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1.  Regulation of lactose-phosphoenolpyruvate-dependent phosphotransferase system and beta-D-phosphogalactoside galactohydrolase activities in Lactobacillus casei. 
Journal of Bacteriology  1983;154(3):1195-1203.
The lactose-phosphoenolpyruvate-dependent phosphotransferase system (lac-PTS) and beta-D-phosphogalactoside galactohydrolase (P-beta-gal) mediate the metabolism of lactose by Lactobacillus casei. Starved cells of L. casei contained a high intracellular concentration of phosphoenolpyruvate, and this endogenous energy reserve facilitated characterization of phosphotransferase system activities in physiologically intact cells. Data obtained from transport studies with whole cells and from in vitro phosphotransferase system assays with permeabilized cells revealed that the lac-PTS had a high affinity for beta-galactosides (e.g., lactose, lactulose, lactobionic acid, and arabinosyl-beta-D-galactoside). lac-PTS and P-beta-gal activities were determined in wild-type strains and strains defective in the glucose-phosphoenolpyruvate-dependent phosphotransferase system after growth on various sugars and in the presence of potential inducers. We found that (i) the lac genes (i.e., the genes coding for the lac-PTS proteins and P-beta-gal) were induced by metabolizable and non-metabolizable beta-galactosides (presumably acting as their phosphorylated derivatives), (ii) galactose 6-phosphate was not an inducer in most strains, (iii) the ratio of lac-PTS activity to P-beta-gal activity in a given strain was not constant, and (iv) inhibition of lac gene expression during growth on glucose was a consequence of glucose-phosphoenolpyruvate-dependent phosphotransferase system-mediated inducer exclusion, repressive effects of a functional glucose-phosphoenolpyruvate-dependent phosphotransferase system and glucose-derived metabolites. The expression of the lac-PTS structural genes and the expression of the P-beta-gal gene are independently regulated and may be subject to both positive control and negative control.
PMCID: PMC217591  PMID: 6406426
2.  Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium. 
Journal of Bacteriology  1980;141(2):603-610.
Inhibition of cellular adenylate cyclase activity by sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system was reliant on the activities of the protein components of this enzyme system and on a gene designated crrA. In bacterial strains containing very low enzyme I activity, inhibition could be elicited by nanomolar concentrations of sugar. An antagonistic effect between methyl alpha-glucoside and phosphoenolpyruvate was observed in permeabilized Escherichia coli cells containing normal activities of the phosphotransferase system enzymes. In contrast, phosphoenolpyruvate could not overcome the inhibitory effect of this sugar in strains deficient for enzyme I or HPr. Although the in vivo sensitivity of adenylate cyclase to inhibition correlated with sensitivity of carbohydrate permease function to inhibition in most strains studied, a few mutant strains were isolated in which sensitivity of carbohydrate uptake to inhibition was lost and sensitivity of adenylate cyclase to regulation was retained. These results are consistent with the conclusions that adenylate cyclase and the carbohydrate permeases were regulated by a common mechanism involving phosphorylation of a cellular constituent by the phosphotransferase system, but that bacterial cells possess mechanisms for selectively uncoupling carbohydrate transport from regulation.
PMCID: PMC293665  PMID: 6245052
3.  Unraveling the evolutionary history of the phosphoryl-transfer chain of the phosphoenolpyruvate:phosphotransferase system through phylogenetic analyses and genome context 
The phosphoenolpyruvate phosphotransferase system (PTS) plays a major role in sugar transport and in the regulation of essential physiological processes in many bacteria. The PTS couples solute transport to its phosphorylation at the expense of phosphoenolpyruvate (PEP) and it consists of general cytoplasmic phosphoryl transfer proteins and specific enzyme II complexes which catalyze the uptake and phosphorylation of solutes. Previous studies have suggested that the evolution of the constituents of the enzyme II complexes has been driven largely by horizontal gene transfer whereas vertical inheritance has been prevalent in the general phosphoryl transfer proteins in some bacterial groups. The aim of this work is to test this hypothesis by studying the evolution of the phosphoryl transfer proteins of the PTS.
