Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus.     

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway.     

Lactose and D0galactose metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation

The pathway by which D-galactose 6-phosphate is degraded in Staphylococcus aureus has been elucidated. Galactose 6-phosphate is isomerized to tagatose 6-phosphate, which is phosphorylated with adenosine 5′-triphosphate, and the resulting tagatose 1,6-diphosphate is cleaved to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The isomerase, kinase, and aldolase that catalyze these reactions are inducible and are distinct from the corresponding enzymes of glucose 6-phosphate metabolism.     

ColE1 hybrid plasmids for Escherichia coli genes of glycolysis and the hexose monophosphate shunt.

The Clarke-Carbon clone bank carrying ColE1-Escherichia coli DNA has been screened by conjugation for complementation of glycolysis and hexose monophosphate shunt mutations. Plasmids were identified for phosphofructokinase (pfkA), triose phosphate isomerase (tpi), phosphoglucose isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf), gluconate-6-phosphate dehydrogenase (gnd), enolase (eno), phosphoglycerate kinase (pgk), and fructose-1,6-P2 aldolase (fda). Enzyme levels for the plasmid-carried gene ranged, for the various plasmids, from 4- to 25-fold the normal level.     

Genetic evidence for the physiological significance of the D-tagatose 6-phosphate pathway of lactose and D-galactose degradation in staphylococcus aureus

Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain.     

Natural and altered induction of the L-fucose catabolic enzymes in Klebsiella aerogenes.

Mutants of Klebsiella aerogenes W70 were isolated that had gained the ability to utilize the uncommon pentose D-arabinose as their sole source of carbon and energy. In contrast to the D-arabinose-negative, parent strain, these mutants were found to be either constitutive for certain enzymes of the L-fucose catabolic pathway or inducible for such enzymes when incubated in the presence of D-arabinose. The mutants used L-fucose isomerase to convert D-arabinose to D-ribulose, which is an intermediate and inducer of the ribitol catabolic pathway. The D-ribulokinase of the ribitol pathway was then induced. This enzyme catalyzed the phosphorylation of D-ribulose at the 5-carbon position. Mutants that were negative for D-ribulokinase could still dissimilate D-arabinose slowly by using all three enzymes, the isomerase, kinase, and aldolase, of the L-fucose pathway. Using condition negative mutants, we were able to demonstrate that the natural induction of the L-fucose pathway enzymes by L-fucose required the activity of a functional L-fucose isomerase and a functional L-fuculokinase but not an L-fuculose-1-phosphate aldolase. A metabolic intermediate, L-fuculose-1-phosphate, was thereby shown to be a probable inducer of at least the isomerase and kinase of the L-fucose catabolic pathway. Similar experiments, with D-arabinose-positive mutants, which were induced for the L-fucose pathway enzymes upon incubation with D-arabinose, revealed that the activities of the L-fucose isomerase and the L-fuculokinase were also required for the induction of the L-fucose enzymes. These D-arabinose-positive mutants apparently produced an altered regulatory protein that accepted both L-fuculose-1-phosphate and D-ribulose-1-phosphate as inducers. Examination of constitutive mutants revealed that L-fucose isomerase and L-fuculokinase were both synthesized constitutively, with the aldolase apparently under separate control.     

Differential selectivity of the Escherichia coli cell membrane shifts the equilibrium for the enzyme-catalyzed isomerization of galactose to tagatose

An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.     

Restriction mapping and localization of the lactose-metabolizing genes of Streptococcus cremoris pDI-21

Summary The conjugative plasmid pDI-21 (63 kb) from Streptococcus cremoris encodes genes which are responsible for lactose utilization and protein degradation. Restriction mapping and hybridization using heterologous probes have localized these genes to regions of 12.5 kb and 4.4 kb. The tagatose-6-phosphate pathway genes were closely linked and were assigned in the order; galactose-6-phosphate isomerase, d-tagatose-6-phosphate kinase and tagatose-1,6-bisphosphate aldolase. The Lac-PTS genes and the phospho--galactosidase gene were mapped approximately 1 kb from the tagatose-6-phosphate genes.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.     

Enzymatic analysis of the pathways of glucose catabolism and gluconeogenesis in Pseudomonas citronellolis

Extracts of Pseudomonas citronellolis cells grown on glucose or gluconate possessed all the enzymes of the Entner-Doudoroff pathway. Gluconokinase and either or both 6-phosphogluconate dehydratase and KDPG aldolase were induced by growth on these substrates. Glucose and gluconate dehydrogenases and 6-phosphofructokinase were not detected. Thus catabolism of glucose proceeds via an inducible Entner-Doudoroff pathway. Metabolism of glyceraldehyde 3-phosphate apparently proceeded via glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase. These same enzymes plus triose phosphate isomerase were present in lactate-grown cells indicating that synthesis of triose phosphates from gluconeogenic substrates also occurs via this pathway. Extracts of lactate grown-cells possessed fructose diphosphatase and phosphohexoisomerase but apparently lacked fructose diphosphate aldolase thus indicating either the presence of an aldolase with unusual properties or requirements or an alternative pathway for the conversion of triose phosphate to fructose disphosphate. Cells contained two species of glyceraldehyde 3-phosphate dehydrogenase, one an NAD-dependent enzyme which predominated when the organism was grown on glycolytic substrates and the other, an NADP-dependent enzyme which predominated when the organism was grown on gluconeogenic substrates.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Enzymes of glycolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection.The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms.The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO₂ into phosphoenol pyruvate to produce oxaloacetate.Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.     

