Intracellular phosphorylation of glucose analogs via the phosphoenolpyruvate: mannose-phosphotransferase system in Streptococcus lactis.

The bacterial phosphoenolpyruvate:sugar-phosphotransferase system (PTS) mediates the vectorial translocation and concomitant phosphorylation of sugars. The question arises of whether the PTS can also mediate the phosphorylation of intracellular sugars. To investigate this possibility in Streptococcus lactis 133, lactose derivatives have been prepared containing 14C-labeled 2-deoxy-glucose (2DG), 2-deoxy-2-fluoro-D-glucose (2FG), or alpha-methylglucoside as the aglycon substituent of the disaccharide. Two of the compounds, beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-D-glucopyranose (2'D-lactose) and beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-2'-fluoro-D-glucopyranose (2'F-lactose), were high-affinity substrates of the lactose-PTS. After translocation, the radiolabeled 2'F-lactose 6-phosphate (2'F-lactose-6P) and 2'D-lactose-6P derivatives were hydrolyzed by P-beta-galactoside-galactohydrolase to galactose-6P and either [14C]2FG or [14C]2DG, respectively. Thereafter, the glucose analogs appeared in the medium, but the rates of sugar exit from mannose-PTS-defective mutants were greater than those determined in the parent strain. Unexpectedly, the results of kinetic studies and quantitative analyses of intracellular products in S. lactis 133 showed that initially (and before exit) the glucose analogs existed primarily in phosphorylated form. Furthermore, the production of intracellular [14C]2FG-6P and [14C]2DG-6P (during uptake of the lactose analogs) continued when the possibility for reentry of [14C]2FG and 2DG was precluded by addition of mannose-PTS inhibitors (N-acetylglucosamine or N-acetylmannosamine) to the medium. By contrast, (i) only [14C]2DG, [14C]2FG, and trace amounts of [14C]2FG-6P were found in cells of a mannose-PTS-defective mutant, and (ii) only [14C]2FG and [14C]2DG were present in cells of a double mutant lacking both mannose-PTS and glucokinase activities. We conclude from these data that the mannose-PTS can effect the intracellular phosphorylation of glucose and its analogs in S. lactis 133.     

Novel phosphoenolpyruvate-dependent futile cycle in Streptococcus lactis: 2-deoxy-D-glucose uncouples energy production from growth.

The addition of 2-deoxy-D-glucose to cultures of Streptococcus lactis 133 that were growing exponentially on sucrose or lactose reduced the growth rate by ca. 95%. Inhibition did not occur with glucose or mannose as the growth sugar. The reduction in growth rate was concomitant with rapid accumulation of the analog in phosphorylated form (2-deoxy-D-glucose 6-phosphate) via the phosphoenolpyruvate-dependent mannose:phosphotransferase system. Within 5 min the intracellular 2-deoxy-D-glucose 6-phosphate concentration reached a steady-state level of greater than 100 mM. After maximum accumulation of the sugar phosphate, the rate of sucrose metabolism (glycolysis) decreased by only 30%, but the cells were depleted of fructose-1,6-diphosphate. The addition of glucose to 2-deoxy-D-glucose 6-phosphate preloaded cells caused expulsion of 2-deoxy-D-glucose and a resumption of normal growth. S. lactis 133 contained an intracellular Mg2+-dependent, fluoride-sensitive phosphatase which hydrolyzed 2-deoxy-D-glucose 6-phosphate (and glucose 6-phosphate) to free sugar and inorganic phosphate. Because of continued dephosphorylation and efflux of the non-metabolizable analog, the maintenance of the intracellular 2-deoxy-D-glucose 6-phosphate pool during growth stasis was dependent upon continued glycolysis. This steady-state condition represented a dynamic equilibrium of: (i) phosphoenolpyruvate-dependent accumulation of 2-deoxy-D-glucose 6-phosphate, (ii) intracellular dephosphorylation, and (iii) efflux of free 2-deoxy-D-glucose. This sequence of events constitutes a futile cycle which promotes the dissipation of phosphoenolpyruvate. We conclude that 2-deoxy-D-glucose functions as an uncoupler by dissociating energy production from growth in S. lactis 133.     

Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.

