Inhibition ofCandida utilis by cycloheximide and acetoxycycloheximide

Cycloheximide and acetoxycyloheximide were similar in their effect on the synthesis of RNA and protein by exponentially growing cells ofCandida utilis. Both antibiotics inhibited the synthesis of protein more than that of RNA. During inhibition there was preferential synthesis of ribosomal protein and some completed ribosomes were formed. However, the synthesis of ribosomal RNA was reduced more than that of transfer RNA. The actions of these drugs onCandida utilis are compared with the effects of other antibiotics whose primary effect on bacteria is to inhibit protein synthesis.     

Fermentation ofCandida utilis for uricase production

Summary Conditions for the production of microbial uricase byCandida utilis were studied. For the selected strain, hypoxanthine proved to be the most effective inducer of uricase formation. The highest values of biomass as well as uricase activity in the mechanically agitated fermentor were obtained under the following conditions: 50 h, rotation impeller speed 7 s^–1, air flow rate 1.25×10^–5 m³s^–1, concentration of inducer 0.1%.List of symbols b width of baffle, m - c length of baffle, m - D diameter of cylindrical fermentor, m - d diameter of impeller, m - d ₁ diameter of impeller disc, m - Fr _m impeller Froud number - g gravitional acceleration, ms^–2 - H height of batch surface above bottom, m - H ₂ height of impeller disc above bottom, m - h height of impeller blade, m - Kp _g flow rate number - L length of impeller blade, m - N rotational speed of impeller, s^–1 - Re _m impeller Reynolds number - T time, h - V volume of batch, m³ - V _g air (gas) flow rate, m³s^–1 - x mass fraction of the dry matter of cells - x ₀ initial value of the mass fraction of the dry matter of cells - _r volume fraction of the dry matter of cells - <eta<₁ viscosity of pure liquid, Pa s - viscosity of batch (suspension of microbial suspension), Pa s - density of batch, kg m^–3     

Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg²⁺ or Mn²⁺, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K_i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.     

Biosynthesis of methionine in an ethionine-resistant strain ofCandida utilis

The strainCandida utilis T 20 adapted to a high concentration of ethionine, excretes considerable amounts of methionine in a synthetic medium, about 40 times as much as the original non-adapted strain. At the same time, the amount of methionine in yeast cells incrncreased, predominantly in the pool (9 times as much as in the control). This ability to produce greater amounts of methionine in the pool or to excrete it into the medium is not permanent, since after 5 passages on agar with ut ethionine the amount of methionine was practically not increased as compared with the original non-adapted strain. An increase in free methionine and of methionine excreted into the medium was found on cultivating the strain in a molasses-containing medium, too.     

Control of the cell cycle ofCandida utilis by external conditions

A mathematical model of the cell cycle ofCandida utilis in a continuous culture was formulated with respect to dilution rate. It makes it possible to express the duration of morphological stages in minutes, separately for mother cells and daughter cells. These values were compared with equivalent parameters in batch cultures. Duration of the morphological stage with buds was much longer in batch cultures as compared with the same value determined for a continuous culture according to the mathematical model. When using cultivation apparatus with a higher aeration capacity the (S + G₂) phase, i.e. the stage bearing the bud, was reduced also in the batch cultures and approached the values determined for the continuous culture by means of the mathematical model.     

External conditions influencing ethanol oxidation by resting cells ofCandida utilis

Effects of ethanol inhibition, initial pH and buffering capacity of media on the catabolic activity of nongrowing cells ofCandida utilis were studied. Effects of external conditions on the kinetic of ethanol oxidation and cell respiration are described by mathematical models. The results revealed a significant influence of both the external pH and the buffering capacity of the medium on the kinetic parameters of catabolic activity. The inhibitory effect results in the bottleneck of one of the reaction of the citrate cycle, glyoxylate cycle or electron transfer in a respiratory chain although the total rate of ethanol dissimilation increases under these conditions.     

