E. Stejskalová

Transport of histone intoEscherichia coli

Histone appears to be transported intoEscherichia coli cells in two ways: by an adsorption-like process and by a system which is temperature-dependent and is inhibited by 2,4-dinitrophenol. The adsorbed component cannot be washed out with a salt solution but can be partly exchanged for labelled histone and can be displaced with Tween 80. The actively transported histone can be replaced with unlabelled histone and with polylysine, and released by a high concentration of Mg2+ or by Tween 80. The active transport is observed only at concentrations greater than 100 μg histone/ml; the adsorption-like process occurring at lower concentrations.

Histone-induced changes of protein synthesis inEscherichia coli

Both arginine- and lysine-rich histones inhibited the inducible synthesis of β-galactosidase in the logarithmic phase ofEscherichia coli ML 30 whereas the synthesis of this enzyme in the stationary phase of growth was stimulated under conditions when the growth rate was unaffected. The inhibitory effect was greatest at the beginning of the logarithmic phase of growth and changed continuously into stimulatory, reaching its maximum 60 min after the beginning of the stationary phase of growth. The effect of arginine-rich histone was more pronounced than that of the lysine-rich histone and no other basic compounds tested exerted a similar influence upon the synthesis of the enzyme. The incorporation of14C uracil and leucine was inhibited resp. stimulated by arginine-rich histone in a similar direction as in the case of β-galactosidase, however a most pronounced inhibitory resp. stimulatory effect was observed on comparing the pulse label incorporation of14C uracil in the logarithmic resp. stationary phase of growth. We anticipate from this result and from the analysis of the early kinetics of induced β-galac-tosidase synthesis that the primary target of histone action might be the process of transcription. However, we also discuss other possibilities.

Selection of constitutive tryptophanase hyperproducing mutants of Escherichia coli

SummaryConstitutive tryptophanase hyperproducing mutants of Escherichia coli were isolated. The specific enzyme activities of these mutants are 3–5 times higher than those of the fully induced wild-type strain and the enzyme synthesis is more resistant to catabolite repression.

Simultaneous and successive induction of synthesis of β-Galactosidase and tryptophanase inEscherichia coli K 12 in the chemostat

Abstractβ-Galactosidase and tryptophanase were induced either simultaneously or successively during continuous cultivation of the inducible strainEscherichia coli K 12 in the chemostat. Growth was limited by glycerol and the dilution rate was 0.1 h−1. During both the simultaneous and successive induction specific rates of synthesis, as well as maximum enzyme levels, were identical with those obtained after independent induction of individual enzymes. As compared with batch cultivation, β-galactosidase reached the same specific rate of synthesis in the chemostat, whereas the specific rate of synthesis of tryptophanase in the chemostat was up to five times higher.

Simultaneous and gradual induction of β-galactosidase and tryptophanase synthesis in anEscherichia coli batch culture

Abstractβ-Galactosidase and tryptophanase can be induced inEscherichia coli simultaneously or gradually during a batch cultivation. In the strainEscherichia coli K 12 and ML 30, in which the synthesis of the two enzymes was induced simultaneously, only the synthesis of tryptophanase partially decreased, whereas the synthesis of β-galactosidase was not influenced. In the strains B 28 and ATCC 9637 the synthesis of both enzymes was partially decreased. On a gradual induction of these enzymes in the strainEscherichia coli E 12 only the synthesis of tryptophanase decreased. Thus, the results obtained here resemble those observed during the simultaneous induction. In addition, it was found that it is not important which of the two enzymes is induced as the first one.

Effect of cyclic adenosine-3′,5′-monophosphate on the simultaneous synthesis of β-galactosidase and tryptophanase inEscherichia coli

When inducing simultaneously β-galactosidase and tryptophanase in a batch culture either the synthesis of tryptophanase or of both enzymes is decreased due to an insufficient cAMP concentration. The addition of this nucleotide can overcome this decrease. In a continuous culture both enzymes are synthesized at the maximum rate, as the amount of cAMP produced during carbon limitation of growth is probably sufficient for the simultaneous synthesis of both enzymes. In the β-galactosidase hyperproduction mutant cultivated continuously the level of β-galactosidase markedly decreases when tryptophanase is simultaneously induced. Also this decrease is caused by cAMP insufficiency and can be overcome by increasing its concentration. cAMP is thus an important regulatory factor of both enzymes and becomes a limiting factor in their simultaneous synthesis; a competition for this regulatory compound apparently occurs and probably also a different mutual affinity of the regulatory complex with the promoter site of the enzyme opérons is involved.

Selection aid properties of hyperproducing D-serine deaminase mutants of Escherichia coli

SummaryHyperproducing constitutive D-serine deaminase mutant E. coli were selected by a chemostat method. The specific activity of the enzyme in these mutants is 25fold higher in comparison with the fully induced parental strain.

Enzyme synthesis ofl-tryptophan

Enzyme synthesis of tryptophan from indole, pyruvate and ammonium salts was studied usingEscherichia coli cells exhibiting a significant tryptophanase activity. In addition to the effect of cultivation medium composition and cultivation conditions, factors affecting the course of the conversion were investigated. Production of 32.4 g/L ofl-tryptophan was reached after 48 h under optimal conditions.

Spores of microorganisms. XXV. The effects of histone on the syntheses of macromolecules in germinating and outgrowing spores of Bacillus cereus.

Histone (final concentration 200 μg/ml) inhibits the synthesis of both proteins and total ribonucleic acid (RNA) in germinated spores ofBacillus cereus. It also blocks a further cytodifferentiation of germinated spores into vegetative cells. The inhibition takes place both in complex media facilitating the differentiation (media with bactopeptone or casamino acids) and in limited minimal medium which permits only the germination and the synthesis of a limited amount of proteins and RNA. At this concentration, the histone inhibits strongly the synthesis of pulse-labelled RNA and brings about a change in the sedimentation constants of ribosomes. The results are interpreted in terms of the strong affinity of the histone towards electronegative peripheral layers and intracellular structures and macromolecules of the germinated spore.

Stability and storage ofEscherichia coli mutants hyperproducing β-galactosidase

SummaryEnzyme activity of the hyperproducing mutants isolated from a chemostat decreases by passaging under nonselective conditions to about one half of the original value, and then remains stable. High activity can be quickly restored by transfer to chemostat selective conditions. The elaborated storage method prevents the decrease of enzyme activity after 2–3 years.