Membrane sites regulating developmental gene expression in Dictyostelium discoideum

Postaggregative gene expression in Dictyostelium discoideum requires cell contact. Polyspecific monovalent antibodies (Fab) prepared from sera raised against membranes of aggregation- and postaggregation-stage cells were used to probe the cell interactions that induce rapid postaggregative synthesis of UDP-glucose pyrophosphorylase. When cells of strain V12M2 were dissociated after 8 hr of development and replated in the presence of immune Fab, both reaggregation and pyrophosphorylase synthesis were blocked. Fab neutralized by incubation with EDTA-high salt extracts of cells developed for 3 hr blocked pyrophosphorylase synthesis but not reaggregation. Therefore, some cell-surface components that regulate pyrophosphorylase synthesis (called E sites) are antigenically distinct from those required for reaggregation. The Fab provides a means to assay E sites during their purification. Addition of 10(-3) M cyclic AMP or cyclic GMP enabled the cells to bypass the blocking of E sites by Fab; pyrophosphorylase was synthesized in the absence of reaggregation. We hypothesize that E sites function by raising the level of intracellular cyclic AMP.     

The effect of epsilon-aminocaproic acid on biochemical changes in the development of the cellular slime mould Dictyostelium discoideum.

epsilon-Aminocaproic acid (EACA) inhibited the development of Dictyostelium discoideum strain AX2 after the aggregation stage. Biochemical changes that occurred early in development (loss of cellular protein, RNA and carbohydrate; increase in the specific activity of N-acetylglucosaminidase, alpha-mannosidase, threonine deaminase and leucine aminopeptidase) were not affected by concentrations of EACA which blocked development; but biochemical changes that occurred later (synthesis of carbohydrate, increase in the specific activity of UDP-glucose pyrophosphorylase) were inhibited. Spores from fruiting bodies formed in the presence of low concentrations of EACA were larger, more spherical and less able to survive heat treatment than spores from fruiting bodies of control (no EACA) cells.     

Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis.

We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.     

Changes in morphogenesis and developmental enzyme levels in Dictyostelium discoideum after gamma irradiation.

Changes in the levels of specific activity of two enzymes believed to be involved in developmental regulation were observed after irradiating differentiating cells of Dictyostelium discoideum. Stimulation of the levels of specific activity of alkaline phosphatase occured after irradiation at the beginning of development and at the end of the aggregation period, but not after irradiation at the beginning of aggregation. A stimulation in UDP-glucose pyrophosphorylase specific activity was also observed, but to a lesser extent and only after irradiation at the end of aggregation. Dose-dependent delays in the appearance of peaks of specific activity were noted. The delay per unit dose was less when irradiation took place at the beginning of development as opposed to the beginning or end of the aggregation period. Radiation-induced delays in progression through visible developmental stages were almost identical to delays in enzyme appearance. Other radiation effects on morphogenesis included the induction of a migratory slug phase.     

Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture

1. The DNA, RNA, protein and carbohydrate contents of myxamoebae of Dictyostelium discoideum strain Ax-2 were measured after growth on bacteria or in various axenic media. 2. Myxamoebae grown in the different axenic media have similar DNA, RNA and protein contents, but there are marked differences in the contents of glycogen and free sugars. The DNA and protein contents of myxamoebae grown on bacteria are different from those in myxamoebae grown axenically. 3. Approximately half the DNA found in myxamoebae grown on bacteria is of bacterial rather than of slime-mould origin. 4. The specific activities of some enzymes (including UDP-glucose pyrophosphorylase) are higher in myxamoebae grown axenically than in myxamoebae grown on bacteria. Nevertheless the characteristic increase in the specific activity of UDP-glucose pyrophosphorylase occurring during differentiation of cells of the wild-type strain NC-4 is also found in cells grown axenically. 5. The rate of amino acid oxidation during axenic growth of the myxamoebae is decreased when the cells are supplied with glucose.     

Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis

A metabolic engineering strategy was successfully applied to engineer the UDP-glucose synthesis pathway in E. coli. Two key enzymes of the pathway, phosphoglucomutase and UDP-glucose pyrophosphorylase, were overexpressed to increase the carbon flux toward UDP-glucose synthesis. When additional enzymes (a UDP-galactose epimerase and a galactosyltransferease) were introduced to the engineered strain, the increased flux to UDP-glucose synthesis led to an enhanced UDP-galactose derived disaccharide synthesis. Specifically, close to 20 mM UDP-galactose derived disaccharides were synthesized in the engineered strain, whereas in the control strain only 2.5 mM products were obtained, indicating that the metabolic engineering strategy was successful in channeling carbon flux (8-fold more) into the UDP-glucose synthesis pathway. UDP-sugar synthesis and oligosaccharide synthesis were shown to increase according to the enzyme expression levels when inducer concentration was between 0 and 0.5 mM. However, this dependence on the enzyme expression stopped when expression level was further increased (IPTG concentration was increased from 0.5 to 1 mM), indicating that other factors emerged as bottlenecks of the synthesis. Several likely bottlenecks and possible engineering strategies to further improve the synthesis are discussed.     