Wild-type Streptomyces clavuligerus NRRL 3585 is unable to utilize glucose. A glucose-utilizing (gut-1) mutant of S. clavuligerus NRRL 3585 has been obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The gut-1 mutant is able to grow on glucose or galactose, while the wild type is unable to catabolize these hexoses. Similar binding affinities of glucose by cells of the wild type and the gut-1 mutant were found, but the wild type was unable to complete glucose transport. A soluble intracellular ATP-dependent (but not phosphoenolpyruvate-dependent) glucokinase activity was found both in the wild type and the gut-1 mutant. The gut-1 mutant has acquired a functional transport system that allows transport of glucose, 2-deoxyglucose, and galactose, as shown by hexose competition experiments. The gut-1 transport system concentrates glucose inside the cell at least 10- to 20-fold and is strongly inhibited by respiratory inhibitors, which prevent the establishment of a proton motive force, and by proton-conducting ionophores, suggesting that it is energized by a proton motive force. The new transport system is not completely sugar specific (transporting galactose and glucose through the same system), as opposed to the hexose-specific system reported in wild-type Streptomyces griseus.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

Regulation of sugar transport and metabolism in lactic acid bacteria

Abstract The phosphoenolpyruvate (PEP)-dependent lactose: phosphotransferase system (PTS), P-β-galactosidase, and enzymes of the d -tagatose-6P pathway, are prerequisite for rapid homolactic fermentation of lactose by Group N ('starter') streptococci. Moreover, the reactions of transport and catabolism constitute an open cycle in which ATP and lactic acid are metabolic products. The efficient and controlled operation of this cycle requires 'fine-control' mechanisms to ensure: (i) tight coupling between transport and energy-yielding reactions, (ii) co-metabolism of both glucose and galactose moieties of the disaccharide, and (iii) coordination of the rate of sugar transport to the rate of sugar catabolism. The elucidation of these fine-control mechanisms in intact cells of Streptococcus lactis has required the isolation of glucokinase (GK) and mannose-PTS defective mutants, the synthesis of novel lactose analogs, and the use of high resolution [³¹P]NMR spectroscopy. It has been established that PEP provides the crucial link between transport and energy-yielding reactions of the PTS: glycolysis cycle, and that both ATP-dependent glucokinase and PEP-dependent mannose-PTS can participate in the phosphorylation of intracellular glucose. Finally, evidence has been obtained in vivo, for modulation of pyruvate kinase activity in response to fluctuation in, concentrations of positive (FDP), and negative (P_i) effectors of the allosteric enzyme. Fine-control of pyruvate kinase activity may in turn regulate: (i) the distribution of PEP to either the PTS or ATP synthesis, (ii) overall activity of the PTS: glycolysis cycle, and (iii) the formation of the endogenous PEP-potential in starved organisms. The accumulation of non-metabolizable PTS sugars (e.g., 2-deoxy- d -glucose) by growing cells can perturb these fine-control mechanisms and, by establishment of a PEP-dissipating futile cycle, may result in bacteriostasis.     

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.     

Regulation of methyl-beta-d-thiogalactopyranoside-6-phosphate accumulation in Streptococcus lactis by exclusion and expulsion mechanisms

Starved cells of Streptococcus lactis ML3 (grown previously on galactose, lactose, or maltose) accumulated methyl-beta-D-thiogalactopyranoside (TMG) by the lactose:phosphotransferase system. More than 98% of accumulated sugar was present as a phosphorylated derivative, TMG-6-phosphate (TMG-6P). When a phosphotransferase system sugar (glucose, mannose, 2-deoxyglucose, or lactose) was added to the medium simultaneously with TMG, the beta-galactoside was excluded from the cells. Galactose enhanced the accumulation of TMG-6P. Glucose, mannose, lactose, or maltose plus arginine, was added to a suspension of TMG-6P-loaded cells of S. lactis ML3, elicited rapid expulsion of intracellular solute. The material recovered in the medium was exclusively free TMG. Expulsion of galactoside required both entry and metabolism of an appropriate sugar, and intracellular dephosphorylation of TMG-6P preceded efflux of TMG. The rate of dephosphorylation of TMG-6P by permeabilized cells was increased two-to threefold by adenosine 5'-triphosphate but was strongly inhibited by fluoride. S. lactis ML3 (DGr) was derived from S. lactis ML3 by positive selection for resistance to 2-deoxy-D-glucose and was defective in the enzyme IIMan component of the glucose:phosphotransferase system. Neither glucose nor mannose excluded TMG from cells of S. lactic ML3 (DGr), and these two sugars failed to elicit TMG expulsion from preloaded cells of the mutant strain. Accumulation of TMG-6P by S. lactis ML3 can be regulation by two independent mechanisms whose activities promote exclusion or expulsion of galactoside from the cell.     

