Differential gene expression in Thermotoga neapolitana in response to growth substrate.

We have previously shown that beta-galactosidase activity expressed in Thermotoga neapolitana cells grown on lactose is subject to repression by glucose when they are grown on both substrates whereas beta-galactosidase and beta-glucosidase activities observed in cells grown on cellobiose are not repressed by growth on both glucose and cellobiose. To examine the differential expression of bgalA, bgalB, bglA and bglB in T. neapolitana, total RNA was isolated from cells growing on either glucose, lactose or cellobiose as the sole source of carbon and transcripts encoding these genes were quantitated by Northern blot analyses. BglA expression was induced by cellobiose while bglB was expressed under all three conditions at a lower level. Expression of the beta-galactosidase genes, bgalA and bgalB, was detected only in lactose-grown cells. beta-Glucosidase enzyme activity was only found in cell extracts of cellobiose-grown cells while beta-galactosidase activity was found in both lactose- and cellobiose-grown cell extracts. Our results show that in cellobiose-grown cells, the high beta-glucosidase activity is likely due to expression of bglA and that neither bgalA nor bgalB is responsible for the beta-galactosidase activity.

Evolution of carbohydrate metabolic pathways.

Current studies of hyperthermophilic archaea and bacteria, the phylogenetically deepest-rooted and slowest-evolving extant organisms known, are allowing new insights into the nature of presumably ancient metabolic pathways. The apparent common occurrence of modified non-phosphorylated Entner-Doudoroff (ED) pathways among saccharolytic archaea and the absence of the conventional Embden-Meyerhof-Parnas (EMP) mode of glycolysis indicate that the ED pathway is the older route of carbohydrate dissimilation. However, gluconeogenesis via the "reversed" EMP route has been found in archaea. Thus, the EMP pathway was probably an anabolic pathway to begin with; its catabolic role came later, with the evolution of fructose phosphate kinases, using ATP, ADP or pyrophosphate as phosphate donors. Similarly, the presence of reductive reactions of the citric acid cycle in anaerobic archaea and the most deeply rooted bacteria, including autotrophs, indicates that the citric acid cycle was originally a reductive biosynthetic pathway.

The role of phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, in the regulation of carbon metabolism in gram-positive bacteria.

HPr of the Gram-positive bacterial phosphotransferase system (PTS) can be phosphorylated by an ATP-dependent protein kinase on a serine residue or by PEP-dependent Enzyme 1 on a histidyl residue. Both phosphorylation events appear to influence the metabolism of non-PTS carbon sources. Catabolite repression of the gluconate (gnt) operon of B. subtilis appears to be regulated by the former phosphorylation event, while glycerol kinase appears to be regulated by the latter phosphorylation reaction. The extent of our understanding of these processes will be described.

Physiological studies on regulation of glycerol utilization by the phosphoenolpyruvate:sugar phosphotransferase system in Enterococcus faecalis.

In vitro studies with purified glycerol kinase from Enterococcus faecalis have established that this enzyme is activated by phosphorylation of a histidyl residue in the protein, catalyzed by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), but the physiological significance of this observation is not known. In the present study, the regulation of glycerol uptake was examined in a wild-type strain of E. faecalis as well as in tight and leaky ptsI mutants, altered with respect to their levels of enzyme I of the PTS. Glycerol kinase was shown to be weakly repressible by lactose and strongly repressible by glucose in the wild-type strain. Greatly reduced levels of glycerol kinase activity were also observed in the ptsI mutants. Uptake of glycerol into intact wild-type and mutant cells paralleled the glycerol kinase activities in extracts. Glycerol uptake in the leaky ptsI mutant was hypersensitive to inhibition by low concentrations of 2-deoxyglucose or glucose even though the rates and extent of 2-deoxyglucose uptake were greatly reduced. These observations provide strong support for the involvement of reversible PTS-mediated phosphorylation of glycerol kinase in the regulation of glycerol uptake in response to the presence or absence of a sugar substrate of the PTS in the medium. Glucose and 2-deoxyglucose were shown to elicit rapid efflux of cytoplasmic [14C]lactate derived from [14C]glycerol. This phenomenon was distinct from the inhibition of glycerol uptake and was due to phosphorylation of the incoming sugar by cytoplasmic phosphoenolpyruvate. Lactate appeared to be generated by sequential dephosphorylation and reduction of cytoplasmic phosphoenolpyruvate present in high concentrations in resting cells. The relevance of these findings to regulatory phenomena in other bacteria is discussed.