We have analyzed the evolutionary history of the PTS phosphoryl transfer chain (PTS-ptc) components in 222 complete genomes by combining phylogenetic methods and analysis of genomic context. Phylogenetic analyses alone were not conclusive for the deepest nodes but when complemented with analyses of genomic context and functional information, the main evolutionary trends of this system could be depicted.
The PTS-ptc evolved in bacteria after the divergence of early lineages such as Aquificales, Thermotogales and Thermus/Deinococcus. The subsequent evolutionary history of the PTS-ptc varied in different bacterial lineages: vertical inheritance and lineage-specific gene losses mainly explain the current situation in Actinobacteria and Firmicutes whereas horizontal gene transfer (HGT) also played a major role in Proteobacteria. Most remarkably, we have identified a HGT event from Firmicutes or Fusobacteria to the last common ancestor of the Enterobacteriaceae, Pasteurellaceae, Shewanellaceae and Vibrionaceae. This transfer led to extensive changes in the metabolic and regulatory networks of these bacteria including the development of a novel carbon catabolite repression system. Hence, this example illustrates that HGT can drive major physiological modifications in bacteria.
PMCID: PMC2405797  PMID: 18485189
4.  Galactose transport systems in Streptococcus lactis. 
Journal of Bacteriology  1980;144(2):683-691.
Galactose-grown cells of Streptococcus lactis ML3 have the capacity to transport the growth sugar by two separate systems: (i) the phosphoenolpyruvate-dependent phosphotransferase system and (ii) an adenosine 5'-triphosphate-energized permease system. Proton-conducting uncouplers (tetrachlorosalicylanilide and carbonyl cyanide-m-chlorophenyl hydrazone) inhibited galactose uptake by the permease system, but had no effect on phosphotransferase activity. Inhibition and efflux experiments conducted using beta-galactoside analogs showed that the galactose permease had a high affinity for galactose, methyl-beta-D-thiogalactopyranoside, and methyl-beta-D-galactopyranoside, but possessed little or no affinity for glucose and lactose. The spatial configurations of hydroxyl groups at C-2, C-4, and C-6 were structurally important in facilitating interaction between the carrier and the sugar analog. Iodoacetate had no inhibitory effect on accumulation of galactose, methyl-beta-D-thiogalactopyranoside, or lactose via the phosphotransferase system. However, after exposure of the cells to p-chloromercuribenzoate, phosphoenolpyruvate-dependent uptake of lactose and methyl-beta-D-thiogalactopyranoside were reduced by 75 and 100%, respectively, whereas galactose phosphotransferase activity remained unchanged. The independent kinetic analysis of each transport system was achieved by the selective generation of the appropriate energy source (adenosine 5'-triphosphate or phosphoenolpyruvate) in vivo. The maximum rates of galactose transport by the two systems were similar, but the permease system exhibited a 10-fold greater affinity for sugar than did the phosphotransferase system.
PMCID: PMC294718  PMID: 6776094
5.  Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants. 
Journal of Bacteriology  1996;178(4):1126-1133.
The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in HPr play important roles in controlling conformational aspects of HPr that influence (i) autophosphohydrolysis, (ii) the interaction of this protein with the sugar permeases of the bacterial phosphotransferase system, and (iii) catalysis of phosphoryl transfer to the IIA domains in these permeases.
PMCID: PMC177775  PMID: 8576048
6.  Structural Studies of ROK Fructokinase YdhR from Bacillus subtilis: Insights into substrates binding and fructose specificity 
Journal of Molecular Biology  2010;406(2):325-342.
The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK) that phosphorylates d-fructose with ATP as a cofactor has been shown to be a member of this secondary system. Bioinformatics analysis has shown that FK is a member of the “ROK” (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/dfructose. All structures show that YdhR is a homo-dimer with a monomer composed of two similar α/βdomains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.
