Opposite regulation by temperature of the intracellular Ca2+-dependent serine proteinase in growing and nongrowingBacillus megaterium

Specific activity of the cytoplasmic Ca²⁺-dependent serine proteinase (ISP1) in exponentially growing cultures decreased with increasing growth temperature. On the other hand, a temperature shift-up applied to a non-growing population incubated in a sporulation medium induced a rise of its activity. The ISP1 activity was assayed in the presence of 30 mmol/L CaCl₂ to release the enzyme inhibition and/or stimulate its processing. Immunoblotting applied to the 1-D SDS-PAGE electrophoretogram detected the ISP1 in growing cells mainly in bands withM of 41 and 38 kDa. The intensity of the latter decreased with increasing growth temperature. In nongrowing cells another intensively reacting band ofM 40 kDa appeared. In contrast to the commonly accepted opinion that starvation brings about a rise of the ISP1 synthesis or activation, its increase during incubation in sporulation medium was found only in cells pregrown at 35 and 42°C, where the enzyme activity in growing culture was low. No increase of the ISP1 specific activity in sporulation medium was detected in cells pregrown at 24 or 31°C, where the activity in growing cells was high.     

Netropsin suppresses development of intracellular serine proteinase activity during postexponential growth ofBacillus megaterium

The antibiotic netropsin decreased only slightly the exponential growth rate in a medium with mineral salts and glucose but its effect was increased during postexponential growth. Protein turnover and the basal intracellular serine proteinase (ISP) activity in the crude cytoplasmic fraction were not significantly affected by netropsin during exponential growth. However, ISP activity increased during postexponential growth and its rise was inhibited by the antibiotic. The population in the postexponential phase was not committed to sporulation.     

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T₀) or at T₃, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.     

Redirection of metabolism during nutrient feeding in fed-batch cultures of Bacillus thuringiensis

During sporulation, Bacillus thuringiensis produces insecticidal crystal inclusions (Cry proteins) encoded by cry genes. In fed-batch cultures (FBCs), spores and Cry protein yields are usually low, so we therefore studied the pattern of metabolic changes occurring in batch cultures and FBCs of a B. thuringiensis strain having a cry1Aa promoter-lacZ fusion, and their effect on sporulation and cry1A gene expression. In FBCs, there was a redirection of bacterial metabolism and a reduction in the specific growth rate during feeding, even when the nutrient concentration was higher than at the beginning of batch culture. These physiological changes suggest that the transition state is set up during feeding and this set-up seems to have a negative effect on both sporulation and cry1Aa expression. When the filtrate of a culture in the transition state was added to a batch culture early in the first exponential growth phase, it delayed sporulation and cry1Aa expression, thus suggesting that a soluble cellular factor that blocked sporulation might be excreted during the transition state. Citrate production usually started during the transition state but, when a medium rich in free amino acids was fed, citrate was produced from the first growth phase and sporulation was nearly blocked.     

Growth of Escherichia coli cells in the presence of cysteine on sulphate-deficient media

Summary The behaviour of E. coli B culture grown on SO₄ ^2--free minimal glucose-salt medium was examined in the presence of exogenous cysteine at various concentrations. This was done by means of using the following parameters: length of lag, growth rate and total population. Up to a concentration of cysteine at 0.2mm the growth sets in without a lag phase, the growth rate is optimal (identical with that of cultures grown on media containing Na₂SO₄ as source of sulphur), only the size of total population being decreased by cysteine. At concentrations of 0.2mm and upwards, after a concentration-dependent lag-period, the cultures were found to increase at various lower growth rates.The toxic effect of cysteine was reduced by leucine itself, as well as by a mixture of leucine, isoleucine, valine and threonine. The anti-cysteine action of these amino acids showed itself in the shortening of the lag period and in the recovery of the growth rate which, however, failed to reach the original level.Cysteamine failed to provide sulphur for cultures of E. coli B grown on the above medium. Neither was the utilization of cysteamine affected by the application of amino acids possessing an anti-cysteine action.We have postulated that beside the inhibition of the biosynthesis of amino acids having an anti-cysteine effect, toxic concentrations of cysteine posses additional sites of action.     

Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.     

