Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant

Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO(2) production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.     

Thialysine- and selenalysine-resistance in a E. coli mutant

A thialysine-resistant mutant of E. coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium. No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Fructose 1,6-bisphosphate-activated pyruvate kinase from E. coli: ligand promoted conformational changes.

The ligand-dependent susceptibility to heat inactivation and to tryptic digestion and the intrinsic fluorescence of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli were investigated in the absence and in the presence of physiological ligands. With respect to the enzyme alone, binding of the allosteric activator fructose 1,6-bisphosphate makes the protein sensitive to tryptic attack and thermolabile, while binding of phosphoenolpyruvate and Mg2+, but not of either ligand separately, induces in the enzyme a highly thermostable conformation, the attainment of which does not require an ordered binding sequence of the two ligands. The apparent loosening of the enzyme structure induced by fructose bisphosphate suggests that the activation it exerts at low phosphoenolpyruvate concentration might be due to an increased accessibility of substrate to the active site.     

Selenalysine utilization for growth and protein synthesis by a lysine requiring E. coli mutant

Summary Selenalysine can be utilized in substitution of lysine by a lysine requiring E. coli mutant. The presence of some lysine in the culture medium is necessary to allow selenalysine utilization for growth; in the presence of an excess of lysine, selenalysine is not utilized. When utilized, selenalysine gives rise to an increase of final growth. However, it shows some toxic effects as demonstrated by the decrease of both growth rate and cell viability. Selenalysine is incorporated into proteins in substitution of lysine. Up to a maximum of 50% of total protein lysine can be substituted. The decrease of cell viability is correlated with the extent of lysine substitution.This paper is dedicated to Professor A. E. Braunstein on his 80th birthday.     

Inhibitory effect of Li+ on cell growth and pyruvate kinase activity of Escherichia coli.

Li+ inhibited growth of Escherichia coli when glucose, galactose, fructose, or glycerol was added as the sole source of carbon. Growth inhibition was not observed when lactate or a mixture of amino acids was used as the carbon source. A mutant possessing elevated activity of Li+ extrusion was not inhibited by Li+. These results suggested that intracellular Li+ inhibited the glycolytic pathway, most likely triose metabolism, without affecting gluconeogenesis. We also found that pyruvate kinase I was inhibited by Li+.     

Aspartokinase III repression in a thialysine-resistant mutant of E. coli

A thialysine-resistant mutant of the E. coli KL16 strain was isolated. It can grow equally well in the presence and in the absence of thialysine. The properties of the two lysine transport systems, of the lysyl-tRNA synthetase and of the aspartokinase III (AK III) were studied in the mutant and in the parent strain. AK III is the first enzyme of the lysine biosynthetic pathway and its activity is involved in the regulation of lysine biosynthesis by feed-back and repression mechanism. No difference between the two strains was evidenced as regards 1) the affinity of the transport systems for lysine and thialysine 2) the activity of the lysyl-tRNA synthetase 3) the allosteric inhibition of the AK III by lysine and thialysine. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the enzyme is much less repressed both by lysine and thialysine. The possible correlation between the activity of AK III and the thialysine-resistance is discussed in this paper.     

Cell length in a wee dnaA mutant of Escherichia coli.

The cell length of the short siblings of dividing pairs formed in the absence of replication by two strains of Escherichia coli, OV-25-9 [dnaA46 wee(Am)] and OV-25-10 [dnaA46 wee(AM) supF] was measured. In the presence of Wee, the length of these cells increased to those values expected for newborn wild-type cells growing under similar conditions. In its absence, cell length remained at values near the minimum unit length possible for newborn cells. Our results show that both cell elongation and the action of Wee are independent of DNA replication, being compatible with the role proposed for Wee in coordination between cell elongation and division.     

Utilization of lysine analogs by a lysine-requiring E. coli mutant

Thialysine and selenalysine cannot substitute lysine as a growth factor for a lysine-requiring E. coli mutant, but can nevertheless be utilized for protein synthesis in the presence of lysine. In order to have information about the effects of lysine on the utilization of the two analogs, the extent of the incorporation of the three aminoacids into newly synthesized proteins has been determined. The analog starts to be utilized by cells growing in a medium containing either analog and lysine when lysine concentration becomes very low. Of the two analogs, thialysine is more easily utilized. In fact thialysine can be utilized when the lysine/thialysine ratio in the medium is 1/25. Selenalysine starts to be utilized when the lysine/selenalysine ratio is 1/200.     

