PfkA locus of Escherichia coli.

pfkA was know, on the basis of three mutants, as the likely locus of phosphofructokinase in Escherichia coli, and the unlinked pfkB1 mutation suppressed these mutations by restoring some enzyme activity (Morrissey and Fraenkel, 1972). We now report a new search for the complete inactivation of pfkA (e.g., by deletion or amber mutation), done to assess whether the pfkB1 suppression is by an independent enzyme, phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973). Ten new phosphofructokinase mutants all were at pfkA, rather than at pfkB or pfkC. One of them (pfkA9) gave temperature-sensitive reverants with heat-labile enzyme. Another (pfkA11) proved genetically to be a nonsense mutation, but showed no restored activity when suppressed by supF. However, even unsuppressed it was found to contain an enzyme related to phosphofructokinase activity 1 kinetically (more allosteric), physically (almot identical subunit), and antigenically. All the pfkA mutants apparently contained cross-reacting material to activity 1. All (including pfkA11) were suppressed by the pfkB1 mutation. Several results support the idea that pfkA is the structural gene for the main phosphofructokinase of E. coli (activity 1), but that there is some restriction to its complete inactivation.     

Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme.

The main phosphofructokinase of Escherichia coli (PFK I) is an extensively studied allosteric enzyme specified by the pfkA gene. A nonallosteric phosphofructokinase was reported (Fraenkel, D.G., Kotlarz, D., and Bluc, H. (1973) J. Biol. Chem. 248, 4865-4866) in strains carrying the pfkB1 mutation, a suppressor of pfkA mutants, and very low levels of this enzyme have also been detected in strains not carrying the suppressor (i.e. pfkB+). The nonallosteric protein has now been prepared pure from three strains, one carrying pfkB1 and pfkA+, one carrying pfkB1 and completely deleted for pfkA, and one carrying pfkB+ and also deleted for pfkA. It is apparently the same enzyme (PFK II) in all three strains, which shows that pfkB1 is a mutation affecting the amount of a normally minor isozyme. PFK II is a tetramer of slightly larger subunit molecular weight than PFK I (36,000 and 34,000, respectively). No immunological cross-reactivity was detected between PFK II and PFK I. Unlike PFK I, PFK II does not show cooperative interactions with fructose-6-P, inhibition by P-enolpyruvate, or activation by ADP. Also unlike PFK I, PFK II is somewhat sensitive to inhibition by fructose-1,6-P2 and can use tagatose-6-P as substrate. Both enzymes can perform the reverse reaction, fructose-6-P + ATP from fructose-1,6-P2 + ADP in vitro, but not in vivo. The normal function of PFK II is not known.     

Tn10 insertions in the pfkB region of Escherichia coli.

The locus pfkB is known to determine expression of a minor phosphofructokinase (Pfk-2). Pfk-2 and pfkB seem to be dispensable, since Tn10 insertions in pfkB, as well as deletions from Tn10 nearby, are obtainable. Strains deleted for both pfkA and pgkB are unable to grow at all on sugars whose primary route of metabolism is via fructose 6-phosphate, confirming earlier reports implicating the low Pfk-2 activity, rather than the pentose-phosphate pathway, as needed for the slow growth on sugars of pfkA pfkB+ strains. The pfkB locus probably contains the structural gene for Pfk-2, since a mutation closely linked to pfkB1, which affects growth on glycerol, is found to alter the enzyme. Partial phenotypic suppression of the pfkA mutant phenotype results from Tn10 insertion very close to the pps gene, ca. 0.5 min from pgkB. The insertion does not clearly affect either Pfk-2 or phosphoenolpyruvate synthetase, and the mechanism of suppression is unclear.     

