Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.     

Genome-scale model for Clostridium acetobutylicum: Part I. Metabolic network resolution and analysis

A genome-scale metabolic network reconstruction for Clostridium acetobutylicum (ATCC 824) was carried out using a new semi-automated reverse engineering algorithm. The network consists of 422 intracellular metabolites involved in 552 reactions and includes 80 membrane transport reactions. The metabolic network illustrates the reliance of clostridia on the urea cycle, intracellular L-glutamate solute pools, and the acetylornithine transaminase for amino acid biosynthesis from the 2-oxoglutarate precursor. The semi-automated reverse engineering algorithm identified discrepancies in reaction network databases that are major obstacles for fully automated network-building algorithms. The proposed semi-automated approach allowed for the conservation of unique clostridial metabolic pathways, such as an incomplete TCA cycle. A thermodynamic analysis was used to determine the physiological conditions under which proposed pathways (e.g., reverse partial TCA cycle and reverse arginine biosynthesis pathway) are feasible. The reconstructed metabolic network was used to create a genome-scale model that correctly characterized the butyrate kinase knock-out and the asolventogenic M5 pSOL1 megaplasmid degenerate strains. Systematic gene knock-out simulations were performed to identify a set of genes encoding clostridial enzymes essential for growth in silico.     

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum.The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H₂O → 1 butyrate^? + 2 HCO₃^? + 3 H⁺ + 2 H₂ (1) Glucose + 4 H₂O → 2 acetate^? + 2 HCO₃^? + 4 H⁺ + 4 H₂ (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases.The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations.The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP⁺.     

Molecular biological detection and characterization of Clostridium populations in municipal landfill sites

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.     

The phosphoenolpyruvate-dependent carbohydrate: Phosphotransferase system enzymes II as chemoreceptors in chemotaxis of Escherichia coli K12

Summary In Escherichia coli K12, eight substrate-specific, membrane-bound enzymes II of the PEP-dependent carbohydrate: phosphotransferase system (PTS), specific for hexoses, hexosamines and hexitols, have been characterised in a series of isogenic and constitutive strains. In such mutants, lacking all but one enzyme II, the transport and vectorial phosphorylation activities as well as the chemotactical response in capillary tube assays have been compared. According to the data obtained, all enzymes II not only are directly involved in the transport and vectorial phosphorylation of their substrates, but they have also a primary role as the chemoreceptors for these substrates: (1) Metabolism of the attractant beyond the phosphorylation step is not a pre-requisite to eliciting positive chemotaxis. (2) Mutants, having only one enzyme II react in the capillary tube assay only to substrates of this enzyme II, but not to substrates of the missing enzymes II. This holds for enzymes II consisting of one membrane-bound protein as well as for systems containing a soluble factor III (FIII). (3) The substrate specificities or affinities, whether tested by transport and chemotaxis assays in vivo or by phosphorylation tests in vitro, are in correpondence. (4) The activities of enzymes II, regulated in a complex way at the level of enzyme synthesis and activity and tested as above, are also in agreement. (5) Mutants lacking the soluble proteins enzyme I or HPr of the PTS no longer respond chemotactically to any substrate taken up and phosphorylated by enzymes II. It is concluded that in PTS enzymes II some functions required for transport and chemotaxis are identical. It is suggested furthermore, that the alternation of intrinsic membrane-bound proteins between a phosphorylated and a dephosphorylated state, rather than binding of the substrate to the enzyme II, is the decisive stimulus in the chemotaxis toward carbohydrates taken up by these transport systems.     

Molecular Biological Detection and Characterization of Clostridium Populations in Municipal Landfill Sites

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.     

The mechanisms of carbon catabolite repression in bacteria

Carbon catabolite repression (CCR) is the paradigm of cellular regulation. CCR happens when bacteria are exposed to two or more carbon sources and one of them is preferentially utilised (frequently glucose). CCR is often mediated by several mechanisms, which can either affect the synthesis of catabolic enzymes via global or specific regulators or inhibit the uptake of a carbon source and thus the formation of the corresponding inducer. The major CCR mechanisms operative in Enterobacteriaceae and Firmicutes are quite different, but in both types of organisms components of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and protein phosphorylation play a major role. PTS-independent CCR mechanisms are operative in several other bacteria.     