PMCID: PMC3365590  PMID: 21185308
Fructokinase; ROK family; metal dependent; ADP and d-fructose binding; reductive methylation
7.  Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants. 
Journal of Bacteriology  1977;129(3):1208-1214.
Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase. Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars. This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP. In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars. No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.
PMCID: PMC235077  PMID: 321416
8.  Regulation of glycolysis and sugar phosphotransferase activities in Streptococcus lactis: growth in the presence of 2-deoxy-D-glucose. 
Journal of Bacteriology  1983;154(2):819-830.
Streptococcus lactis K1 has the capacity to grow on many sugars, including sucrose and lactose, in the presence of high levels (greater than 500 mM) of 2-deoxy-D-glucose. Initially, growth of the organism was transiently halted by the addition of comparatively low concentrations (less than 0.5 mM) of the glucose analog to the culture. Inhibition was coincident with (i) rapid accumulation of 2-deoxy-D-glucose 6-phosphate (ca. 120 mM) and preferential utilization of phosphoenolpyruvate via the mannose:phosphotransferase system, (ii) depletion of phosphorylated glycolytic intermediates, and (iii) a 60% reduction in intracellular ATP concentration. During the 5- to 10-min period of bacteriostasis the intracellular concentration of 2-deoxy-D-glucose 6-phosphate rapidly declined, and the concentrations of glycolytic intermediates were restored to near-normal levels. When growth resumed, the cell doubling time (Td) and the steady-state levels of 2-deoxy-D-glucose 6-phosphate maintained by the cells were dependent upon the medium concentration of 2-deoxy-D-glucose. Resistance of S. lactis K1 to the potentially toxic analog was a consequence of negative regulation of the mannose:phosphotransferase system by two independent mechanisms. The first, short-term response occurred immediately after the initial "overshoot" accumulation of 2-deoxy-D-glucose 6-phosphate, and this mechanism reduced the activity (fine control) of the mannose:phosphotransferase system. The second, long-term mechanism resulted in repression of synthesis (coarse control) of enzyme IImannose. The two regulatory mechanisms reduced the rate of 2-deoxy-D-glucose translocation via the mannose:phosphotransferase system and minimized the activity of the phosphoenolpyruvate-dependent futile cycle of the glucose analog (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982). Phosphoenolpyruvate was thus conserved for transport of the growth sugar and for generation of ATP required for biosynthetic and work functions of the growing cell.
PMCID: PMC217534  PMID: 6404888
9.  Characterization of factor IIIGLc in catabolite repression-resistant (crr) mutants of Salmonella typhimurium. 
Journal of Bacteriology  1982;149(2):576-586.
crr mutants of Salmonella typhimurium are thought to be defective in the regulation of adenylate cyclase and a number of transport systems by the phosphoenolpyruvate-dependent sugar phosphotransferase system, crr mutants are also defective in the enzymatic activity of factor IIIGlc (IIIGlc), a protein component of the phosphotransferase system involved in glucose transport. Therefore, it has been proposed that IIIGlc is the primary effector of phosphotransferase system-mediated regulation of cell metabolism. We characterized crr mutants with respect to the presence and function of IIIGlc by using an immunochemical approach. All of the crr mutants tested had low (0 to 30%) levels of IIIGlc compared with wild-type cells, as determined by rocket immunoelectrophoresis. The IIIGlc isolated from one crr mutant was investigated in more detail and showed abnormal aggregation behavior, which indicated a structural change in the protein. These results supported the hypothesis that a crr mutation directly affects IIIGlc, probably by altering the structural gene of IIIGlc. Several crr strains which appeared to be devoid of IIIGlc in immunoprecipitation assays were still capable of in vitro phosphorylation and transport of methyl alpha-glucoside. This phosphorylation activity was sensitive to specific anti-IIIGlc serum. Moreover, the membranes of crr mutants, as well as those of wild-type cells, contained a protein that reacted strongly with our anti-IIIGlc serum. We propose that S. typhimurium contains a membrane-bound form of IIIGlc which may be involved in phosphotransferase system activity.
