Glycerol-specific revertants of a phosphoenolpyruvate phosphotransferase mutant: suppression by the desensitization of glycerol kinase to feedback inhibition

Glycerol-specific revertants were isolated from a phosphoenolpyruvate phosphotransferase mutant lacking enzyme I activity. Sixteen of the eighteen separately derived revertants were found to synthesize a fully active glycerol kinase no longer subject to feedback inhibition by fructose 1,6-diphosphate. The suppressor mutation mapped at the known glpK locus. When the fructose, 1,6-diphosphate-insensitive kinase allele was transduced into a strain producing the glp enzymes constitutively, cells of the resultant strain were susceptible to killing by glycerol if this compound was added to a culture growing exponentially in casein hydrolysate. This phenomenon had been previously described for a strain which had a constitutive glycerol kinase refractory to feedback inhibition, but isolated by a different procedure. It is suggested that the suppression of the growth defect on glycerol in the enzyme I(-) mutant by the fructose 1,6-diphosphate-insensitive kinase is achieved by increasing the in vivo catalytic potential of glycerol kinase. This increased activity would allow more rapid conversion of glycerol to l-alpha-glycerophosphate, the true inducer of the glp system. The enzyme I defect in the parental strain impaired the inducibility of the glp system so that the normal basal catalytic activity of the kinase was insufficient to insure induction by glycerol.     

Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator.

Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein. We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.     

Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.     

Properties of a revertant of Escherichia coli viable in the presence of spermidine accumulation: increase in L-glycerol 3-phosphate.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.     

Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Interaction between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and glycerol kinase of Salmonella typhimurium.

Purified IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system of Salmonella typhimurium inhibits glycerol kinase. Phosphorylation of IIIGlc via phosphoenolpyruvate, enzyme I, and HPr abolishes this inhibition. The glycerol facilitator is not inhibited by IIIGlc. It is proposed that regulation of glycerol metabolism by the phosphoenolpyruvate:sugar phosphotransferase system is at the level of glycerol kinase.     

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms

We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.     

Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product.

The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.     

Purification and characterization of glpX-encoded fructose 1, 6-bisphosphatase, a new enzyme of the glycerol 3-phosphate regulon of Escherichia coli

In Escherichia coli, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was dimeric, dependent on Mn(2+) for activity, and exhibited an apparent K(m) of 35 microM for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.     

Comparison of the Frequencies of Spontaneous and Chemically-Induced 5-Bromodeoxyuridine-Resistance Mutations in Wild-Type and Revertant Bhk-21/13 Cells

5-bromodeoxyuridine resistance mutations induced by mutagenesis were studied. The average expression time for induced mutations varied with the concentration of the mutagen ethyl methanesulfonate (EMS). However, a constant number of two generation times was necessary for half maximal expression of induced mutations. Also, induced mutation rates were compared under optimal expression conditions for bromodeoxyuridine, fluorodeoxyuridine and azaguanine resistance markers. Ten independent bromodeoxy-uridine-resistant clones were tested for reversion. Two clones reverted-one spontaneously and the other after mutagenesis. The spontaneous rate of mutation to bromodeoxyuridine resistance, estimated by the fluctuation test, was high in revertant clones (4 x 10(-6) / cell / generation) and low in the wild-type cells (< 3.5 x 10(-8) / cell / generation). A comparison of induced mutation frequencies at variable EMS concentrations showed a single-hit curve for revertant clones and a multihit curve for the wild-type cells. Thymidine kinase activities of resistant clones were usually less than 2% of that of the wild-type clone. Inducibility, thermal stability and intracellular localization of the thymidine kinases of the wild-type cells and of a revertant clone were identical. A low, but significant (P < 0.10), Km discrepancy was observed between enzyme extracts of these lines. The genetic implications of these results are discussed.     

Genetic Characterization of the SufJ Frameshift Suppressor in SALMONELLA TYPHIMURIUM

A new suppressor of +1 frameshift mutations has been isolated in Salmonella typhimurium. This suppressor, sufJ, maps at minute 89 on the Salmonella genetic map between the argH and rpo(rif) loci, closely linked to the gene for the ochre suppressor tyrU(supM). The suppressor mutation is dominant to its wild-type allele, consistent with the suppressor phenotype being caused by an altered tRNA species. The sufJ map position coincides with that of a threonine tRNA(ACC/U) gene; the suppressor has been shown to read the related fourbase codons ACCU, ACCC, ACCA.--The ability of sufJ to correct one particular mutation depends on the presence of a hisT mutation which causes a defect in tRNA modification. This requirement is allele specific, since other frameshift mutations can be corrected by sufJ regardless of the state of the hisT locus.--Strains carrying both a sufJ and a hisT mutation are acutely sensitive to growth inhibition by uracil; the inhibition is reversed by arginine. This behavior is characteristic of strains with mutations affecting the arginine-uracil biosynthetic enzyme carbamyl phosphate synthetase. The combination of two mutations affecting tRNA structure may reduce expression of the structural gene for this enzyme (pyrA).     

