Expulsion of preaccumulated methyl-beta-D-thiogalactoside-phosphate (TMG-P) from Streptococcus pyogenes is a two-step process comprising intracellular dephosphorylation of TMG-P followed by rapid efflux of the intracellularly formed free galactoside (J. Reizer, M.J. Novotny, C. Panos, and M.H. Saier, Jr., J. Bacteriol. 156:354-361, 1983). The present study identifies the mechanism and the order and characterizes the temperature dependency of the efflux step. Unidirectional efflux of the intracellularly formed [14C]TMG was only slightly affected when measured in the presence of unlabeled TMG (25 to 400 mM) in the extracellular medium. In contrast, pronounced inhibition of net efflux was observed in the presence of relatively low concentrations (1 to 16 mM) of extracellular [14C]TMG. Since net efflux was nearly arrested when the external concentration of [14C]TMG approached the intracellular concentration of this sugar, we propose that a facilitated diffusion mechanism is responsible for efflux and equilibration of TMG between the intracellular and extracellular milieus. The exit reaction was markedly dependent upon temperature, exhibited a high energy of activation (23 kcal [ca. 96 kJ] per mol), and followed first-order kinetics, indicating that the permease mediating this efflux was not saturated under the conditions of expulsion employed.
The gut operon was subcloned into various plasmid vectors (M. Yamada and M. H. Saier, Jr., J. Bacteriol. 169:2990-2994, 1987). Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth. Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds. On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism. Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene. A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity. Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase. These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.
The mechanism by which enzyme IIIglc of the bacterial phosphotransferase system regulates the activity of crystalline glycerol kinase from Escherichia coli has been studied, and the inhibitory effects have been compared with those produced by fructose-1,6-diphosphate. It was shown that the free, but not the phosphorylated, form of enzyme IIIglc inhibits the kinase. Mutants of Salmonella typhimurium were isolated which were resistant to inhibition by either enzyme IIIglc (glpKr mutants) or fructose-1,6-diphosphate (glpKi mutants), and each mutant type was shown to retain full sensitivity to inhibition by the other regulatory agent. Other mutants were fully or partially resistant to regulation by both agents. The two regulatory sites on the kinase are evidently distinct but must overlap or interact functionally. Kinetic analyses have revealed several mechanistic features of the regulatory interactions. (i) Inhibition by both allosteric regulatory agents is strongly pH dependent, with maximal inhibition occurring at ca. pH 6.5 under the assay conditions employed. (ii) Binding of enzyme IIIglc to glycerol kinase is also pH dependent, the Ki being near 4 microM at pH 6.0 but near 10 microM at pH 7.0. (iii) Whereas fructose-1,6-diphosphate inhibition apparently requires that the enzyme exist in a tetrameric state, both the dimer and the tetramer appear to be fully sensitive to enzyme IIIglc inhibition. (iv) Inhibition by enzyme IIIglc (like that by fructose-1,6-diphosphate) is noncompetitive with respect to both substrates. (v) The inhibitory responses of glycerol kinase to fructose-1, 6-diphosphate and enzyme IIIglc show features characteristic of positive cooperativity at low inhibitor concentration. (vi) Neither agent inhibits completely at high inhibitor concentration. (vii) Apparent negative cooperativity with respect to ATP binding is observed with purified E. coli glycerol kinase, with glycerol kinase in crude extracts of wild-type S. typhimurium cells, and with glpKr and glpKi mutant forms of glycerol kinase from S. typhimurium. These results serve to characterize the regulatory interactions which control the activity of glycerol kinase by fructose-1,6-diphosphate and by enzyme IIIglc of the phosphotransferase system.
This article examines in a broad perspective entropy and some examples of its relationship to evolution, genetic instructions and how we view diseases. Many knowledge gaps abound, hence our understanding is still fragmented and incomplete. Living organisms are programmed by functional genetic instructions (FGI), through cellular communication pathways, to grow and reproduce by maintaining a variety of hemistable, ordered structures (low entropy). Living organisms are far from equilibrium with their surrounding environmental systems, which tends towards increasing disorder (increasing entropy). Organisms must free themselves from high entropy (high disorder) to maintain their cellular structures for a period of time sufficient enough to allow reproduction and the resultant offspring to reach reproductive ages. This time interval varies for different species. Bacteria, for example need no sexual parents; dividing cells are nearly identical to the previous generation of cells, and can begin a new cell cycle without delay under appropriate conditions. By contrast, human infants require years of care before they can reproduce. Living organisms maintain order in spite of their changing surrounding environment, that decreases order according to the second law of thermodynamics. These events actually work together since living organisms create ordered biological structures by increasing local entropy. From a disease perspective, viruses and other disease agents interrupt the normal functioning of cells. The pressure for survival may result in mechanisms that allow organisms to resist attacks by viruses, other pathogens, destructive chemicals and physical agents such as radiation. However, when the attack is successful, the organism can be damaged until the cell, tissue, organ or entire organism is no longer functional and entropy increases.