Phenotypic suppression of phosphofructokinase mutations in Escherichia coli by constitutive expression of the glyoxylate shunt

Fructose-6-phosphate kinase (pfkA) mutants have impaired growth on carbon sources which enter glycolysis at or above the level of fructose-6-phosphate, but the degree of impairment depends on the carbon source (e.g., growth on glucose is very much slower than growth on glucose-6-phosphate). The present report contains considerable data on this complicated growth phenotype and derives mainly from the finding of a class of partial revertants which grow as fast on glucose as on glucose-6-phosphate; the reversion mutation is shown to be constitutivity of the glyoxylate shunt (iclR(c)). iclR(c) does not increase the fructose-6-phosphate kinase level in the mutants, and the exact mechanism of the partial phenotypic suppression is not understood. However, iclR(c) was already known to suppress some mutations which affected phosphoenolpyruvate levels, and H. L. Kornberg and J. Smith have suggested (1970) that the growth phenotype of pfkA mutants might be related to pathways of phosphoenolpyruvate formation. Surprisingly, the hexose-monophosphate shunt is not necessary for the suppression, which therefore must act to restore metabolism via the residual phosphofructokinase activity present in all pfkA mutants. A mutant totally lacking phosphofructokinase activity was not suppressed.     

An alteration in phosphofructokinase 2 of Escherichia coli which impairs gluconeogenic growth and improves growth on sugars

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.     

Analysis of mutations affecting the dissmilation of galactitol (dulcitol) in Escherichia coli K 12.

Summary Three mutations clustered at 45.5 min of the genetic map of E. coli K12 have been shown previously (Lengeler, 1975a) to affect specifically galactitol transport via an enzyme II-complex^Gat (gatA) of the PEP dependent phosphotransferase system and a soluble, NAD dependent dehydrogenase (gatD). In the present report data are given further supporting the existence of a gat operon, made up by a control gene gatC and the structural genes gatA and gatD. The enzyme II-complex^Gat is shown to catalyze the formation of galactitol-1-P and the dehydrogenase to catalyze the reversible conversion of galactitol-1-P and D-tagatose-6-P. Loss of a phosphofructokinase activity controlled by the gene pfkA prevents growth on galactitol and concomitantly the formation of D-tagatose-1,6-P₂, while the suppressing mutation pfkB-1 restores a phosphofrucokinase activity and growth on galactitol.As shown further the erratic growth behaviour of E. coli K12, B and C on galactitol is apparently due to a temperature sensitive ketose-bis-phosphate aldolase inactive at temperatures >35° C. This enzyme reacts with D-tagatose-1,6-P₂ and to a lesser extent with D-fructose-1,6-P₂ and thus is able to suppress fda mutations. It is controlled by a new gene locus kba located within 1 min of the marker argG, remoted from the gat operon and the gene fda. Galactitol dissimilation in E. coli K12 thus seems to be via galactitol-1-P-D-tagatose-6-P-D-tagatose-1,6-P₂ to dihydroxyacetone-P+glyceraldehyde-P, controlled by the genetic loci gatC A D, pfkA, pfkB-1 and kba respectively.     

Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources

As the key obligatory step in the glycolytic pathway, the regulation of phosphofructokinase (PFK-1) has been the focus of study of several laboratories. While standard cloning procedures have opened the door to study PFK from a vast array of sources, a good pfk knockout Escherichia coli strain has not previously been developed. Many laboratories rely on DF1020 or similar derivatives for PFK expression. Unfortunately, DF1020 grows poorly and does not have an inherent means for controlling expression of genes from plasmids. More importantly, however, DF1020 has a tendency to grow on minimal media when glucose is used as the sole carbon source. In this study, a new E. coli PFK expression strain lacking both PFK-1 and PFK-2 has been engineered using lambda-red mediated chromosomal deletion. The resulting strain has been designated RL257. In addition to having both pfkA and pfkB deleted, RL257 contains the lacI(q) allele, which allows for inducible expression when coupled with an expression vector containing either the lac or tac promoter.     

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.     