Evidence for the presence of heat-stable protein (HPr) and ATP-dependent HPr kinase in heterofermentative lactobacilli lacking phosphoenolpyruvate:glycose phosphotransferase activity.

An analysis of the biochemical basis for the lack of phosphoenolpyruvate:glycose phosphotransferase activity in heterofermentative lactobacilli was carried out. Extracts of Lactobacillus brevis and Lactobacillus buchneri failed to reconstitute phosphotransferase activity of extracts of Staphylococcus aureus mutants impaired in the phosphotransferase system due to the absence of enzyme I, enzyme IILac, or enzyme IIILac activity, suggesting that these lactobacilli lack those phosphotransferase system components. In contrast, complementation tests with an extract of a S. aureus mutant deficient in heat-stable protein (HPr) indicated the presence of HPr activity in heterofermentative lactobacilli. The HPr of L. brevis was purified and shown to have properties similar to those of a typical HPr. In addition, L. brevis possesses an ATP-dependent protein kinase that phosphorylates a serine residue of the endogenous HPr as well as other HPrs of Gram-positive origin. The kinase activity is markedly stimulated by phosphorylated compounds related to sugar metabolism and is negatively modulated by orthophosphate, pyrophosphate, or arsenate and by a low molecular weight endogenous factor. In keeping with the idea of a regulatory role for the phosphorylation of HPr in lactobacilli, a HPr[Ser(P)] phosphatase activity in L. brevis was also demonstrated. On the basis of the finding of HPr and a system for its reversible covalent modification in an organism devoid of a functional phosphotransferase system we propose that, in lactobacilli, HPr has a role in the regulation of pathways other than the phosphotransferase system.

Regulation of beta-galactoside transport and accumulation in heterofermentative lactic acid bacteria.

Galactose-grown cells of the heterofermentative lactic acid bacteria Lactobacillus brevis and Lactobacillus buchneri transported methyl-beta-D-thiogalactopyranoside (TMG) by an active transport mechanism and accumulated intracellular free TMG when provided with an exogenous source of energy, such as arginine. The intracellular concentration of TMG resultant under these conditions was approximately 20-fold higher than that in the medium. In contrast, the provision of energy by metabolism of glucose, gluconate, or glucosamine promoted a rapid but transient uptake of TMG followed by efflux that established a low cellular concentration of the galactoside, i.e., only two- to fourfold higher than that in the medium. Furthermore, the addition of glucose to cells preloaded with TMG in the presence of arginine elicited a rapid efflux of the intracellular galactoside. The extent of cellular TMG displacement and the duration of the transient effect of glucose on TMG transport were related to the initial concentration of glucose in the medium. Exhaustion of glucose from the medium restored uptake and accumulation of TMG, providing arginine was available for ATP generation. The nonmetabolizable sugar 2-deoxyglucose elicited efflux of TMG from preloaded cells of L. buchneri but not from those of L. brevis. Phosphorylation of this glucose analog was catalyzed by cell extracts of L. buchneri but not by those of L. brevis. Iodoacetate, at a concentration that inhibits growth and ATP production from glucose, did not prevent efflux of cellular TMG elicited by glucose. The results suggested that a phosphorylated metabolite(s) at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular TMG from the cells. Counterflow experiments suggested that glucose converted the active uptake of TMG in L. brevis to a facilitated diffusion mechanism that allowed equilibrium of TMG between the extra- and intracellular milieux. The means by which glucose metabolites elicited this vectorial regulation is not known, but similarities to the inducer expulsion that has been described for homofermentative Streptococcus and Lactobacillus species suggested the involvement of HPr, a protein that functions as a phosphocarrier protein in the phosphotransferase system, as well as a presumptive regulator of sugar transport. Indeed, complementation assays wit extracts of Staphylococcus aureus ptsH mutant revealed the presence of HPr in L. brevis, although this lactobacillus lacked a functional phaosphoenolpyruvate-dependent phosphortransferase system for glucose, 2-deoxyglucose, or TMG.