PMCID: PMC216545  PMID: 7035434
10.  Regulation of the putative bglPH operon for aryl-beta-glucoside utilization in Bacillus subtilis. 
Journal of Bacteriology  1995;177(19):5590-5597.
The expression of the putative operon bglPH of Bacillus subtilis was studied by using bglP'-lacZ transcriptional fusions. The bglP gene encodes an aryl-beta-glucoside-specific enzyme II of the phosphoenolpyruvate sugar:phosphotransferase system, whereas the bglH gene product functions as a phospho-beta-glucosidase. Expression of bglPH is regulated by at least two different mechanisms: (i) carbon catabolite repression and (ii) induction via an antitermination mechanism. Distinct deletions of the promoter region were created to determine cis-acting sites for regulation. An operatorlike structure partially overlapping the -35 box of the promoter of bglP appears to be the catabolite-responsive element of this operon. The motif is similar to that of amyO and shows no mismatches with respect to the consensus sequence established as the target of carbon catabolite repression in B. subtilis. Catabolite repression is abolished in both ccpA and ptsH1 mutants. The target of the induction by the substrate, salicin or arbutin, is a transcriptional terminator located downstream from the promoter of bglP. This structure is very similar to that of transcriptional terminators which regulate the induction of the B. subtilis sacB gene, the sacPA operon, and the Escherichia coli bgl operon. The licT gene product, a member of the BglG-SacY family of antitermination proteins, is essential for the induction process. Expression of bglP is under the negative control of its own gene product. The general proteins of the phosphoenolpyruvate-dependent phosphotransferase system are required for bglP expression. Furthermore, the region upstream from bglP, which reveals a high AT content, exerts a negative regulatory effect on bglP expression.
PMCID: PMC177369  PMID: 7559347
11.  Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system 
The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source. In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports glucose when this sugar is present at concentrations like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport, therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes. Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates.
PMCID: PMC1156936  PMID: 15904518
12.  Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation. 
Journal of Bacteriology  1980;141(2):476-484.
In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.
PMCID: PMC293650  PMID: 6988384
13.  BglF, the Escherichia coli β-Glucoside Permease and Sensor of the bgl System: Domain Requirements of the Different Catalytic Activities 
Journal of Bacteriology  1999;181(2):462-468.
The Escherichia coli BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities. In the absence of β-glucosides, it phosphorylates BglG, a positive regulator of bgl operon transcription, thus inactivating BglG. In the presence of β-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports β-glucosides into the cell and phosphorylates them. BglF is composed of two hydrophilic domains, IIAbgl and IIBbgl, and a membrane-bound domain, IICbgl, which are covalently linked in the order IIBCAbgl. Cys-24 in the IIBbgl domain is essential for all the phosphorylation and dephosphorylation activities of BglF. We have investigated the domain requirement of the different functions carried out by BglF. To this end, we cloned the individual BglF domains, as well as the domain pairs IIBCbgl and IICAbgl, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo. We show here that the IIB and IIC domains, linked to each other (IIBCbgl), are required for the sugar-driven reactions, i.e., sugar phosphotransfer and BglG activation by dephosphorylation. In contrast, phosphorylated IIBbgl alone can catalyze BglG inactivation by phosphorylation. Thus, the sugar-induced and noninduced functions have different structural requirements. Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl which occur upon the interaction of BglF with the sugar.
PMCID: PMC93399  PMID: 9882659
14.  Deletion Mapping of the Genes Coding for HPr and Enzyme I of the Phosphoenolpyruvate: Sugar Phosphotransferase System in Salmonella typhimurium 
Journal of Bacteriology  1972;112(1):17-29.