Metabolic analysis of adaptive evolution for in silico-designed lactate-producing strains

Experimental evolution is now frequently applied to many biological systems to achieve desired objectives. To obtain optimized performance for metabolite production, a successful strategy has been recently developed that couples metabolic engineering techniques with laboratory evolution of microorganisms. Previously, we reported the growth characteristics of three lactate-producing, adaptively evolved Escherichia coli mutant strains designed by the OptKnock computational algorithm. Here, we describe the use of (13)C-labeled experiments and mass distribution measurements to study the evolutionary effects on the fluxome of these differently designed strains. Metabolic flux ratios and intracellular flux distributions as well as physiological data were used to elucidate metabolic responses over the course of adaptive evolution and metabolic differences among strains. The study of 3 unevolved and 12 evolved engineered strains as well as a wild-type strain suggests that evolution resulted in remarkable improvements in both substrate utilization rate and the proportion of glycolytic flux to total glucose utilization flux. Among three strain designs, the most significant increases in the fraction of glucose catabolized through glycolysis (>50%) and the glycolytic fluxes (>twofold) were observed in phosphotransacetylase and phosphofructokinase 1 (PFK1) double deletion (pta- pfkA) strains, which were likely attributed to the dramatic evolutionary increase in gene expression and catalytic activity of the minor PFK encoded by pfkB. These fluxomic studies also revealed the important role of acetate synthetic pathway in anaerobic lactate production. Moreover, flux analysis suggested that independent of genetic background, optimal relative flux distributions in cells could be achieved faster than physiological parameters such as nutrient utilization rate.     

Ca2+-mediated activation of phosphofructokinase in perfused rat heart.

Perfusion of the isolated rat heart with Ca2+ concentrations exceeding 3 mM activated phosphofructokinase and phosphorylase, and decreased the concentration of cyclic AMP. Half-maximal activation of phosphofructokinase occurred at 5 mM-CaCl2; significant activation of phosphorylase did not occur until the concentration of CaCl2 exceeded 12 mM. The time course for the activation of phosphofructokinase at 12 mM-CaCl2 indicated that maximal activation occurred within 2 min; when the perfusion-medium Ca2+ concentration was re-adjusted to 3 mM, the phosphofructokinase activity returned to pre-activation values within 30 s. The addition of Ca2+ to extracts of heart did not activate phosphofructokinase. The activation of phosphofructokinase by sub-maximal doses of adrenaline and Ca2+ were not additive. The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol and by 1 microM-isoprenaline was inhibited by high concentrations of K+ (22-56 mM). The activation of phosphofructokinase by 1 microM-adrenaline + 10 microM-propranolol, 12 mM-CaCl2 and by 1 microM-isoprenaline was blocked by the slow Ca2+-channel blocker nifedipine. These findings suggest that both the beta- and alpha-adrenergic mechanisms for the activation of rat heart phosphofructokinase involve an increase in the myoplasmic Ca2+ concentration. This increase may result from an inhibition of Ca2+ efflux or a stimulation of Ca2+ influx.     

Rapid inhibition by intragastric triolein of the re-activation of glucose utilization and lipogenesis in the mammary gland during the starved-refed transition in lactating rats. Evidence for a direct effect of oral lipid on mammary tissue.

1. Oral administration of triacylglycerol (triolein) to starved/chow-refed lactating rats suppressed the lipogenic switch-on in the mammary gland in vivo. 2. A time-course study revealed that triolein, administered at 30 min after the onset of refeeding, had no influence on lipogenic rate in the mammary gland between 30 and 60 min, but markedly decreased it between 60 and 90 min. Glucose uptake by the mammary gland (arteriovenous difference) increased by 30 min of refeeding, as did lactate production. Between 30 and 90 min glucose uptake remained high in the control animals, but glucose uptake and net C3-unit uptake were decreased in the triolein-loaded animals by 90 min. 3. Triolein increased [glucose 6-phosphate] in the gland and simultaneously decreased [fructose 1,6-bisphosphate], indicative of a decrease in phosphofructokinase activity. This cross-over occurred at 60 min, i.e. immediately before the inhibition of lipogenesis, and by 90 min had reached 'starved' values. 4. Triolein had no effect on plasma [insulin] nor on whole-blood [glucose], [lactate] or [3-hydroxybutyrate]; a small increase in [acetoacetate] was observed. 5. Infusion of the lipoprotein lipase inhibitor, Triton WR1339, abolished the suppression of mammary-gland lipogenesis by triolein and the increase in the [glucose 6-phosphate]/[fructose 1,6-bisphosphate] ratio, suggesting a direct influence of dietary lipid on mammary-gland glucose utilization and phosphofructokinase activity.     