Characterization, Production, and Purification of Leucocin H, a Two-Peptide Bacteriocin from Leuconostoc MF215B

Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H. The two peptides were termed leucocin Hα and leucocin Hβ. When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. Production of leucocin H in growth medium takes place at temperatures down to 6°C and at pH below 7. The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase. The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography. Upon purification, specific activity increased 10⁵-fold, and the final specific activity was 2 × 10⁷ BU/OD₂₈₀. Amino acid composition analyses of leucocin Hα and leucocin Hβ indicated that both peptides consisted of around 40 amino acid residues. Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin Hβ did not respond to Cyanogen Bromide (CNBr) cleavage. Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation. From leucocin Hα, the sequence of 20 amino acids was obtained; from leucocin Hβ the sequence of 28 amino acid residues was obtained. No sequence homology to other known bacteriocins could be demonstrated. It also appeared that the two peptides themselves shared little or no sequence homology. The presence of soy oil did not affect the activity of leucocin H in agar. Received: 10 February 1999 / Accepted: 15 March 1999     

Cell viability and protein turnover in nongrowingBacillus megaterium at sporulation suppressing temperature

InBacillus megaterium, a temperature that suppresses sporulation (43°C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40–41°C). Here we show that, when cells grown at 35°C and transferred to a sporulation medium, were subjected to shifts between 35°C and the sporulation suppressing temperature (SST, 43°C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35°C for 2–3 h (T₂–T₃), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T₄–T₅. Protein turnover rate increased again after T₂ and up to T₈ 60–70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T₃ no asymmetric septa were observed and the amount of heat-resistant spores at T₈ was by 4–5 orders lower than at 35°C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T₃ but then its velocity sharply dropped and the amount of degraded protein at T₈ corresponded to slightly more than one-half of that found at 35°C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T_2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35°C. A later (T₄) shift to SST had no effect on the sporulation process.     

Incorporation of Glycine and Serine into Sporulating Cells of Bacillus subtilis

The changes during growth and sporulation in activities of cells of Bacillus subtilis to incorporate various amino acids were investigated with wild-type strain and its asporogenous mutant. In the case of wild type strain the uptake of valine, phenylalanine, and proline was largest during the logarithmic growth period. The uptake of these amino acids decreased rapidly during the early stationary phase. The uptake of valine and cysteine increased again to some extent just prior to the forespore stage. The uptake of glycine and serine, however, was largest at the forespore stage at which the formation of spore coat took place. From these observed phenomena it was assumed that the remarkable incorporation of glycine and serine into the wild type strain during sporulation was closely related to the formation of spore coat.     

Characterization of the submerged growth ofMoraxella bovis

A submerged batch cultivation ofMoraxella bovis in a medium containing enzymic casein hydrolyzate and supplemented with dialyzed ram blood was described. Up to the growth limitation the bacteria grew exponentially without a lag phase and with a doubling period of 64 min. During the exponential phase no significant decrease of viable cells and cell autolysis were observed. Amino acids were the limiting factor. At the end of growth glutamie acid, threonine and serine were detected at the lowest concentrations. Substrate limitation led to an irreversible decrease of the metabolic activity. Growth yield on oxygen was 2.3 × 10⁹ cells per mg O₂. The fraction of respiration required for growth-uncoupled processes was negligible. During the cultivation ammonia was produced and the pH increased but it was not the inhibitory factor. Respiration was not limited by oxygen at concentrations higher than 0.63 μmol O₂ per L. Sufficient pili were produced.     

Induction of sporulation inBlastocladiella emersonii: Influence of nutritional variables

We have studied the induction of zoosporangial sporulation inBlastocladiella emersonii by determining the influence of nutritional variables on the formation of the basal septum. This is an easy-to-monitor structure that has proved to be a useful marker of initiation of this cellular differentiation process. At any time before formation of the septum, the cells returned to the growth phase when incubated in the presence of 2% casamino acids. However, after septum formation has occurred, the cells were committed to sporulation. Certain of the amino acid components of the defined growth medium, DM₂, either prevent (tyrosine, phenylalanine, tryptophan, histidine, and threonine) or delay (valine, serine, arginine, and methionine) formation of the septum when added to the cultures together with the sporulation solution (buffered 1 mM CaCl₂). Sugars do not prevent the formation of the septum, although they can interrupt sporulation at later developmental phases. Glucosamine and acetylglucosamine can prevent formation of the discharge papilla and glucose can block completion of zoospore production. Glutamine, adenosine, and guanosine also caused long delays in septum formation. Taken together, these data favor (a) the hypothesis that amino acid starvation is one of the most important variables with respect to the induction of sporulation, and (b) the use of certain environmental chemical conditions that block sporulation at different steps as developmental tools for the study of this cellular differentiation process.     