Clostridial strain degeneration

Abstract: Strain degeneration, the loss of the capacity to produce solvents and form spores, typically occurs when Clostridium acetobutylicum and related clostridia are repeatedly subcultured in batch culture or grown in continuous culture, as opposed to being grown from germinated, heat-treated spores. Several mechanisms for degeneration have been identified thus far. (i) Degeneration can be caused by excessive acidification of the culture during exponential growth. We present data interpreted to mean that C. beijerinckii (formerly C. acetobutylicum ) NCIMB 8052 cells ferment glucose to acetic and butyric acids at an uncontrolled rate, so that, during rapid growth, the rate of acid production can exceed the rate of induction of the solventogenic pathway enzymes. As a result, the medium pH drops to bactericical levels, and the cells cannot switch to solventogenesis and sporulation. The clostridia seem to be poised either to produce excess acids, or to initiate solventogenesis, depending on small differences in the rates of growth. (ii) We have isolated transposon-insertion mutants of C. beijerinckii NCIMB 8052 that are resistant to degeneration, suggesting the involvement of a regulatory region of the clostridial chromosome. (iii) Involvement of a global regulatory gene has been inferred in C. beijerinckii NCIMB 8052 which degenerates irreversibly in chemostat culture. (iv) Impairment of butanol formation due to a defect in NADH generation has been reported in an oligosporogenous strain which can revert to the non-degenerate phenotype. (v) In continuous culture, degenerate cells may be selected because they continue to divide, while the non-degenerate cells stop dividing and start differentiating.     

Metabolic regulation in solventogenic clostridia: regulators,mechanisms and engineering

Solventogenic clostridia, a group of important industrial microorganisms, have exceptional substrate and product diversity, capable of producing a series of two-carbon and even long-chain chemicals and fuels by using various substrates, including sugars, cellulose and hemicellulose, and C1 gases. For the sake of in-depth understanding and engineering these anaerobic microorganisms for broader applications, studies on metabolic regulation of solventogenic clostridia had been extensively carried out during the past ten years, based on the rapid development of various genetic tools. To date, a number of regulators that are essential for cell physiological and metabolic processes have been identified in clostridia, and the relevant mechanisms have also been dissected, providing a wealth of valuable information for metabolic engineering. Here, we reviewed the latest research progresses on the metabolic regulation for chemical production and substrate utilization in solventogenic clostridia, by focusing on three typical Clostridium species, the saccharolytic C. acetobutylicum and C. beijerinckii, as well as the gas-fermenting C. ljungdahlii. On this basis, future directions in the study and remodeling of clostridial regulation systems, were proposed for effective modification of these industrially important anaerobes.     

Metabolism of adenylylated nucleotides in Clostridium acetobutylicum.

In response to the stresses imposed by temperature upshift or addition of butanol, Clostridium acetobutylicum cultures accumulated diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A) and adenosine 5'-P1,P4-tetraphospho-5'-guanosine (Ap4G) to high levels. The two adenylylated nucleotides were also accumulated in batch culture in the absence of imposed stresses when the clostridia switched from the acidogenic phase of growth to the solventogenic phase. Most of the adenylylated nucleotides were extracellular. The intracellular concentrations of these compounds were low throughout batch growth and in cells stressed by added butanol. In contrast to other procaryotes, these clostridia did not possess enzymes to degrade the dinucleotides, as shown with both intact cells and cell-free preparations. Our findings are consistent with the hypothesis that endogenously produced solvents are stressful to the cells, stimulating the synthesis of adenylylated nucleotides. The nucleotides accumulate extracellularly because they cannot be degraded and because the cell membranes are permeabilized by the solvents produced.     