diseases; entropy; evolution; genetic instructions; laws of biology; microorganisms
D-Mannitol-1-phosphate dehydrogenase (EC 188.8.131.52) and D-glucitol-6-phosphate dehydrogenase (EC 184.108.40.206) were purified to apparent homogeneity in good yields from Escherichia coli. The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined. Both enzymes showed absolute specificities for their substrates. The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively. It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer. These two proteins differed in several fundamental respects.
In enteric bacteria, chromosomally encoded permeases specific for lactose, maltose, and melibiose are allosterically regulated by the glucose-specific enzyme IIA of the phosphotransferase system. We here demonstrate that the plasmid-encoded raffinose permease of enteric bacteria is similarly subject to this type of inhibition.
Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a fructose-specific enzyme II complex which generates fructose-1-phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-phosphate kinase then converts the product of the PTS reaction to fructose-1,6-bisphosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a fructose-1,6-bisphosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus.
A genetic locus designated fruR, previously mapped to min 3 on the Salmonella typhimurium chromosome, gave rise to constitutive expression of the fructose (fru) regulon and pleiotropically prevented growth on all Krebs cycle intermediates. Regulatory effects of fruR were independent of cyclic AMP and its receptor protein and did not prevent uptake of Krebs cycle intermediates. Instead, the phosphotransferase system appeared to regulate gluconeogenesis by controlling the activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.
Positive selection procedures were developed for the isolation of mutants defective in components of the glucitol-specific catabolic enzyme system in Salmonella typhimurium. gutA (enzyme IIgut-negative), gutB (enzyme IIIgut-negative), and gutC (constitutive for the glucitol operon) mutants were isolated and characterized biochemically and genetically. The gene order was shown to be gutCAB.
Mutants expressing a novel enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, termed enzyme I, were isolated from strains of Salmonella typhimurium which were deleted for the HPr and enzyme I structural genes. The mutations lay in a newly defined gene, termed ptsJ, which mapped on the S. typhimurium chromosome between the ptsHI operon and the cysA gene.
Approximately 60 mutants of Salmonella typhimurium were isolated which exhibited altered levels of the activities of the mannitol enzyme II. The mutants were grouped into six distinct categories based on their mannitol fermentation, transport, chemotaxis, and phosphorylation activities.
Vectorial transphosphorylation of hexitols, catalyzed by enzymes II of the bacterial phosphotransferase system, was studied in intact cells and membrane vesicles of Escherichia coli. In strains depleted of phosphoenolpyruvate and unable to metabolize the internal hexitol phosphate, internal mannitol-1-phosphate stimulated uptake of extracellular [14C]mannitol, whereas external mannitol stimulated release of [14C]mannitol from the intracellular [14C]mannitol-1-phosphate pool. The stoichiometry of mannitol uptake to mannitol release was 1:1. Glucitol did not promote release of [14C]mannitol from the mannitol phosphate pool but stimulated release of [14C]glucitol from internal glucitol phosphate pools when the glucitol enzyme II was induced to high levels. In E coli cells and membrane vesicles, both vectorial and nonvectorial transphosphorylation reactions of hexitols and hexoses were demonstrated. The nonvectorial reactions, but not the vectorial reactions, catalyzed by the mannitol and glucose enzymes II, were inhibited by p-chloromercuriphenyl sulfonate, a membrane-impermeable sulfhydryl reagent which inactivates enzymes II. Similarly, glucose-6-sulfate, an inhibitor of the glucose enzyme II-catalyzed transphosphorylation reaction, specifically inhibited the nonvectorial reaction. This compound was shown to be a noncompetitive inhibitor of methyl alpha-glucoside phosphorylation employing phospho-HPr as the phosphate donor. It apparently exerts its inhibitory effect by exclusive binding to the sugar phosphate binding site on the enzyme II complex. The results are consistent with the conclusion that enzymes II can exist in two distinct dispositions in the membrane, one of which catalyzes vectorial transphosphorylation, and the other catalyzes nonvectorial transphosphorylation.
Ancalomicrobium adetum possesses a membrane-associated phosphoenolpyruvate:sugar phosphotransferase system, the components of which exhibited enzymatic cross-reactivity with those from Salmonella typhimurium.
