for genes that may be putative PTS-targets or that may operate in general carbon regulation revealed a possible regulatory gene encoding an antiterminator protein downstream from ptsG. Furthermore, genes were detected encoding glycerol kinase, glucose kinase, and a homologue of the global activator of carbon catabolite repression in Escherichia coli, CAP. The possible significance of these observations in carbon metabolism and the novel features of the detected genes are discussed.
Chapter 12
Corynebacterium glutamicum: a Dissection of the PTS
Stephan Parche, Andreas Burkovski, Georg A. Sprenger, Brita Weil, Reinhard Krämer, and Fritz Titgemeyer
Abstract
The high-GC Gram-positive actinomycete Corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. Despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. We have addressed this issue by analyzing the phosphotransferase system (PTS), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. The general PTS phosphotransferases enzyme I (EI) and HPr were characterized by demonstration of PEP-dependent phosphotransferase activity. An EI mutant exhibited a pleiotropic negative phenotype in carbon utilization. The role of the PTS as a major sugar uptake system was further demonstrated by the finding that glucose and fructose negative mutants were deficient in the respective enzyme II PTS permease activities. These carbon sources also caused repression of glutamate uptake, which suggests an involvement of the PTS in carbon regulation. The observation that no HPr kinase/phosphatase could be detected suggests that the mechanism of carbon regulation in C. glutamicum is different to the one found in low-GC Gram-positive bacteria.
Chapter 13
Evidence for a Dimerisation State of the Bacillus subtilis Catabolite Repression HPr-Like Protein, Crh
François Penin, Adrien Favier, Roland Montserret, Bernhard Brutscher, Josef Deutscher, Dominique Marion and Anne Galinier
Abstract
The Bacillus subtilis catabolite repression HPr (Crh) exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system. We report here that Crh preparations contain a mixture of monomers and homodimers, whereas HPr is known to be monomeric in solution. The dissociation rate of dimers is very slow (t1/2 of about 10 hours), and the percentage of dimers in Crh preparations increases with rising temperature or protein concentration. However, at temperatures above 25°C and a protein concentration of 10 mg/ml, Crh dimers slowly aggregate. Typically, NMR spectra recorded at 25°C showed the coexistence of both forms of Crh, while in Crh solutions kept at 35°C, almost exclusively Crh monomers could be detected. Circular dichroism analysis revealed that the monomeric and dimeric forms of Crh are well folded and exhibit the same overall structure. The physiological significance of the slow Crh monomer/dimer equilibrium remains enigmatic.
Chapter 14
Catabolite Regulation of the Cytochrome c550-Encoding Bacillus subtilis cccA Gene
Vicente Monedero, Grégory Boël, and Josef Deutscher
Abstract
In Gram-positive bacteria, catabolite control protein A (CcpA)-mediated catabolite repression or activation regulates not only the expression of a great number of catabolic operons, but also the synthesis of enzymes of central metabolic pathways. We found that a constituent of the Bacillus subtilis respiratory chain, the small cytochrome c550 encoded by the cccA gene, was also submitted to catabolite repression. Similar to most catabolite-repressed genes and operons, the Bacillus subtilis cccA gene contains a potential catabolite response element cre, an operator site recognized by CcpA. The presumed cre overlaps the -35 region of the cccA promoter. Strains carrying a cccA'-lacZ fusion formed blue colonies when grown on rich solid medium, whereas white colonies were obtained when glucose was present. b-Galactosidase assays with cells grown in rich medium confirmed the repressive effect of glucose on cccA'-lacZ expression. Introduction of a ccpA or hprK mutation or of a mutation affecting the presumed cccA cre relieved the repressive effect of glucose during late log phase. An additional glucose repression mechanism was activated during stationary phase, which was not relieved by the ccpA, hprK or cre mutations. An interaction of the repressor/corepressor complex (CcpA/seryl-phosphorylated HPr (P-Ser-HPr)) with the cccA cre could be demonstrated by gel shift experiments. By contrast, a DNA fragment carrying mutations in the presumed cccA cre was barely shifted by the CcpA/P-Ser-HPr complex. In footprinting experiments, the region corresponding to the presumed cccA cre was specifically protected in the presence of the CcpA/P-Ser-HPr complex.
Chapter 15
Phosphotransfer Functions of Mutated Bacillus subtilis HPr-Like Protein Crh Carrying a Histidine in the Active Site
Emmanuelle Darbon, Anne Galinier, Dominique Le Coq, and Josef Deutscher
Abstract
The Bacillus subtilis protein Crh exhibits strong similarity to HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). HPr phosphorylated at His-15 can transfer its phosphoryl group to several EIIAs of the PTS for sugar transport and phosphorylation. In addition, it phosphorylates and activates transcriptional regulators containing PTS regulation domains (PRDs). In Gram-positive bacteria, it also controls the enzyme glycerol kinase. Since in Crh the active site His-15 of HPr is replaced with a glutamine, Crh was not able to carry out the catalytic and regulatory functions mediated by P~His-HPr. However, when Gln-15 of Crh was replaced with a histidine, Crh gained most of the catalytic and regulatory functions exerted by HPr. To allow CrhQ15H to efficiently phosphorylate and activate the PRD-containing antiterminator LicT, which controls the expression of the bglS gene and the bglPH operon, it was sufficient to express the crhQ15H allele under control of the spac promoter in monocopy. By contrast, to phosphorylate and activate glycerol kinase and to allow a ptsH deletion strain (devoid of HPr) to slowly grow on the non-PTS substrate glycerol and to efficiently utilize the PTS sugars glucose and mannitol, the crhQ15H allele had to be expressed from a multicopy plasmid.
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