Sugars transported by a bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) require two soluble proteins: HPr, a low-molecular-weight phosphate-carrier protein, and enzyme I. The structural genes coding for HPr (ptsH) and Enzyme I (ptsI) are shown to be cotransducible in Salmonella typhimurium. The gene order of this region of the Salmonella chromosome is cysA-trzA-ptsH-ptsI...(crr). A method for the isolation of trzA-pts deletion is described. One class of pts deletions extends through ptsH and into ptsI; a second class includes both ptsH and ptsI and extends into or through the crr gene. The crr gene either codes for or regulates the synthesis of a third PTS protein (factor III) which is sugar-specific. A hypothesis is presented for a mechanism of deletion formation.
PMCID: PMC251376  PMID: 4562394
15.  Initial Characterization of Hexose and Hexitol Phosphoenolpyruvate-Dependent Phosphotransferases of Staphylococcus aureus 
Journal of Bacteriology  1977;130(3):991-999.
The phosphoenolpyruvate sugar phosphotransferases of Staphylococcus aureus were surveyed biochemically to determine substrate range, inducibility and constitutivity, and requirements for soluble sugar-specific proteins. The substrate range is similar to that of the phosphotransferases of enteric bacteria, but the staphylococcal mannose and sorbitol systems are very inefficient. In addition, S. qureus has phosphotransferase activities for lactose and sucrose. The systems tested fell into two broad classes. Sugars for which there was substantial constitutive activity (fructose, mannose, sucrose, and glucose and its nonmetabolized analogues) did not require sugar-specific soluble factors for phosphorylation. Only in the case of fructose did growth in the presence of these constitutive sugars induce the corresponding phosphotransferase activity to higher levels. Kinetic experiments with each of these constitutive sugars yielded biphasic Hofstee plots; i.e., the kinetics were not characteristic of single enzymes. Preliminary experiments suggest that sucrose phosphorylation may involve the glucose and/or fructose systems. Truly inducible sugar phosphotransferase systems represent a second class; those for lactose and mannitol are the only members thus far identified. These systems are absent from uninduced cells, require soluble sugar-specific factors, and exhibit linear Hofstee plots. Sorbitol is apparently transported very poorly by intact cells but is an inducer of the mannitol system; it is phosphorylated efficiently in vitro by extracts of cells grown on either hexitol, but is taken up by intact cells at 0.1% of the mannitol rate.
PMCID: PMC235319  PMID: 863862
16.  Genetic Dissection of Specificity Determinants in the Interaction of HPr with Enzymes II of the Bacterial Phosphoenolpyruvate:Sugar Phosphotransferase System in Escherichia coli▿  
Journal of Bacteriology  2007;189(13):4603-4613.
The histidine protein (HPr) is the energy-coupling protein of the phosphoenolpyruvate (PEP)-dependent carbohydrate:phosphotransferase system (PTS), which catalyzes sugar transport in many bacteria. In its functions, HPr interacts with a number of evolutionarily unrelated proteins. Mainly, it delivers phosphoryl groups from enzyme I (EI) to the sugar-specific transporters (EIIs). HPr proteins of different bacteria exhibit almost identical structures, and, where known, they use similar surfaces to interact with their target proteins. Here we studied the in vivo effects of the replacement of HPr and EI of Escherichia coli with the homologous proteins from Bacillus subtilis, a gram-positive bacterium. This replacement resulted in severe growth defects on PTS sugars, suggesting that HPr of B. subtilis cannot efficiently phosphorylate the EIIs of E. coli. In contrast, activation of the E. coli BglG regulatory protein by HPr-catalyzed phosphorylation works well with the B. subtilis HPr protein. Random mutations were introduced into B. subtilis HPr, and a screen for improved growth on PTS sugars yielded amino acid changes in positions 12, 16, 17, 20, 24, 27, 47, and 51, located in the interaction surface of HPr. Most of the changes restore intermolecular hydrophobic interactions and salt bridges normally formed by the corresponding residues in E. coli HPr. The residues present at the targeted positions differ between HPrs of gram-positive and -negative bacteria, but within each group they are highly conserved. Therefore, they may constitute a signature motif that determines the specificity of HPr for either gram-negative or -positive EIIs.