Gaseous CO2 signal initiates growth of butyric-acid-producing Clostridium butyricum in both pure culture and mixed cultures with Lactobacillus brevis

Microbial strains produce numerous volatile substances in the anaerobic conditions of the human intestines. The availability of CO(2) is known to be a prerequisite for bacterial growth in general. In experiments with anaerobic Lactobacillus brevis and Clostridium butyricum bacteria in the Portable Microbial Enrichment Unit (PMEU) it was shown that these strains interact; this interaction being mediated by CO(2) emission. CO(2) promoted clostridial growth in pure cultures and mixed cultures with lactobacilli. The growth of C. butyricum in pure cultures was much delayed or did not start at all without CO(2) from outside. Conversely, the onset of growth was provoked by a short (15 min) CO(2) burst. In mixed cultures the presence of lactobacilli in equal numbers speeded up the onset of clostridial growth by 10 h. If C. butyricum cultures designated as PMEU 1, 2, and 3 in cultivation syringes were chained by connecting the gas flow thereby allowing the volatiles of the preceding syringe culture to bubble to the next one, the growth started in 20, 10, or 6 h, respectively. This effect of gaseous emissions from other cultures speeding up the bacterial growth initiation was abolished if the gas was passed through sodium hydroxide to remove the CO(2). The positive contribution of lactobacilli to the growth of butyric-acid-producing clostridia documented in this simulation experiment with PMEU has in vivo implications and indicates molecular communication between the species. CO(2) is a necessary signal for the growth of clostridia, and lactobacilli can promote clostridial growth in mixed cultures where both bacteria grow well with mutual benefit.     

Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns

The initial proteolytic events in the hydrolysis of rat tendon type I collagen by the class I and II collagenases from Clostridium histolyticum have been investigated at 15 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used to detect the initial cleavage fragments of both the alpha 1(I) and alpha 2 chains, which migrate at different rates in the buffer system employed. Experiments with the class I collagenases indicate that the first cleavage occurs across all three chains of the triple helix close to the C-terminus to produce fragments whose alpha chains have molecular weights of approximately 88,000. The second cleavage occurs near the N-terminus to reduce the molecular weight of the alpha chains to 80,000. Initial proteolysis by the class II collagenases occurs across all three chains at a site in the interior of the collagen triple helix to give N- and C-terminal fragments with alpha-chain molecular weights of 35,000 and 62,000, respectively. The C-terminal fragment is subsequently cleaved to give fragments with alpha-chain molecular weights of 59,000. These results indicate that type I collagen is degraded at several hyperreactive sites by these enzymes. Thus, initial proteolysis by these bacterial collagenases occurs at specific sites, much like the mammalian collagenases. These results with the individual clostridial collagenases provide an explanation for earlier data which indicated that collagen is degraded sequentially from the ends by a crude clostridial collagenase preparation.     

Two Uptake Systems for Fructose in Lactococcus lactis subsp. cremoris FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation

Fructose transport in lactococci is mediated by two phosphotransferase systems (PTS). The constitutive mannose PTS has a broad specificity and may be used for uptake of fructose with a fructose saturation constant (K_Fru) of 0.89 mM, giving intracellular fructose 6-phosphate. The inducible fructose PTS has a very small saturation constant (K_Fru, <17 μM), and the fructose 1-phosphate produced enters the Embden-Meyerhof-Parnas (EMP) pathway as fructose 1,6-diphosphate. Growth in batch cultures of Lactococcus lactis subsp. cremoris FD1 in a yeast extract medium with fructose as the only sugar is poor both with respect to specific growth rate and biomass yield, whereas the specific lactic acid production rate is higher than those in similar fermentations on other sugars metabolized via the EMP pathway, e.g., glucose. In fructose-limited chemostat cultures, the biomass concentration exhibits a strong correlation with the dilution rate, and starting a continuous culture at the end of a batch fermentation leads to large and persistent oscillations in the biomass concentration and specific lactic acid production rate. Two proposed mechanisms underlying this strange growth pattern follow. (i) Fructose transported via the fructose PTS cannot be converted into essential biomass precursors (glucose 6-phosphate or fructose 6-phosphate), because L. lactis subsp. cremoris FD1 is devoid of fructose 1,6-diphosphatase activity. (ii) The fructose PTS apparently produces a metabolite (presumably fructose 1-phosphate) which exerts catabolite repression of both mannose PTS and lactose PTS. Since the repressed mannose PTS and lactose PTS are shown to have identical maximum molar transport rates, the results indicate that it is the general PTS proteins which are repressed.     