Enzyme I of the bacterial phosphotransferase system catalyzes transfer of the phosphoryl moiety from phosphoenolpyruvate to both of the heat-stable phosphoryl carrier proteins of the phosphotransferase system, HPr and FPr. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-pressure liquid chromatography, we demonstrated the existence of covalently cross-linked enzyme I dimers and trimers. Enzyme I exchange assays and phosphorylation experiments with [32P]phosphoenolpyruvate showed that covalent dimers and trimers are catalytically active. Inhibitors of the enzyme I-catalyzed phosphoenolpyruvate-pyruvate exchange block the phosphorylation of enzyme I dimers and trimers. Inhibition of the activity of enzyme I by N-ethylmaleimide, but not that by p-chloromercuriphenylsulfonate, could be overcome by high concentrations of enzyme, suggesting that N-ethylmaleimide modification changes the associative properties of enzyme I. We present evidence for two distinct classes of sulfhydryl groups in enzyme I.
A total of 1,911 proteins with N-terminal methionyl residues were computer screened for potential N-terminal alpha-helices with strong amphipathic character. By the criteria of D. Eisenberg (Annu. Rev. Biochem. 53:595-623, 1984), only 3.5% of nonplastid, nonviral proteins exhibited potential N-terminal alpha-helices, 18 residues in length, with hydrophobic moment values per amino acyl residue ([muH]) in excess of 0.4. By contrast, 10% of viral proteins exhibited corresponding [muH] values in excess of 0.4. Of these viral proteins with known functions, 55% were found to interact functionally with nucleic acids, 30% were membrane-interacting proteins or their precursors, and 15% were structural proteins, primarily concerned with host cell interactions. These observations suggest that N-terminal amphipathic alpha-helices of viral proteins may (i) function in nucleic acid binding, (ii) facilitate membrane insertion, and (iii) promote host cell interactions. Analyses of potential amphipathic N-terminal alpha-helices of cellular proteins are also reported, and their significance to organellar or envelope targeting is discussed.
Enteric bacteria have been previously shown to regulate the uptake of certain carbohydrates (lactose, maltose, and glycerol) by an allosteric mechanism involving the catalytic activities of the phosphoenolpyruvate-sugar phosphotransferase system. In the present studies, a ptsI mutant of Bacillus subtilis, possessing a thermosensitive enzyme I of the phosphotransferase system, was used to gain evidence for a similar regulatory mechanism in a gram-positive bacterium. Thermoinactivation of enzyme I resulted in the loss of methyl alpha-glucoside uptake activity and enhanced sensitivity of glycerol uptake to inhibition by sugar substrates of the phosphotransferase system. The concentration of the inhibiting sugar which half maximally blocked glycerol uptake was directly related to residual enzyme I activity. Each sugar substrate of the phosphotransferase system inhibited glycerol uptake provided that the enzyme II specific for that sugar was induced to a sufficiently high level. The results support the conclusion that the phosphotransferase system regulates glycerol uptake in B. subtilis and perhaps in other gram-positive bacteria.
Hydrolysis of sugar phosphates by crude and purified preparations of periplasmic hexose phosphatase from Salmonella typhimurium followed Michaelis-Menten kinetics. The enzyme bound glucose 1-phosphate with high affinity (Km = 10 microM) but bound glucose 6-phosphate with low affinity (Km = 2,000 microM). The order of substrate affinities was glucose 1-phosphate greater than mannose 1-phosphate = galactose 1-phosphate greater than fructose 1-phosphate greater than glucose 6-phosphate. These results and others suggest that the physiological function of the enzyme is the periplasmic hydrolysis of hexose 1-phosphates.
Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl beta-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly effluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylase serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of Bacillus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.
DNA sequence and expressional analyses of the gcd gene of Escherichia coli K-12 W3110 revealed that two promoters that were detected were regulated negatively by cyclic AMP and positively by oxygen. Sequence conservation of the gcd gene between E. coli K-12 W3110 and PPA42 suggests that glucose dehydrogenase is required for the E. coli cells, even though it ordinarily exists as an apoprotein.
Streptococcus pyogenes accumulated thiomethyl-beta-galactoside as the 6-phosphate ester due to the action of the phosphoenolpyruvate:lactose phosphotransferase system. Subsequent addition of glucose resulted in rapid efflux of the free galactoside after intracellular dephosphorylation (inducer expulsion). Efflux was shown to occur in the apparent absence of the galactose permease, but was inhibited by substrate analogs of the lactose enzyme II and could not be demonstrated in a mutant of S. lactis ML3 which lacked this enzyme. The results suggest that the enzymes II of the phosphotransferase system can catalyze the rapid efflux of free sugar under appropriate physiological conditions.