Physiological changes ofCandida utilis in transient state during continuous cultivation

In a continuous culture ofCandida utilis, the air supply was interrupted for 15 min at 1-d intervals after the steady state had been reached. Analysis of morphology and physiology of the cell showed that after this intervention the indicators of the physiological state changed their values with different time delays and needed different times to resume their steady-state values. Another consequence of the interrupted aeration was a higher degree of the culture synchronization. The possibility to bring about a transient (i. e. unsteady) state offers a tool for a directed control of mutual proportion of intracellular components.     

Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.     

Continuous growth ofCandida utilis under periodic change of growth-limiting substrate

A periodic change of limitation by glucose and ammonia was effected during continuous cultivation ofCandida utilis. Values of feed parameters providing periodic nitrogen limitation were established. Cell biomass yield, macromolecular composition and parameters of individual cells (cell mass, budding percentage and cell-wall polysaccharide per surface square unit) were examined. The cyclic regime was found to result in culture synchrony. Parametêrs obtained on the basis of cell counts displayed a high sensitivity to changes in limitation where as the remaining parameters were less sensible.     

Continuous culture ofCandida utilis growing under cell-cycle-period changes of aeration

Periodic interruption of oxygen supply in a cell-cycle period decreases the RNA content in the biomass ofCandida utilis. The effect was observed in a mineral medium in slowly as well as in rapidly growing cultures.     

Population study of cell cycle in a continuous culture ofCandida utilis

The mean lengths of G1, S, G2 and M phases of the cell cycle were determined on the basis of the population distribution ofCandida utilis grown in a continuous culture under steady-state conditions by using an original mathematical method. The length of the G2 phase was proportional to that of G1; the length of M was effectively independent of the growth rate. The length of S was proportional to the mean number of mitochondria in the cell.     

Regulation of homoserine dehydrogenase inAzotobacter species

The regulation of homoserine dehydrogenase activity was studied in nineAzotobacter strains belonging to five different species. In all the species the enzyme is subject to feedback inhibition byl-threonine andl-isoleucine, the first being much more active as inhibitor. The inhibition byl-threonine is noncompetitive with respect to NADPH and of mixed type with respect to aspartate-Β-semialdehyde; the inhibition byl-isoleucine is noncompetitive with respect to both substrates. The synthesis of homoserine dehydrogenase inAzotobacter chroococcum I.P. is somewhat repressed by 1mm l-methionine and 5mm l-isoleucine. In all the strains examined either NADPH or NADH can serve as cofactors for this activity, though the ratio of activity with the two pyridine nucleotides (NADPH/NADH) shows higher values (3.3–3.8) in the speciesmacrocytogenes andinsignis than in thechroococcum, beijerinckii andvinelandii group (1.5–1.6). The pattern of control of this enzyme in the genusAzotobacter is discussed in relation to other bacterial homoserine dehydrogenases. We are grateful to Dr. G. N. Cohen, Service de Physiologie Microbienne, Institut Pasteur, Paris, for helpful discussions and encouragements.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Saccharomyces cerevisiae homoserine kinase is homologous to prokaryotic homoserine kinases

The Saccharomyces cerevisiae gene (THR1) encoding homoserine kinase (HK; EC 2.7.1.39) was cloned by complementation in yeast. Disruption of the THR1 gene results in threonine auxotrophy in yeast. Comparison of the amino acid sequences of yeast and bacterial HKs reveals substantial similarity.     

Protoplast fusion between a petite strain ofCandida utilis andSaccharomyces cerevisiae respiratory-competent cells

Prototrophic RD mutant cells ofCandida utilis NRRL-Y-1084 and auxotrophic mutant respiratory-competent cells ofSaccharomyces cerevisiae 4003-5Ba his ₄ leu ₂ can ^S meth ₂ trp ₅ ade ₁ ura ₃ gal were turned into protoplasts to be further fused with the aid of polyethylene glycol (PEG) and Ca²⁺ ions. Minimal medium containing glycerol as the carbon source was employed for fusion product selection. The respiratory-competent fusion products, mainly oval cells, resembledCandida utilis and had the fermentative abilities of this strain (dextrose, sucrose, raffinose). Five fusion products were analyzed as to their ability to metabolize dextrose, xylose, cellobiose, trehalose, glycerol, succinic acid, citric acid, salycin, and maltose. Fusion products partially restored the respiratory-competentCandida utilis capacity to grow by use of these carbon compounds, and none of theSaccharomyces cerevisiae fermenting abilities were found. Our results would suggest either a partial recombination between parental mitochondria or some occurring phenomenon affecting the cell, membrane function after somatic fusion without concomitant nuclear fusion.     