The enzymology of the bacterial phosphoenolpyruvate-dependent sugar transport systems

Summary The phosphoenolpyruvate-dependent sugar transport system (PTS) is present in a large variety of bacteria. It catalyzes transport and phosphorylation of hexoses and hexitols at the expense of phosphoenolpyruvate. Only three of four enzymes are required for this entire sequence. Each component has been isolated and purified to the homogeneity from one bacterial species or another allowing recent investigations intomechanistic aspects of energy coupling, energy conservation, transport and regulation using well-characterized enzymes. In each case the phosphorylation of the enzyme is a key element in that enzymes function.The initial step in the energy conversion process is the E_I catalyzed conversion of phosphoenolpyruvate to pyruvate and P-HPr. E_II is a metal requiring hydrophobic enzyme which is active only as a dimer. Kinetic and gel filtration data confirm that it forms functional ternary complexes with HPr or P-Hpr and phosphoenolpyruvate or pyruvate which influence both the degree of dimerization and the specific activity of the dimer. The dimer appears to carry only one phosphoryl group suggesting that negative cooperativity or a flip-flop mechanism may be involved in the sequence of phosphoryl group transfer.Many of the PTS phosphoenzyme intermediates carry the phosphoryl group as a phospho-histidine. A general mechanism for the transfer of the phosphoryl group to and from the active site histidine residue in each protein has been established with high resolution ¹H NMR data. At physiological pH the active site histidine is deprotonated, whereas the phosphohistidine is protonated. Consequently the histidine, as a strong nucleophile, can abstract the phosphoryl group from the donor while protonation destabilizes the phosphohistidine facilitating passage of the phosphoryl group to the following enzyme intermediate. The change in protonation state accompanies a phosphorylation induced conformational change in the carrier.The ability of the PTS to regulate the activity of other permeases and catabolic enzymes has been attributed to E_III ^Glc. Data obtained with mutants suggest that changes in the phosphorylation state alter the regulatory properties of the enzyme. The nonphosphorylated species blocks various permeases and suppresses adenylate cyclase activity thereby inhibiting the synthesis of catabolic enzyme systems. The phosphorylated species stimulates adenylate cyclase and permits the uptake of inducers leading to the initiation of catabolic enzyme synthesis. Experiments with the isolated E_III ^Glc confirm that a phosphoenzyme intermediate exists.Transport and phosphorylation of the sugar are catalyzed by a membrane-bound E_II via a phosphoenzyme intermediate which can be reached from P-HPr, P-E_III or sugar-P. The phosphorylation state controls the affinity of the enzyme for its substrates. E_II is high affinity for P-HPr or P-E_III and low affinity for sugar. P-E_II is high affinity for sugar and low affinity for P-HPr or P-E_III. The affinity of the enzyme for sugar substrates is controlled by the oxidation state of a dithiol. The reduced, dithiol form is high affinity for sugar substrates. The oxidized, disulfide form, is low affinity. Phosphorylation of the enzyme chould shift the affinity for substrates by altering the oxidation state of the enzyme.     

Regulation of the lic Operon of Bacillus subtilis and Characterization of Potential Phosphorylation Sites of the LicR Regulator Protein by Site-Directed Mutagenesis

The lic operon of Bacillus subtilis is required for the transport and degradation of oligomeric beta-glucosides, which are produced by extracellular enzymes on substrates such as lichenan or barley glucan. The lic operon is transcribed from a sigma(A)-dependent promoter and is inducible by lichenan, lichenan hydrolysate, and cellobiose. Induction of the operon requires a DNA sequence with dyad symmetry located immediately upstream of the licBCAH promoter. Expression of the lic operon is positively controlled by the LicR regulator protein, which contains two potential helix-turn-helix motifs, two phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and a domain similar to PTS enzyme IIA (EIIA). The activity of LicR is stimulated by modification (probably phosphorylation) of both PRD-I and PRD-II by the general PTS components and is negatively regulated by modification (probably phosphorylation) of its EIIA domain by the specific EII(Lic) in the absence of oligomeric beta-glucosides. This was shown by the analysis of licR mutants affected in potential phosphorylation sites. Moreover, the lic operon is subject to carbon catabolite repression (CCR). CCR takes place via a CcpA-dependent mechanism and a CcpA-independent mechanism in which the general PTS enzyme HPr is involved.