Accumulation of UDP-glucose pyrophosphorylase by axenically grown amoebae of Dictyostelium discoideum.

Amoebae of the slime mould Dictyostelium discoideum AX2 possess only low UDP-glucose pyrophosphorylase activity when grown on autoclaved Klebsiella aerogenes (approx. 30 units/mg of protein), but accumulate the enzyme to approx. 150-200 units/mg of protein during vegetative growth in axenic medium. The vegetative accumulation of UDP-glucose pyrophosphorylase by axenically grown cells is prevented if autoclaved K. aerogenes are included in the axenic medium, suggesting the absence of a specific inducer. Affinity chromatography using anti-(UDP-glucose pyrophosphorylase) antibody and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicate that the enzyme accumulated during axenic growth and that normally accumulated during development are immunologically cross-reactive and that both are composed of two subunits with mol.wts. 55,600 and 57,500 present in approximately equal amounts in the active enzyme.     

Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei

UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides.     

Protein kinases of Dictyostelium discoideum, strain AX-2.

In cell homogenates of Dictyostelium discoideum, strain AX-2, four major soluble protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) and one membrane-associated protein kinase activity were identified. The enzymes showed high affinity for casein. One of the enzymes was purified by affinity chromatography on casein-coated Sepharose. The soluble high molecular weight enzymes phosphorylated histones, whereas the low molecular weight enzymes did not. The same protein kinase species were present in vegetative and aggregation-competent cells. Their specific activity, however, changed during the development to aggregation competence. None of the enzymes was stimulated by cyclic AMP or cyclic GMP, regardless of their origin from vegetative or aggregation-competent cells.     

Functional Analysis of the Lactococcus lactis galU and galE Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis

We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal⁻ ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

The effects of cyclic AMP on disaggregation-induced changes in activities of developmentally regulated enzymes in Dictyostelium discoideum.

The addition of cyclic AMP to the shaking medium of cells disaggregated from pseudoplasmodia of $\text{Dictyostelium discoideum}$ suppressed the accumulation of cell-bound phosphodiesterase which normally occurs (1) after disaggregation. The suppression was not secondarily brought about by its possible inhibitory effect of cyclic AMP on protein synthesis or by its stimulating effect on the release of the enzyme into the medium. The effect was reversible and specific to cyclic AMP. On the other hand, the inhibitory effect of cyclic AMP on the disaggregation-induced inactivation of UDP-galactose transferase was not apparent in the initial period, but thereafter it slowed down the decrease in the enzyme activity. These results indicate that exogenous cyclic AMP mimics at least in part the regulatory effects of cell-to-cell contact on certain enzymes.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal- ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Leishmania UDP-sugar Pyrophosphorylase: THE MISSING LINK IN GALACTOSE SALVAGE?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.     

Specific binding proteins for cyclic AMP and cyclic GMP in Dictyostelium discoideum.

In Dictyostelium discoideum both cyclic AMP and cyclic GMP are regulated by chemotactic stimuli. Binding proteins specific for cAMP and cGMP have been found in aggregation competent cells as well as in cells harvested during growth. The activity of binding proteins was, on the average, lower in the growth phase cells. cAMP binding proteins were separated into 3 fractions, whereas the cGMP binding activity appeared in 1 major peak both on DEAE-cellulose and Sephadex G-200. Protein kinase activity was present in most but not all cyclic necleotide binding fractions; evidence for a relationship is however missing.     

Ecto-cyclic AMP-receptor in goat epididymal intact spermatozoa and its change in activity during forward motility

Goat epididymal intact spermatozoa have been shown to possess on the external surface specific receptors that bind with high affinity to exogenous [8-3H]cyclic AMP. The ecto-cyclic AMP-receptor activity was not due to contamination of broken or "leaky" cells, if any. The binding reaction of [3H]cyclic AMP with the receptors was extremely rapid. Uptake of the labeled cyclic AMP to the sperm cytosolic fraction was undetectable. There was little leakage of cyclic AMP-receptors from intact spermatozoa during the binding assays. The binding reaction was proportional to cell concentration, specific and saturable at 250 nM cyclic AMP. The binding of the labelled cyclic nucleotide was nearly completely displaced at saturating concentrations (2.5 microM) of the unlabelled nucleotide. The ecto-receptors showed high specificity for binding to cyclic AMP. The Kd of the binding sites was approximately 1.7 X 10(-8) M. The binding interaction was highly sensitive to treatment with proteolytic enzymes: trypsin, chymotrypsin, or pronase (125 micrograms/ml). Sonication caused a nearly 450% increase of the ecto-receptor activity. The specific activity of the ecto-cyclic AMP-receptor was approximately twofold higher in the vigorously forwardly motile spermatozoa than in the "composite" cells, suggesting that the ecto-receptors may have a role in modulating flagellar motility.     