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function.

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.

d-Glucose Transport System of Zymomonas mobilis.

The properties of the d-glucose transport system of Zymomonas mobilis were determined by measuring the uptake of nonmetabolizable analogs (2-deoxy-d-glucose and d-xylose) by wild-type cells and the uptake of d-glucose itself by a mutant lacking glucokinase. d-Glucose was transported by a constitutive, stereospecific, carrier-mediated facilitated diffusion system, whereby its intracellular concentration quickly reached a plateau close to but not above the external concentration. d-Xylose was transported by the d-glucose system, as evidenced by inhibition of its uptake by d-glucose. d-Fructose was not an efficient competitive inhibitor of d-glucose uptake, indicating that it has a low affinity for the d-glucose transport system. The apparent K(m) of d-glucose transport was in the range of 5 to 15 mM, with a V(max) of 200 to 300 nmol min mg of protein. The K(m) of Z. mobilis glucokinase (0.25 to 0.4 mM) was 1 order of magnitude lower than the K(m) for d-glucose transport, although the V(max) values for transport and phosphorylation were similar. Thus, glucose transport cannot be expected to be rate limiting at concentrations of extracellular glucose normally used in fermentation processes, which greatly exceed the K(m) for the transport system. The low-affinity, high-velocity, nonconcentrative system for d-glucose transport described here is consistent with the natural occurrence of Z. mobilis in high-sugar environments and with the capacity of Z. mobilis for rapid conversion of glucose to metabolic products with low energetic yield.

D-glucose transport by membrane vesicles from quiescent, serum-stimulated, and SV40-transformed mouse 3T3 cells.

Mixed membrane vesicle preparations from mouse embryo fibroblasts (Swiss 3T3) exhibited a facilitated diffusion transport system for D-glucose that showed many of the characteristics of the D-glucose transport system of whole cells: stereospecificity, counterflow, Michaelis-Menten kinetics with an apparent Km similar to that of whole cells, and sensitivity to inhibition by cytochalasin B. Comparison of the stereospecific D-glucose transport activities of membrane vesicles from quiescent, serum-stimulated, and SV40 virus-transformed 3T3 cells showed no significant differences in rates of D-glucose uptake or efflux. This is in contrast to whole cells; quiescent 3T3 cells transported 6-deoxy-D-glucose at a significantly lower rate than serum-stimulated or SV40-transformed cells. These results indicate that D-glucose transport in quiescent vs. actively growing cells is regulated by cellular factors that are not retained in membrane vesicle preparations.

Facilitated diffusion of 6-deoxy-D-glucose in bakers' yeast: evidence against phosphorylation-associated transport of glucose.

6-Deoxy-D-glucose, a structural homomorph of D-glucose which lacks a hydroxyl group at carbon 6 and thus cannot be phosphorylated, is transported by Saccharomyces cerevisiae via a facilitated diffusion system with affinity equivalent to that shown with D-glucose. This finding supports the facilitated diffusion mechanism for glucose transport and contradicts theories of transport-associated phosphorylation which hold that sugar phosphorylation is necessary for high-affinity operation of the glucose carrier.

Effect of glucose uptake on growth rate of mouse 3T3 cells.

The objective of this investigation was to determine whether the rate of glucose uptake by mouse 3T3 cells was a primary determinant of growth rate. The experimental approach was to control the rate of glucose uptake into intracellular pools by supplying this sugar at varying concentration in minimal Eagle's medium with dialyzed serum in the absence and presence of 6-deoxy-D-glucose, a metabolically inert homomorphic analog of D-glucose that competitively inhibits the uptake of D-glucose. Total hexose (D-glucose and 6-deoxy-D-glucose) concentration was maintained at the physiological concentration of 5.5 mM, in order to maintain saturation and maximum activity of the D-glucose transport system; thus the flux of D-glucose into the cell was controlled by adjusting its concentration relative to its competing nonmetabolizable analog. It was found that even when the concentration of D-glucose was reduced to 0.7 mM, one eighth of the "normal" level of 5.5 mM, and 6-deoxy-D-glucose was present in sevenfold excess (4.8 mM), conditions under which glucose uptake was reduced to 20% of that shown by cells in the presence of 5.5 mM D-glucose, and intracellular pools of glucose and phosphorylated sugars derived from glucose were reduced to approximately 14% of normal, there was not a significant decrease in growth rate. These data support the view that the rate of glucose uptake is not a primary determinant of growth rate under the usual conditions of cell culture.