Nucleotide sequence of gene pfkB encoding the minor phosphofructokinase of Escherichia coli K-12

The nucleotide sequence of a 1.3-kb DNA fragment containing the entire pfkB gene which codes for Pfk-2 of Escherichia coli, a minor phosphofructokinase (Pfk) enzyme, is reported. The Pfk-2 protein subunit is encoded by 924 bp, has 308 amino acids and an Mr of 33 000. Like other weakly expressed E. coli genes the codon usage in the pfkB gene is random; there is no strong bias for the usage of major tRNA isoaccepting species, and the codon preference rules of Grosjean and Fiers [Gene, 18 (1982) 199-209] are followed. This is the first report of the complete gene sequence of a phosphofructokinase.     

Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli

The closely linked structural genes of phosphofructokinase (pfkA) and triosephosphate isomerase (tpi) of Escherichia coli were separately cloned onto plasmid pBR322. By gene dosage effects, transformed cells of E. coli C600 with these pBR322 hybrid plasmids showed 7- and 16-fold increases in the specific activities of phosphofructokinase and triosephosphate isomerase, respectively, over the specific activities in C600. Dried preparations of E. coli cells dosed with these genes showed appreciably high ATP-regenerating activity.     

Two new genes (smhA and lytE) apparently functionally related to the murH gene of Escherichia coli

The murH mutant of Escherichia coli exhibits temperature-sensitive growth and lysis at the restrictive temperature. Temperature-resistant derivatives of the mutant occurred at a frequency of about 3 X 10(-6). All of the seven independent isolates examined were shown to be pseudorevertants carrying extragenic suppressors of murH, which mapped at 24.5 min on the linkage map. One allele, apparently representing a new locus, designated smhA, was characterized further. The smhA mutation by itself conferred no recognizable phenotype. However, smhA suppressed the temperature-sensitive lysis phenotype of the murH mutant. The smhA mutant acquired a spontaneous mutation in another new gene, designated lytE, which was mapped at 25 min. The lytE mutation by itself conferred a temperature-sensitive lysis phenotype indistinguishable from that of the murH mutant. The lytE mutation was suppressed by smhA as well as by another suppressor of murH designated smhB. The suppressor activity of smhA was apparently relatively specific in that smhA failed to prevent lysis caused by either mutational or antibiotic-induced blocks in peptidoglycan synthesis. The possibility that the smhA and lytE genes are functionally related to murH is considered.     

Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other gram-negative bacteria.

The Escherichia coli pfkA gene has been cloned in the non-self-transmissible vector pVK101 from hybrid plasmids obtained from the Clarke and Carbon clone bank, resulting in the plasmids pAS300 and pAS100; the latter plasmid also encoded the E. coli tpi gene. These plasmids were transferred by conjugation to mutants of Alcaligenes eutrophus which are unable to grow on fructose and gluconate due to lack of 2-keto-3-deoxy-6-phosphogluconate aldolase activity. These transconjugants recovered the ability to grow on fructose and harbored pAS100 or pAS300. After growth on fructose, the transconjugants contained phosphofructokinase at specific activities between 0.73 and 1.83 U/mg of protein, indicating that the E. coli pfkA gene is readily expressed in A. eutrophus and that the utilization of fructose occurs via the Embden-Meyerhof pathway instead of the Entner-Doudoroff pathway. In contrast, transconjugants of the wild type of A. eutrophus, which are potentially able to catabolize fructose via both pathways, grew at a decreased rate on fructose and during growth on fructose did not stably maintain pAS100 or pAS300. Indications for a glycolytic futile cycling of fructose 6-phosphate and fructose 1,6-bisphosphate are discussed. Plasmid pA 100 was also transferred to 14 different species of gram-negative bacteria. The pfkA gene was expressed in most of these species. In addition, most transconjugants of these strains and of A. eutrophus exhibited higher specific activities of triosephosphate isomerase than did the corresponding parent strains.