Characterization of a glycerol kinase mutant of Aspergillus niger

A glycerol-kinase-deficient mutant of Aspergillus niger was isolated. Genetic analysis revealed that the mutation is located on linkage group VI. The phenotype of this mutant differed from that of a glycerol kinase mutant of Aspergillus nidulans in its ability to utilize dihydroxyacetone (DHA). The weak growth on glycerol of the A. niger glycerol kinase mutant showed that glycerol phosphorylation is an important step in glycerol catabolism. The mutant could still grow normally on DHA because of the presence of a DHA kinase. This enzyme, probably in combination with an NAD(+)-dependent glycerol dehydrogenase, present only in the mutant, is responsible for the weak growth of the mutant on glycerol. Enzymic analysis of both the mutant and the parental strain showed that at least three different glycerol dehydrogenases were formed under different physiological conditions: the NAD(+)-dependent enzyme described above, a constitutive NADP(+)-dependent enzyme and a D-glyceraldehyde-specific enzyme induced on D-galacturonate. The glycerol kinase mutant showed impaired growth on D-galacturonate.     

A single amino acid change in Escherichia coli glycerol kinase abolishes glucose control of glycerol utilization in vivo.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in glucose control of glycerol metabolism. Its catalytic activity is inhibited allosterically by the glycolytic intermediate, fructose 1,6-biphosphate, and by the phosphotransferase system phosphocarrier protein, IIIGlc (also known as IIAGlc). These inhibitors provide mechanisms by which glucose blocks glycerol utilization in vivo. We report here the cloning and sequencing of the glpK22 gene isolated from E. C. C. Lin strain 43, a strain that shows the loss of glucose control of glycerol utilization. DNA sequencing shows a single missense mutation that translates to the amino acid change Gly-304 to Ser (G-304-S) in glycerol kinase. The effects of this substitution on the functional and physical properties of the purified mutant enzyme were determined. Neither of the allosteric ligands inhibits it under conditions that produce strong inhibition of the wild-type enzyme, which is sufficient to explain the phenotype of strain 43. However, IIIGlc activates the mutant enzyme, which could not be predicted from the phenotype. In the wild-type enzyme, G-304 is located 1.3 nm from the active site and 2.5 nm from the IIIGlc binding site (M. Feese, D. W. Pettigrew, N. D. Meadow, S. Roseman, and S. J. Remington, Proc. Natl. Acad. Sci. USA 91:3544-3548, 1994). It is located in the same region as amino acid substitutions in the related protein DnaK which alter its catalytic and regulatory properties and which are postulated to interfere with a domain closure motion (A. S. Kamath-Loeb, C. Z. Lu, W.-C. Suh, M. A. Lonetto, and C. A. Gross, J. Biol. Chem. 270:30051-30059, 1995). The global effect of the G-304-S substitution on the conformation and catalytic and regulatory properties of glycerol kinase is consistent with a role for the domain closure motion in the molecular mechanism for glucose control of glycerol utilization.     

Isolation and Properties of Selenomethionine-Resistant Mutants of NEUROSPORA CRASSA

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.     

Glycerol kinase, the pacemaker for the dissimilation of glycerol in Escherichia coli

The activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. A mutant strain producing a glycerol kinase resistant to inhibition by fructose-1,6-diphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon and energy. The amount of intracellular fructose-1,6-diphosphate was determined for wild-type cells growing exponentially on glycerol. The water content of such cells was also determined, allowing calculation of the intracellular concentration of fructose-1,6-diphosphate. This value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type glycerol kinase. The desensitization of glycerol kinase to feedback inhibition also enhances the power of glycerol to exert catabolite repression, both on the enzymes of the glycerol system itself and on those of the lactose system. However, desensitization of glycerol kinase alone does not eliminate the phenomenon of diauxic growth in a glucose-glycerol medium. Biphasic growth in such a medium is abolished if the altered enzyme is produced constitutively. The constitutive production of the mutant kinase at high levels, however, renders the cells vulnerable to glycerol. Thus, when the cells have been grown on a carbon source with a low power for catabolite repression, e.g., succinate, sudden exposure to glycerol leads to overconsumption of the nutrient and cell death.     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.     

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.     

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.