6-deoxy-D-glucose and D-xylose: analogs for the study of D-glucose transport by mouse 3T3 cells.

6-Deoxy-D-glucose and D-xylose, structural homomorphs of D-glucose that lack a 6-hydroxyl group or a 6-hydroxymethyl group, respectively, are transported efficiently by mouse 3T3 cells, with good affinity and high specificity for the D-glucose transport system. Since these analogs lack the 6-hydroxyl group, which is the site of phosphorylation of glucose by hexokinase, they are taken up and are recoverable from cells in an unchanged state. Thus, 6-deoxy-D-glucose and D-xylose offer advantages as transport substrates over 2-deoxy-D-glucose, which is phosphorylated by intercellular hexokinases, and 3-O-methyl-D-glucose, which shows a lower specificity for the D-glucose transport system.

Transport of 6-deoxy-D-glucose and D-xylose by untransformed and SV40-transformed 3T3 cells.

Transport rates of the nonphosphorylated D-glucose analogs 6-deoxy-D-glucose and D-xylose were measured in quiescent and serum-stimulated cultures of mouse 3T3 cells, in SV40-transformed 3T3 cells (SV101), and in a density revertant cell line derived from SV101 (FI-SV101). Initial rates of both entry and exit of 6-deoxy-D-glucose and D-xylose were more than threefold higher in serum-stimulated 3T3 and in SV101 cells than they were in quiescent 3T3 cells, but transport rates were not higher in the transformed cells (SV101) than they were in serum-stimulated 3T3. Confluent cultures of FI-SV101 showed lower rates of transport than serum-stimulated FI-SV101, but not as low as quiescent 3T3 cells enter the quiescent G0 state, but emphasize that SV40-transformed 3T3 cells do not show higher activity of the D-glucose carrier than do actively growing 3T3 cells. Thus, enhanced glucose transport appears not to be a specific consequence of transformation, but a reflection of the active growth state of the cell.

Energization of glucose transport by Pseudomonas fluorescens.

We have measured the capacity of Pseudomonas fluorescens to transport the glucose analog 2-deoxy-d-glucose and the amino acids l-alanine and alpha-aminoisobutyric acid under conditions in which the cells could generate (i) both a membrane proton motive force and high-energy phosphate compounds, (ii) a proton motive force but not high-energy phosphate compounds, and (iii) neither a proton motive force nor high-energy phosphate compounds. This was done by depleting cells of adenosine triphosphate stores by treatment with sodium arsenate and then suspending them in a phosphate-free medium, where they could generate a proton motive force but not phosphate bond energy, or in a phosphate-containing medium, where they could generate both a proton motive force and phosphate bond energy. Inclusion of the proton-conducting ionophore carbonyl cyanide-m-chlorophenyl hydrazone under either condition precluded the generation of both a proton motive force and phosphate bond energy. The amino acids l-alanine and alpha-aminoisobutyric acid were transported independently of phosphate bond energy and required only a proton motive force. 2-Deoxy-d-glucose was transported only under conditions in which phosphate bond energy could be generated. These results are consistent with the findings of others that Pseudomonas aeruginosa produces an inducible shock-sensitive glucose-binding protein and conform to the generalization that binding protein-associated transport systems are energized by phosphate bond energy.

Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria.

A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.

Is glucose transport enhanced in virus-transformed mammalian cells? A dissenting view.