PMCID: PMC1913440  PMID: 17449611
17.  Novel phosphoenolpyruvate-dependent futile cycle in Streptococcus lactis: 2-deoxy-D-glucose uncouples energy production from growth. 
Journal of Bacteriology  1982;151(3):1454-1465.
The addition of 2-deoxy-D-glucose to cultures of Streptococcus lactis 133 that were growing exponentially on sucrose or lactose reduced the growth rate by ca. 95%. Inhibition did not occur with glucose or mannose as the growth sugar. The reduction in growth rate was concomitant with rapid accumulation of the analog in phosphorylated form (2-deoxy-D-glucose 6-phosphate) via the phosphoenolpyruvate-dependent mannose:phosphotransferase system. Within 5 min the intracellular 2-deoxy-D-glucose 6-phosphate concentration reached a steady-state level of greater than 100 mM. After maximum accumulation of the sugar phosphate, the rate of sucrose metabolism (glycolysis) decreased by only 30%, but the cells were depleted of fructose-1,6-diphosphate. The addition of glucose to 2-deoxy-D-glucose 6-phosphate preloaded cells caused expulsion of 2-deoxy-D-glucose and a resumption of normal growth. S. lactis 133 contained an intracellular Mg2+-dependent, fluoride-sensitive phosphatase which hydrolyzed 2-deoxy-D-glucose 6-phosphate (and glucose 6-phosphate) to free sugar and inorganic phosphate. Because of continued dephosphorylation and efflux of the non-metabolizable analog, the maintenance of the intracellular 2-deoxy-D-glucose 6-phosphate pool during growth stasis was dependent upon continued glycolysis. This steady-state condition represented a dynamic equilibrium of: (i) phosphoenolpyruvate-dependent accumulation of 2-deoxy-D-glucose 6-phosphate, (ii) intracellular dephosphorylation, and (iii) efflux of free 2-deoxy-D-glucose. This sequence of events constitutes a futile cycle which promotes the dissipation of phosphoenolpyruvate. We conclude that 2-deoxy-D-glucose functions as an uncoupler by dissociating energy production from growth in S. lactis 133.
PMCID: PMC220427  PMID: 6286601
18.  The Phosphoenolpyruvate Phosphotransferase System Regulates Vibrio cholerae Biofilm Formation through Multiple Independent Pathways▿  
Journal of Bacteriology  2010;192(12):3055-3067.
The bacterial phosphoenolpyruvate phosphotransferase system (PTS) is a highly conserved phosphotransfer cascade that participates in the transport and phosphorylation of selected carbohydrates and modulates many cellular functions in response to carbohydrate availability. It plays a role in the virulence of many bacterial pathogens. Components of the carbohydrate-specific PTS include the general cytoplasmic components enzyme I (EI) and histidine protein (HPr), the sugar-specific cytoplasmic components enzymes IIA (EIIA) and IIB (EIIB), and the sugar-specific membrane-associated multisubunit components enzymes IIC (EIIC) and IID (EIID). Many bacterial genomes also encode a parallel PTS pathway that includes the EI homolog EINtr, the HPr homolog NPr, and the EIIA homolog EIIANtr. This pathway is thought to be nitrogen specific because of the proximity of the genes encoding this pathway to the genes encoding the nitrogen-specific σ factor σ54. We previously reported that phosphorylation of HPr and FPr by EI represses Vibrio cholerae biofilm formation in minimal medium supplemented with glucose or pyruvate. Here we report two additional PTS-based biofilm regulatory pathways that are active in LB broth but not in minimal medium. These pathways involve the glucose-specific enzyme EIIA (EIIAGlc) and two nitrogen-specific EIIA homologs, EIIANtr1 and EIIANtr2. The presence of multiple, independent biofilm regulatory circuits in the PTS supports the hypothesis that the PTS and PTS-dependent substrates have a central role in sensing environments suitable for a surface-associated existence.