Changes in activity of enzymes associated with prespore differentiation in a suspension culture of Dictyostelium discoideum

It has been shown (Okamoto, K. (1981) J. Gen. Microbiol. in the press) that Dictyostelim discoideum cells dissociated at early aggregation can differentiate into prespore cells in a suspension containing glucose, albumin, EDTA and cyclic AMP. Strict requirement of cyclic AMP in this process has also been demonstrated. In the present paper, changes in activity of eight developmentally regulated enzymes were examined in this culture system and compared to those occuring in the normal course of development on the solid substratum. The results show that (a) formation in this medium is not accompanied by increases in activity of UDPglucose pyrophosphorylase and trehalose phosphate synthetase, unlike the case of the normal development, (b) among the enzymes examined, only UDPgalactose: polysaccharide galactosyl transferase can be regarded as a specific marker of the prespore formation, and (c) development in this system does not proceed beyond the slug stage of the normal development, in the case of a wild-type strain NC4.     

UDP-glucose pyrophosphorylase from potato tuber: purification and characterization

UDP-glucose pyrophosphorylase from potato tuber was purified 243-fold to a nearly homogeneous state with a recovery of 30%. The purified enzyme utilized UDP-glucose, but not ADP-glucose, as the substrate, and was not activated by 3-phosphoglyceric acid. Product inhibition studies revealed the sequential binding of UDP-glucose and MgPPi and the sequential release of glucose-1-phosphate and MgUTP, in this order. Analyses of the effects of Mg2+ on the enzyme activity suggest that the MgPPi and MgUTP complexes are the actual substrates for the enzyme reaction, and that free UTP acts as an inhibitor. The enzyme exists probably as the monomer of an approximately 50-kDa polypeptide with a blocked amino terminus. For structural comparison, 29 peptides isolated from a tryptic digest of the S-carboxymethylated enzyme were sequenced. The results show that the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold, but not to ADP-glucose pyrophosphorylase from Escherichia coli, and provide structural evidence that UDP-glucose and ADP-glucose pyrophosphorylase are two different protein entities.     

Enzyme activity changes during cyclic AMP-induced stalk cell differentiation in P4, a variant of Dictyostelium discoideum.

The P4 variant of Dictyostelium discoideum is characterized by the production of fruiting structures in which the overall proportion of stalk to spore material is increased, relative to the wild type. The altered morphology of the mutant is due to increased sensitivity to cyclic AMP which promotes stalk cell differentiation. In the presence of 10-4 M-cyclic AMP the entire population of P4 amoebae forms clumps of stalk cells on the surface of the dialysis membrane support. Measurement of changes in activity of a range of developmentally-regulated enzymes during the development of P4 in the presence and absence of cyclic AMP has allowed us to identify three classes of enzyme: (i) Those, such as beta-glucosidase II, trehalose-6-phosphate synthetase and uridine diphosphogalactose-4-epimerase, which are required for the production of spores. (ii) Enzymes, primarily but perhaps not exclusively, required during stalk cell formation. Typical of these are N-acetylglucosaminidase and alkaline phosphatase. (iii) General enzymes, such as threonine dehydrase, alpha-mannosidase and uridine diphosphoglucose pyrophyosphorylase, which are present inboth pre-stalk and pre-spore cells and appear to be necessary for the development of both cell types.     

UDP-galactose 4-epimerase: a key enzyme in exopolysaccharide formation by Lactobacillus casei CRL 87 in controlled pH batch cultures

AIMS: To evaluate the relationship between exopolysaccharide (EPS) production and the sugar nucleotide biosynthetic enzymes in Lactobacillus casei CRL 87 under optimum growth conditions for polymer formation: controlled pH on galactose or glucose. Studies with an EPS mutant were carried out to determine the key enzymes in EPS synthesis under the above culture conditions. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulphuric acid method, while the activities of the biosynthetic enzymes were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. An environmental pH of 5.0, using galactose as carbon source, markedly improved not only polymer production and yield but also, cell growth and lactic acid production. Analysis of the activities of the EPS precursor-forming enzymes revealed that polysaccharide synthesis was correlated with uridine-diphosphate (UDP)-glucose pyrophosphorylase and UDP-galactose 4-epimerase under these growth conditions. CONCLUSIONS: EPS synthesis by Lact. casei CRL 87 was considerably improved at a controlled pH of 5.0 with galactose as carbon source, and was correlated with the activity of UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase. The results obtained with the wild-type and EPS- strains suggest that UDP-galactose 4-epimerase plays an essential role in EPS formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Unravelling the key enzymes involved in EPS biosynthesis under optimum culture conditions for polymer production provides important information for the design of strategies, via genetic engineering, to enhance polysaccharide formation.