Towards Enhanced Galactose Utilization by Lactococcus lactis

Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.Lactococcus lactis is a lactic acid bacterium widely used in the dairy industry for the production of fermented milk products. Because of its economic importance, L. lactis has been studied extensively in the last 40 years. A small genome, a large set of genetic tools, a wealth of physiological knowledge, and a relatively simple metabolic potential render L. lactis an attractive model with which to implement metabolic engineering strategies (reviewed in references 21 and 57).In the process of milk fermentation by L. lactis, lactose is taken up and concomitantly phosphorylated at the galactose moiety (C-6) by the lactose-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS^Lac), after which it is hydrolyzed to glucose and galactose 6-phosphate (Gal6P) (64). The glucose moiety enters the glycolytic pathway upon phosphorylation via glucokinase to glucose 6-phosphate (G6P), whereas Gal6P is metabolized to triose phosphates via the d-tagatose 6-phosphate (Tag6P) pathway, encompassing the steps catalyzed by galactose 6-phosphate isomerase (LacAB), Tag6P kinase (LacC), and tagatose 1,6-bisphosphate aldolase (LacD) (Fig. ​(Fig.1).1). Curiously, during the metabolism of lactose by L. lactis, part of the Gal6P is dephosphorylated and excreted into the growth medium, while the glucose moiety is readily used (2, 7, 51, 56, 60).Open in a separate windowFIG. 1.Schematic overview of the alternative routes for galactose uptake and further catabolism in L. lactis. Galactose can be imported by the non-PTS permease GalP and metabolized via the Leloir pathway (galMKTE) to α-G1P, which is converted to the glycolytic intermediate G6P by α-phosphoglucomutase (pgmH). Alternatively, galactose can be imported by PTS^Lac (lacFE) and further metabolized to triose phosphates by the Tag6P pathway (lacABCD). Here, we propose a new uptake route consisting of galactose translocation via the galactose PTS, followed by dephosphorylation of the internalized Gal6P to galactose, which is further metabolized via the Leloir pathway (highlighted in the gray box). galP, galactose permease; galM, galactose mutarotase; galK, galactokinase; galT, galactose 1-phosphate uridylyltransferase; galE, UDP-galactose-4-epimerase; pgmH, α-phosphoglucomutase; lacAB, galactose 6-phosphate isomerase; lacC, Tag6P kinase; lacD, tagatose 1,6-bisphosphate aldolase; lacFE, PTS^Lac; PTS^Gal, unidentified galactose PTS; Phosphatase; unidentified Gal6P-phosphatase; pgi, phosphoglucose isomerase; pfk, 6-phosphofructo-1-kinase; fba, fructose 1,6-bisphosphate aldolase; tpi, triose phosphate isomerase; α-Gal1P, α-galactose 1-phosphate; α-G1P, α-glucose 1-phosphate; UDP-gal, UDP-galactose; UDP-glc, UDP-glucose; G6P, glucose 6-phosphate; Gal6P, galactose 6-phosphate; Tag6P, tagatose 6-phosphate; TBP, tagatose 1,6-bisphosphate; FBP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate. The dotted arrow represents the conversions of GAP to pyruvate via the glycolytic pathway. Steps essential to improve galactose consumption are shown in black boxes.As a result of incomplete lactose utilization, some fermented dairy products contain significant residual amounts of galactose. The presence of galactose has been associated with shoddier qualities of the fermented product (6, 27, 43). In particular, galactose is a major contributor to the browning that occurs when dairy products (e.g., yogurt and mozzarella, Swiss, and cheddar cheese) are cooked or heated in the manufacture of pizzas, sauce preparation, or processed cheese. In addition, availability of residual galactose may result in production of CO₂ by heterofermentative starters and, consequently, in textural defects such as the development of slits and fractures in cheeses. Therefore, the availability of starter strains with improved galactose utilization capacity is desirable to develop higher-quality dairy products. Additionally, strains with increased galactose metabolism could provide galactose-free foods for individuals and, in particular, children suffering from the rare disease galactosemia (36). To this end, a comprehensive understanding of galactose catabolism is essential.Galactose metabolism in L. lactis was thoroughly studied in the past and has been and still is the subject of some controversy. Indeed, conflicting results regarding the type of PTS involved in galactose uptake have been published. Some authors advocated that galactose is exclusively transported via the plasmid-encoded PTS^Lac, whereas others proposed transport via a galactose-specific PTS (PTS^Gal) to the extreme of questioning the contribution of the PTS^Lac (17, 20, 50, 59). However, a gene encoding PTS^Gal has never been identified in L. lactis. Independently of the nature of the PTS, it is generally accepted that the resulting Gal6P is metabolized via the Tag6P pathway (lac operon) (Fig. ​(Fig.1).1). On the other hand, galactose translocated via the highly specific galactose permease (GalP) is metabolized via the Leloir pathway to α-glucose 1-phosphate (α-G1P) through the sequential action of galactose mutarotase (GalM), galactokinase (GalK), and galactose 1-phosphate uridylyltransferase (GalT)/UDP-galactose-4-epimerase (GalE) (gal operon). Entry in glycolysis is preceded by the α-phosphoglucomutase (α-PGM)-catalyzed isomerization of α-G1P to G6P. The use of the Leloir and/or the Tag6P pathway for galactose utilization is currently viewed as being strain dependent (9, 16, 25, 32, 33, 58), but the relative efficacy in the degradation of the sugar has not been established.The ultimate aim of this study was to engineer L. lactis for improved galactose-fermenting capacity as a means to minimize the galactose content in dairy products. To gain insight into galactose catabolism via the Leloir (gal genes) and the Tag6P (lac genes) pathways, a series of L. lactis subsp. cremoris NZ9000 isogenic gal and lac mutants were constructed. Carbon 13 labeling experiments coupled with nuclear magnetic resonance (NMR) spectroscopy were used to investigate galactose metabolism in the gal and lac strains. The data obtained revealed a novel route for galactose dissimilation and provided clues to further enhance galactose utilization.     