Much of the literature on the uptake of glucose by untransformed and transformed animal cells is based on experiments carried out with 2-deoxy-D-glucose (2-DOG). Results obtained with this analog can be ambiguous, since 2-DOG can be phosphorylated by hexokinases of animal cells. An intracellular trapping mechanism is thus provided. Therefore, the total flux of 2-DOG into the cell is a resultant of both transport and hexokinase action, and the measurement of total 2-DOG incorporation is a valid measurement of transport only if 2-DOG is phosphorylated as rapidly as it enters the cell. Evidence is presented here that this is not necessarily the case, significant levels of free intracellular 2-DOG approaching external concentrations were found in untransformed and transformed mouse 3T3 cells even at early times during uptake. Differences in total intracellular 2-DOG between untransformed and transformed cells were accounted for entirely by 2-deoxyglucose phosphate. Thus, it appears the apparent increase of 2-DOG uptake accompanying transformation in these cell lines is not due to an effect on the transport process, but on enhanced phosphorylation, which is a reflection of an alteration in the regulation of glycolysis. The ambiguity introduced by phosphorylation can be oviated by the use of an analog that cannot be phosphorylated, such as 3-O-methyl-D-glucose. The rate of transport and efflux of this sugar was not found to be different in untransformed versus transformed 3T3 cells. Moreover, deficiencies of this analog as a substrate for the glucose transport system are pointed out.

The effect of serum on the transport and phosphorylation of 2-deoxyglucose by untransformed and transformed mouse 3T3 cells.

Serum starvation of growing and nongrowing (density-inhibited) mouse 3T3 cells resulted in decreased phosphorylation of 2-deoxy--D-glucose, while the time course of transport of this sugar remained unchanged. Serum starvation of SV40 transformed 3T3 cells (SV101) and spontaneously transformed 3T6 cells did not alter either the time course of transport, or phosphorylation of the sugar. Treatment of SV101 cells with 10(-4) M dibutyryl adenosine cyclic 3':5' monophosphate and 10(-3) M theophylline did not restore the capacity to regulate 2-deoxy-D-glucose phosphorylation when these cells were serum deprived. We conclude that serum factors are involved in the modulation of phosphorylation of 2-deoxy-D-glucose in 3T3 cells rather than its transport. This regulation is operative both in growing as well as nongrowing 3T3 cells. In contrast, transformed cells do not respond to this regulation of 2-deoxy-D-glucose phosphorylation.

Phosphorylation but not transport of sugars is enhanced in virus-transformed mouse 3T3 cells.

The transport and phosphorylation of 2-deoxy-D-glucose are separate and sequential events in both normal and virus-transformed 3T3 cells. The apparent enhancement of 2-dOG uptake by 3T3 cells accompanying virus transformation is not due to an effect on the transport process but to enhanced phosphorylation by intracellular kinases. Phosphorylation of 3-O-methyl-D-glucose does not occur in these cells. Both the rate and extent of transport of this glucose analog is the same in normal cells, SV40 virus-transformed cells and sarcoma virus-transformed cells. The appropriateness of using 3-O-MeG for studies of the glucose transport system of animal cells is examined and discussed.

Regulation of the Thiobacillus intermedius glucose uptake system by thiosulfate.

Cells of the mixotrophic chemolithotroph (facultative autotroph) Thiobacillus intermedius which have been grown on a glucose-yeast extract medium, a condition in which glucose is used as a source of energy, accumulate the non-metabolizable analogue 2-deoxy-d-glucose against a concentration gradient in a predominantly unchanged state. On the other hand, cells grown mixotrophically on a thiosulfate-glucose medium, a condition in which glucose provides cell carbon but is not used extensively for energy, and in which enzymes of the Entner-Doudoroff pathway are repressed, do not accumulate 2-deoxy-d-glucose significantly. Similarly, cells grown chemolithotrophically on thiosulfate-carbonate do not take up this sugar. Transfer of thiosulfate-yeast extract-grown cells, which lack the capacity to accumulate 2-deoxy-d-glucose, to a glucose-yeast extract medium results in the induction of the concentrative sugar uptake system. The capacity of induced cells to take up 2-deoxy-d-glucose is inhibited by thiosulfate. Thus, the transport system for glucose appears to be regulated in this organism so that the sugar is accumulated only under conditions where it is utilized as a source of energy, and the presence of the preferred energy source leads to both repression and inhibition of the uptake system.