PMCID: PMC2901703  PMID: 20400550
19.  Comparative Genomic Analyses of the Bacterial Phosphotransferase System 
We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer.
PMCID: PMC1306802  PMID: 16339738
20.  Widespread N-Acetyl-d-Glucosamine Uptake among Pelagic Marine Bacteria and Its Ecological Implications 
Applied and Environmental Microbiology  2002;68(11):5554-5562.
Dissolved free and combined N-acetyl-d-glucosamine (NAG) is among the largest pools of amino sugars in the ocean. NAG is a main structural component in chitin and a substantial constituent of bacterial peptidoglycan and lipopolysaccharides. We studied the distribution and kinetics of NAG uptake by the phosphoenolpyruvate:NAG phosphotransferase systems (PTS) in marine bacterial isolates and natural bacterial assemblages in near-shore waters. Of 78 bacterial isolates examined, 60 took up 3H-NAG, while 18 showed no uptake. No systematic pattern in NAG uptake capability relative to phylogenetic affiliation was found, except that all isolates within Vibrionaceae took up NAG. Among 12 isolates, some showed large differences in the relationship between polymer hydrolysis (measured as chitobiase activity) and uptake of the NAG, the hydrolysis product. Pool turnover time and estimated maximum ambient concentration of dissolved NAG in samples off Scripps Pier (La Jolla, Calif.) were 5.9 ± 3.0 days (n = 10) and 5.2 ± 0.9 nM (n = 3), respectively. Carbohydrate competition experiments indicated that glucose, glucosamine, mannose, and fructose were taken up by the same system as NAG. Sensitivity to the antibiotic and NAG structural analog streptozotocin (STZ) was developed into a culture-independent approach, which demonstrated that approximately one-third of bacteria in natural marine assemblages that were synthesizing DNA took up NAG. Isolates possessing a NAG PTS system were found to be predominantly facultative anaerobes. These results suggest the hypothesis that a substantial fraction of bacteria in natural pelagic assemblages are facultative anaerobes. The adaptive value of fermentative metabolism in the pelagic environment is potentially significant, e.g., to bacteria colonizing microenvironments such as marine snow that may experience periodic O2-limitation.
PMCID: PMC129920  PMID: 12406749
21.  Regulation of carbohydrate transport activities in Salmonella typhimurium: use of the phosphoglycerate transport system to energize solute uptake. 
Journal of Bacteriology  1980;141(2):611-617.
The phosphoglycerate transport system was employed to supply energy-depleted, lysozyme-treated Salmonella typhimurium cells with a continuous intracellular source of phosphoenolpyruvate. When the cells had been induced to high levels of the phosphoglycerate transport system, a low extracellular concentration of phosphoenolpyruvate (0.1 mM) half maximally stimulated uptake of methyl alpha-glucoside via the phosphoenolpyruvate:sugar phosphotransferase system. If the phosphoglycerate transport system was not induced before energy depletion, 100 times this concentration of phosphoenolpyruvate was required for half-maximal stimulation. Phosphoenolpyruvate could not be replaced by other energy sources if potassium fluoride (an inhibitor of enolase) was present. Inhibition of [14C]-glycerol uptake into energy-depleted cells by methyl alpha-glucoside was demonstrated. A concentration of phosphoenolpyruvate which stimulated methyl alpha-glucoside accumulation counteracted the inhibitory effect of the glucoside. In the presence of potassium fluoride, phosphoenolpyruvate could not be replaced by other energy sources. Inhibition of glycerol uptake by methyl alpha-glucoside in intact untreated cells was also counteracted by phosphoenolpyruvate, but several energy sources were equally effective; potassium fluoride was without effect. These and other results were interpreted in terms of a mechanism in which the relative proportions of the phosphorylated and nonphosphorylated forms of a cell constituent influence the activity of the glycerol transport system.