The adaptation ofCandida utilis to dense recycled molasses mashes under continuous cultivation

A two-phase metabolism ofCandida utilis occurred during batch cultivation in a molasses mash. It was characterized by intensive accumulation of biomass without a lag, utilization of glucose, formation of acetate and ethanol and their conversion to ethyl acetate during the first phase. In the second phase the accumulation of biomass continued and was accompanied by simultaneous utilization of ethyl acetate or amino acids, contained in molasses or produced by the culture during the first phase. A content of betaine, ash, non-assimilable nitrogen and reducing compounds as well as osmotic pressure increased with increasing density in separated mashes. The culture adapted to this medium during a two-stage continuous cultivation divided according to the two-phase nature of the metabolism. In the course of the adaptation the culture developed the ability to utilize succinate, glutamate, citrate and other originally non-assimilable compounds. A specific growth rate and productivity of the system increased proportionally with the increased concentration of assimilable substrates during a transition from one steady state to another. The adaptation in batch culture was not successful.     

New phenolic inhibitors of yeast homoserine dehydrogenase

A relatively unexploited potential target for antimicrobial agents is the biosynthesis of essential amino acids. Homoserine dehydrogenase, which reduces aspartate semi-aldehyde to homoserine in a NAD(P)H-dependent reaction, is one such target that is required for the biosynthesis of Met, Thr, and Ile from Asp. We report a small molecule screen of yeast homoserine dehydrogenase that has identified a new class of phenolic inhibitors of this class of enzyme. X-ray crystal structural analysis of one of the inhibitors in complex with homoserine dehydrogenase reveals that these molecules bind in the amino acid binding region of the active site and that the phenolic hydroxyl group interacts specifically with the backbone amide of Gly175. These results provide the first nonamino acid inhibitors of this class of enzyme and have the potential to be exploited as leads in antifungal compound design.     

Incorporation of phosphorus into phased cells ofCandida utilis using32P and33P

Changes in phosphorus metabolism were studied by examining the incorporation of³²P and³³P into cells ofCandida utilis growing in phased culture during a 6 h cell cycle and a post-cycle period of 6 h. Three different chemically defined media were used; these were phosphorus, nitrogen and carbon limited. The patterns of incorporation of phosphorus into RNA, DNA, lipid and cold water extractable phosphate fractions showed a non-uniform behaviour during both cell cycle and post-cycle periods. The patterns were different in all three types of media. The results showed that a cell can grow and develop at a fixed growth rate in different ways: so that the pattern of behaviour during a cell cycle is not stereotyped for a given doubling time, but largely depends upon the nutrient environment in which the cell exists.     

Sporulation and regulation of homoserine dehydrogenase in Bacillus subtilis

Homoserine dehydrogenase in dialyzed cell extracts of Bacillus subtilis 168 was studied, particularly with regard to inhibition, repression, and level of activity as a function of stage of development (growth and sporulation). It was assayed in the "forward direction" using L-aspartic semialdehyde and NADPH as substrates. Of the potentials inhibitors tested, only cysteine and NADP were found to be effective. Both L- and D-cysteine were equally effective. Therefore, the physiological significance of cysteine as an inhibitor is somewhat questionable. Amino acids involved in repression of homoserine dehydrogenase included methionine, isoleucine, possibly threonine, and one or more unidentified components of Casamino acids. The specific activity of homoserine dehydrogenase was highest during the exponential phase of growth and declined steadily during the stationary phase of growth. The low specific activity during late sporulation may favor preferential funnelling of L-aspartic semialdehyde into the lysine pathway, where it is needed for synthesis of large amounts of dipicolinic acid and diaminopimelic acid.