Distinct galactose phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus lactis.

Lactose-negative (Lac-) mutants were isolated from a variant of Streptococcus lactis C2 in which the lactose plasmid had become integrated into the chromosome. These mutants retained their parental growth characteristics on galactose (Lac- Gal+). This is in contrast to the Lac- variants obtained when the lactose plasmid is lost from S. lactis, which results in a slower growth rate on galactose (Lac- Gal+). The Lac- Gal+ mutants were defective in [14C]thiomethyl-beta-D-galactopyranoside accumulation, suggesting a defect in the lactose phosphoenolpyruvate-dependent phosphotransferase system, but still possessed the ability to form galactose-1-phosphate and galactose-6-phosphate from galactose in a ratio similar to that observed from the parental strain. The Lac- Gald variant formed only galactose-1-phosphate. The results imply that galactose is not translocated via the lactose phosphoenolpyruvate-dependent phosphotransferase system, but rather by a specific galactose phosphoenolpyruvate-dependent phosphotransferase system for which the genetic locus is also found on the lactose plasmid in S. lactis.     

Characterization of lactose-fermenting revertants from lactose-negative Streptococcus lactis C2 mutants

Partial lactose-fermenting revertants from lactose-negative (lac(-)) mutants of Streptococcus lactis C2 appeared on a lawn of lac(-) cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac(+)S. lactis C2, the revertants were defective in the accumulation of [(14)C]thiomethyl-beta-d-galactoside, indicating that they were devoid of a transport system. Hydrolysis of o-nitrophenyl-beta-d-galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-beta-d-galactosidase (P-beta-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac(+)S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac(+)S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac(-), respectively. This suggested that the lac(-) and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-beta-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.     

Involvement of phosphoenolpyruvate in lactose utilization by group N streptococci

The effect of sodium fluoride on lactose metabolism and o-nitrophenyl-beta-d-galactopyranoside (ONPG) hydrolysis by Streptococcus lactis strains 7962 and C(2)F suggested that different mechanisms of lactose utilization existed in the two strains. Sodium fluoride prevented lactose utilization and ONPG hydrolysis by whole cells of S. lactis C(2)F but had no effect on S. lactis 7962. Although hydrolysis of ONPG by toluene-treated cells of S. lactis 7962 occurred without addition of phospho-enolpyruvate (PEP), toluene-treated cells of S. lactis C(2)F required the presence of this cofactor. Concentrated cell extracts of S. lactis C(2)F hydrolyzed ONPG; this hydrolysis was inhibited by NaF, but the addition of PEP, in the presence of NaF, restored maximal activity. Addition of acetyl-phosphate, carbamyl-phosphate, adenosine-5'-triphosphate, guanosine-5'-triphosphate, or uridine-5'-triphosphate did not stimulate activity. The presence of cofactors did not stimulate and NaF did not inhibit the hydrolysis in extracts of S. lactis 7962. To confirm the operation of two mechanisms, S. lactis 7962 was shown to hydrolyze lactose to glucose and galactose, whereas S. lactis C(2)F was unable to split the disaccharide. In addition, whole cells of S. lactis C(2)F rapidly accumulated a phosphorylated derivative of thiomethyl-beta-d-galactoside (TMG) which behaved chromatographically and electrophoretically like TMG-PO(4). Unexpectedly, S. lactis 7962 also accumulated a TMG derivative, although the rate was extremely low. These data indicate that different mechanisms of lactose utilization exist in the two strains, with a phosphorylation step dependent on PEP involved in S. lactis C(2)F.     

Properties of a Streptococcus lactis strain that ferments lactose slowly

Streptococcus lactis 7962, which ferments lactose slowly, has a lactose phosphoenolpyruvate-dependent phosphotransferase system and low phospho-beta-galactosidase activity, in addition to high beta-galactosidase activity. Lactose 6'-phosphate accumulated to a high concentration (greater than 100 mM) in cells growing on lactose. In contrast, lactic streptococci, which ferment lactose rapidly and use only the lactose-phosphotransferase system for uptake, contained high phospho-beta-galactosidase activity and low concentrations (0.9 to 1.6 mM) of lactose 6'-phosphate. It is concluded that rate-limiting phospho-beta-galactosidase activity is primarily responsible for defective lactose metabolism in S. lactis 7962.     

Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus

To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing Streptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The pgmA gene, encoding PGM in S. thermophilus LY03, was identified and cloned. The gene was functional in Escherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.     

Loss of Lactose Metabolism in Lactic Streptococci

Lactose-negative mutants occurred spontaneously in broth cultures of Streptococcus lactis C(2)F. Instability of lactose metabolism was noted in other strains of S. lactis, in strains of S. cremoris, and in S. diacetilactis. Colonies of S. lactis C(2)F grown with lactose as the carbohydrate source also possessed lac(-) cells. Treatment of lactic streptococci with the mutagen acriflavine (AF) increased the number of non-lactose-fermenting variants. The effect of AF on growth and on loss of lactose-fermenting ability in S. lactis C(2)F was consequently further examined. The presence of AF appears to favor competitively the growth of spontaneously occurring lactose-negative cells and appears to act in the conversion of lactose-positive to non-lactose-fermenting cells. The lactose-negative mutants partially revert to lactose-positive variants which remain defective in lactose metabolism and remain unable to coagulate milk. The lactose-negative cells become dominant in continuous culture growth and provide evidence that alterations in the characteristics of starter strains can be produced by continuous culture, in this case, the complete loss in ability to ferment lactose.     

Identification and characterization of a novel glucose-phosphorylating enzyme in Kluyveromyces lactis

Recent data suggest that hexokinase KlHxk1 (Rag5) represents the only glucose-phosphorylating enzyme of Kluyveromyces lactis, which also is required for glucose signalling. Long-term growth studies of a K. lactis rag5 mutant, however, reveal slow growth on glucose, but no growth on fructose. Isolation of the permissive glucose-phosphorylating enzyme, mass spectrometric tryptic peptide analysis and determination of basic kinetic data identify a novel glucokinase (KlGlk1) encoded by ORF KLLA0C01,155g. In accordance with the growth characteristics of the rag5 mutant, KlGlk1 phosphorylates glucose, but fails to act on fructose as a sugar substrate. Multiple sequence alignment indicates the presence of at least one glucokinase gene in all sequenced yeast genomes.     

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.     

Evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101

The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type C. beijerinckii NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.     

Regulation of glycolysis and sugar phosphotransferase activities in Streptococcus lactis: growth in the presence of 2-deoxy-D-glucose.

Streptococcus lactis K1 has the capacity to grow on many sugars, including sucrose and lactose, in the presence of high levels (greater than 500 mM) of 2-deoxy-D-glucose. Initially, growth of the organism was transiently halted by the addition of comparatively low concentrations (less than 0.5 mM) of the glucose analog to the culture. Inhibition was coincident with (i) rapid accumulation of 2-deoxy-D-glucose 6-phosphate (ca. 120 mM) and preferential utilization of phosphoenolpyruvate via the mannose:phosphotransferase system, (ii) depletion of phosphorylated glycolytic intermediates, and (iii) a 60% reduction in intracellular ATP concentration. During the 5- to 10-min period of bacteriostasis the intracellular concentration of 2-deoxy-D-glucose 6-phosphate rapidly declined, and the concentrations of glycolytic intermediates were restored to near-normal levels. When growth resumed, the cell doubling time (Td) and the steady-state levels of 2-deoxy-D-glucose 6-phosphate maintained by the cells were dependent upon the medium concentration of 2-deoxy-D-glucose. Resistance of S. lactis K1 to the potentially toxic analog was a consequence of negative regulation of the mannose:phosphotransferase system by two independent mechanisms. The first, short-term response occurred immediately after the initial "overshoot" accumulation of 2-deoxy-D-glucose 6-phosphate, and this mechanism reduced the activity (fine control) of the mannose:phosphotransferase system. The second, long-term mechanism resulted in repression of synthesis (coarse control) of enzyme IImannose. The two regulatory mechanisms reduced the rate of 2-deoxy-D-glucose translocation via the mannose:phosphotransferase system and minimized the activity of the phosphoenolpyruvate-dependent futile cycle of the glucose analog (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982). Phosphoenolpyruvate was thus conserved for transport of the growth sugar and for generation of ATP required for biosynthetic and work functions of the growing cell.