PMCID: PMC293666  PMID: 6988388
22.  Comparative Signature-Tagged Mutagenesis Identifies Pseudomonas Factors Conferring Resistance to the Pulmonary Collectin SP-A 
PLoS Pathogens  2005;1(3):e31.
The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A+/+) and SP-A-deficient (SP-A−/−) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A.
Everyday, normal breathing deposits numerous microorganisms on the surfactant membrane that lines the air-exchanging surfaces of the lung. Surfactant protein SP-A, a component of the surfactant membrane, helps to maintain the lung in a germ-free state by aggregating inhaled microorganisms and facilitating their ingestion by immune cells, and by increasing the permeability of their cell membranes. However, the bacterial pathogen Pseudomonas aeruginosa is resistant to SP-A-mediated membrane disruption.
Using a genetic tool called comparative signature-tagged mutagenesis, the authors have identified two P. aeruginosa genes, pch and ptsP, that are required to resist SP-A-mediated membrane permeabilization. Molecular analyses indicate that the pch gene encodes an enzyme that synthesizes salicylate, a compound utilized by bacteria to acquire essential metal ions. The ptsP gene encodes an enzyme called phosphoenolpyruvate-protein-phosphotransferase. The loss of salicylate and phosphoenolpyruvate-protein-phosphotransferase weakens the P. aeruginosa cell membrane, which allows SP-A to poke holes on the membrane and kill the bacteria.
This is the first known report of the roles played by salicylate and phosphoenolpyruvate-protein-phosphotransferase in maintenance of bacterial membrane, and consequently, protecting bacteria from killing by SP-A, through disruption of membrane integrity.
PMCID: PMC1291351  PMID: 16322768
23.  Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium. 
Journal of Bacteriology  1987;169(6):2810-2818.
Histidine-containing phosphocarrier protein (HPr) is common to all of the phosphoenolpyruvate:sugar phosphotransferase systems (PTS) in Escherichia coli and Salmonella typhimurium, except the fructose-specific PTS. Strains which lack HPr activity (ptsH) have been characterized in the past, and it has proved difficult to delineate between tight and leaky mutants. In this study four different parameters of ptsH strains were measured: in vitro sugar phosphorylation activity of the mutant HPr; detection of 32P-labeled P-HPr; ability of monoclonal antibodies to bind mutant HPr; and sensitivity of ptsH strains to fosfomycin. Tight ptsH strains could be defined; they were fosfomycin resistant and produced no HPr protein or completely inactive mutant HPr. All leaky ptsH strains were fosfomycin sensitive, usually produced normal amounts of mutant HPr protein, and had low but measurable activity, and HPr was detectable as a phosphoprotein. This indicates that the regulatory functions of the PTS require a very low level of HPr activity (about 1%). The antibodies used to detect mutant HPr in crude extracts were two monoclonal immunoglobulin G antibodies Jel42 and Jel44. Both antibodies, which have different pIs, inhibited PTS sugar phosphorylation assays, but the antibody-HPr complex could still be phosphorylated by enzyme I. Preliminary evidence suggests that the antibodies bind to two different epitopes which are in part located in a beta-sheet structure.
PMCID: PMC212188  PMID: 2438269
24.  The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions 
The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.
PMCID: PMC4054256  PMID: 24847021
25.  Enzyme III stimulation of cyclic AMP synthesis in an Escherichia coli crp mutant. 
Journal of Bacteriology  1984;157(3):940-941.
Cyclic AMP (cAMP) synthesis in Escherichia coli is altered in cAMP receptor protein mutants and in phosphoenolpyruvate:sugar phosphotransferase transport system mutants. The stimulation of cAMP synthesis observed in cAMP receptor protein-deficient mutants is largely dependent upon enzyme III of the phosphoenolpyruvate:sugar phosphotransferase transport system. The phosphoenolpyruvate:sugar phosphotransferase transport system enzyme I is not required for elevated cAMP synthesis. These results suggest that enzyme III plays an important role in regulating adenylate cyclase activity.
PMCID: PMC215351  PMID: 6321447

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