Intracytoplasmic membrane production inEscherichia coli O111a1: Nutritional parameters

Escherichia coli O111a₁ ceased growth prematurely and accumulated intracytoplasmic membrane at 42°C in an amino acids-mineral salts medium. The amount of membrane formed appeared to be proportional to the concentration of amino acids in the medium—the greater the concentration of amino acids in the medium, the greater the membrane production.E. coli O111a₁, did not grow at 42°C in glucose-, glycerol- or acetate-mineral salts medium, but mesosome-like structures were produced in glucose-grown cells and some intracytoplasmic membrane in cells grown on glycerol and acetate. Supplementation of the glucose medium with pantothenate and/or thiamine permitted normal growth. The vitamins did not restore growth of the mutant in glycerol or acetate, but intracytoplasmic membrane production was increased, especially in glycerol. Amino acids plus glucose supported normal growth with no membrane production. Glycerol and acetate had no effect on the growth in the amino acids medium, but stimulated the accumulation of membrane.     

RELATIONSHIP OF NUTRITIONAL FACTORS TO IN VITRO TUMOR CELL GROWTH AND CYTOTOXICITY PRODUCED BY CYTOSINE ARABINOSIDE

The in vitro relationship between nutritional factors, proliferative status of tumor cells, and the cytotoxic action of cytosine arabinoside (ara-C) was investigated. The reduction in the concentration of only one essential amino acid, L-isoleucine, in the growth medium of A(T₁)Cl-3 hamster fibrosarcoma cells decreased DNA synthesis in this cell population and slowed the rate of progression of G₁ phase cells into S phase of the cell cycle. The complete omission of isoleucine from the growth medium blocked the progression of G₁ phase cells into S phase and prevented the cytotoxic action of ara-C. The addition of isoleucine to the isoleucine-deprived cells permitted these cells to enter the S phase and restored their sensitivity to the cytotoxic action of ara-C. When G₁ phase cells were placed in a medium containing reduced levels of all the amino acids and vitamins there was a prolongation of the G₁ phase. Since medium with low levels of amino acids produced a delay in the entry of G₁ phase cells into the S phase, the time interval in which these cells were most sensitive to the cytotoxic action of ara-C was different for G₁ phase cells placed in medium with adequate levels of all the amino acids. These in vitro data indicate that nutritional factors can markedly effect the proliferation of tumor cells and the cytotoxic action of ara-C.     

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus.

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.     

Characteristics of amino acid uptake in barley

Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten kinetics. The affinity constant (K _m) was in the range 19.6–33.2 μM. These K _m values are comparable to reported values for soil micro-organisms.     

Farnesol and geranylgeraniol modulate the structural properties of phosphatidylethanolamine model membranes

The biological activity of farnesol (FN) and geranylgeraniol (GG) and their isoprenyl groups is related to membrane-associated processes. We have studied the interactions of FN and GG with 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes using DSC and X-ray diffraction. Storage of samples at low temperature for a long time favors a multidomain system formed by a lamellar crystalline (Lc) phase and isoprenoids (ISPs) aggregates. We demonstrate that ISPs alter the thermotropic behavior of DEPE, thereby promoting a H_II growth in a lamellar Lc phase with a reduced degree of hydration. The H_II phase occurs with the same repeat distance (d_HII=5.4 nm) as the Lc phase and upon heating it expands considerably (δd/δT≈0.22 nm/°C). The dimensional stabilization of this H_II phase coincides with the transition temperature of the Lc to L_α phase. Thereafter, the system DEPE/ISP will progress by increasing the nonlamellar-forming propensity and reaching a single H_II phase at high temperature. The cooling scan followed a similar structural path, except that the system went into a stable gel phase L_β with a repeat distance, d_Lβ=6.5 nm, in co-existence with a H_II phase. The formation of ISP microdomains in model PE membranes substantiates the importance of the isoprenyl group in the binding of isoprenylated proteins to membranes and in lipid–lipid interactions through modulation of the membrane structure.     