Cell surface proteins of E. coli

Cell surface proteins of $\text{E. coli}$ K12 have been labelled with ¹²⁵I using a lactoperoxidase method. Results suggest that most outer membrane proteins so far characterised appear to have at least part of their polypeptide chain on the cell surface. These include major outer membrane protains I and II¹, the maltose and vitamin B₁₂ binding proteins and proteins involved in iron transport. The labelling of an antibiotic sensitive mutant and its parent were compared but their labelling patterns did not appear to differ in any way which would suggest the cause of the permeability difference between these two strains.     

A new RNA synthesis mutant of E. coli

A temperature-sensitive mutant of E. coli is described. At the nonpermissive temperature, the capacity for RNA and protein synthesis decreases logarithmically in the mutant. The mutant is unable to support the growth of f2 or T7 virus, even at the permissive temperature. The temperature-sensitive mutation maps approximately 1 away from rif ^r in E. coli and therefore affects a gene previously undescribed. The temperature sensitivity is suppressed by sublethal concentrations of rifampicin. Moreover, in rif ^r T^s double mutants, the T ^s mutation suppresses rif ^r and vice versa. The partially purified RNA polymerases from mutant and wild-type cells have different temperature and salt optima.This research was supported by Public Health Service grant GM-14368 from the National Institute of General Medical Sciences and by grant IN-29 from the American Cancer Society. One of us (D.P.) is a predoctoral trainee, supported by a National Science Foundation Graduate Traineeship Program and by a National Institutes of Health Predoctoral Research Fellowship. S. Marshall is supported by LASBAU.     

Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032

To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum.     

Enhanced recombinant protein production in pyruvate kinase mutant of Bacillus subtilis

Previous work demonstrated that acetate production was substantially lower in pyruvate kinase (pyk) mutant of Bacillus subtilis. The significantly lower acetate production in the pyk mutant is hypothesized to have positive effect on recombinant protein production either by lifting the inhibitory effect of acetate accumulation in the medium or redirecting the metabolic fluxes beneficial to biomass/protein synthesis. In this study, the impact of the pyk mutation on recombinant protein production was investigated. Green fluorescent protein (GFP+) was selected as a model protein and constitutively expressed in both the wild-type strain and a pyk mutant. In batch cultures, the pyk mutant produced 3-fold higher levels of recombinant protein when grown on glucose as carbon source. Experimental measurements and theoretical analysis show that the higher protein yield of the mutant is not due to removal of an acetate-associated inhibition of expression or gene dosage or protein stability but a much lower acetate production in the mutant allows for a greater fraction of carbon intake to be directed to protein synthesis.     

Expression and characterisation in E. coli of mutant forms of saporin

In the present communication, we report on the expression and characterisation in Escherichia coli of mutant derivatives of saporin, a type 1 ribosome-inactivating protein from Saponaria officinalis L. The effects of substitution of Glu 176 with Lys and those of deletion of 19 amino acids at the C-terminal were evaluated both in vivo, testing the influence of expressed proteins on bacterial growth and in vitro measuring their N-glycosidase and supercoiled DNA relaxation activities. Results indicate that both modifications of the wild-type protein abolish its toxicity to bacterial cells and impair its enzymatic activity on polynucleotide substrates, either RNA or DNA.     

Purification and molecular properties of the AMP-activated pyruvate kinase from Escherichia coli.

The AMP-activated pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Escherichia coli has been purified 200 times through a three-step procedure which gives a homogeneous preparation with a specific activity of 110. The enzyme appears to be a tetramer of molecular weight 190 000. Subunits (molecular weight 51 000) show a single amino-terminal amino acid (serine) and appear as a single band in polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The enzyme crystallizes in conditions of reduced dielectric constant of the solvent in the pH range 6.5-7.5. Kinetic and regulatory properties of the purified enzyme are similar to those described for crude preparations of the enzyme.