Alginic acid synthesis in Pseudomonas aeruginosa mutants defective in carbohydrate metabolism.

Mutant cells of mucoid Pseudomonas aeruginosa isolated from cystic fibrosis patients were examined for their ability to synthesize alginic acid in resting cell suspensions. Unlike the wild-type strain which synthesizes alginic acid from glycerol, fructose, mannitol, glucose, gluconate, glutamate, or succinate, mutants lacking specific enzymes of carbohydrate metabolism are uniquely impaired. A phosphoglucose isomerase mutant did not synthesize the polysaccharide from mannitol, nor did a glucose 6-phosphate dehydrogenase mutant synthesize the polysaccharide from mannitol or glucose. Mutants lacking the Entner-Doudoroff pathway dehydrase or aldolase failed to produce alginate from mannitol, glucose, or gluconate, as a 3-phosphoglycerate kinase or glyceraldehyde 3-phosphate dehydrogenase mutant failed to produce from glutamate or succinate. These results demonstrate the primary role of the Entner-Doudoroff pathway enzymes in the synthesis of alginate from glucose, mannitol, or gluconate and the role of glyceraldehyde 3-phosphate dehydrogenase reaction for the synthesis from gluconeogenic precursors such as glutamate. The virtual absence of any activity of phosphomannose isomerase in cell extracts of several independent mucoid bacteria and the impairment of alginate synthesis from mannitol in mutants lacking phosphoglucose isomerase or glucose 6-phosphate dehydrogenase rule out free mannose 6-phosphate as an intermediate in alginate biosynthesis.     

Kinetics of metabolic pathways. A system in vitro to study the control of flux.

A method for determining Control Coefficients is proposed for systems studied in vitro and applied to a model pathway. Rat liver extract, which converts glucose into glycerol 3-phosphate, was used with the addition to the incubation mixture of fructose-bisphosphate aldolase, triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase as 'auxiliary' enzymes, which leaves all the control on the first three enzymes. The flux of the metabolic pathway was recorded by assaying NADH decay. Flux Control Coefficients (CJE) of hexokinase, glucose-6-phosphate isomerase and phosphofructokinase were calculated by titration of the system with increasing quantities of extraneous enzymes. It is shown that the summation property is fulfilled. The applicability of this procedure to study the control in any metabolic pathway is discussed. Possible relevance of the method to conditions in vivo and its limitations are considered.     

Biochemistry of Coxiella burnetii: Embden-Meyerhof pathway

Purified preparations of Coxiella burnetii were examined for enzymes of the glycolytic pathway. Glucose-phosphate isomerase, fructose-1,6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase were shown to be present in C. burnetii extracts. Heat-killed C. burnetii purified with normal yolk sacs demonstrated no activity after disruption. Aldolase was shown to be of the class II type by complete inhibition of activity in the presence of 8 x 10(-3)m ethylenediaminetetraacetic acid. The host enzyme activity (normal and infected yolk sacs) was not affected by the same treatment. When cellulose acetate electrophoresis was performed on the extracts, aldolase from both normal and infected yolk sacs exhibited five isozyme bands, whereas aldolase from the C. burnetii extract appeared as a single band.     

The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.     

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.