Amino acid consumption by Chloroflexus aurantiacus in batch and continuous cultures

Amino acid consumption was studied with batch and continuous chemostat cultures of Chloroflexus aurantiacus grown phototrophically in complex medium with casamino acids (Pierson and Castenholz 1974). Amino acids like Arg, Asx, Thr, Ala, Tyr, which were utilized during the early exponential phase by cells grown in batch cultures were consumed in chemostat cultures essentially at any of the dilution rates employed (0.018–0.104 h^-1). Those amino acids which were taken up during subsequent phases of growth were consumed in chemostat cultures preferentially at low dilution rates. For example, the consumption of Glx was enhanced during the late exponential phase and at low dilution rates. At high dilution rates Glx was not consumed at all. Since Glx utilization largely paralleled bacteriochlorophyll formation, it is discussed that formation of the photopigment depends on the intracellular availability of Glu as the exclusive precursor for tetrapyrrole synthesis.     

Netropsin suppresses the rise of activity of an intracellular proteolytic system in sporulatingBacillus megaterium

Intracellular catabolism of proteins labeled at the end of the exponential growth proceeded in two phases during sporulation. The first phase was induced by starvation and took place also in cells whose sporulation was inhibited by netropsin. The second phase of degradation, which was triggered at the onset of the irreversible sporulation phase, was inhibited by netropsin. Intracellular proteolytic activity determined in disintegrated cells, i.e., primarily the activity of the cytoplasmic Ca²⁺-dependent serine proteinase(s) at the first place, was increasing throughout the sporulation process and reached its maximum during the irreversible sporulation phase. Its increase was suppressed by netropsin. Fractionation of the cell sap by HPLC revealed a similar distribution of proteolytic activities in the extract from control and netropsin-inhibited cells. The antibiotic thus probably affected the activation, not the formation of the cytoplasmic serine proteinase(s). Netropsin also inhibited an increase of proteolytic activity in the membrane fraction, probably owing to the presence of two different proteolytic enzymes.     

Amino acid composition of azolla as affected by strains and population density

The total nitrogen and amino acid composition of seven Azolla strains were compared at four different growth phases. Total nitrogen content of the individual strains ranged from 2.6% to 5.7% of dry matter and was not significantly influenced by growth phase or population density. The concentration of the sixteen amino acids determined was maximal during the linear growth stage and specific differences occurred among Azolla strains. AnAzolla microphylla strain was the best source of amino acids and anA. filiculoides strain the poorest under the cultural conditions used. The chemical index score demonstrated the potential of some species, such as theA. microphylla strain, as contributor of protein for animals. Strains of other species, such asA. filiculoides, had several limiting amino acids and appear more suited for use solely as a green manure. All Azolla strains contained a similar proportion of essential (55%) and non-essential (45%) amino acids. Leucine, lysine, arginine and phenylanine+tyrosine were the predominant essential amino acids whereas the sulfur containing amino acids (methionine and cystine) were present in smaller amounts.     

Effect of Sm3+ ion on growth of Bacillus thuringiensis by microcalorimetry

By using an LKB-2277 Bioactivity Monitor, cycle-flow method, the thermogenic curves of aerobic growth for Bacillus thuringiensis cry II strain at 28°C have been obtained. The metabolic thermogenic curves of B. thuringiensis cry II contained two distinct patterns: the first reflects the changes during the bacterial growth phase and the second corresponds to the sporulation phase. From these thermogenic curves in the absence and presence of Sm³⁺ ions, the thermokinetic parameters such as the growth rate constants k, the interval time τ_I, the maximum power P _{max 1} and heat-output Q _log for log phase, the maximum power P _{max 2} and heat-output Q _stat for stationary phase, the heat-output Q _spor for sporulation phase and total heat effects Q _T are calculated. Sm³⁺ ion has promoting action on the growth of B. thuringiensis cry II in its lower concentration range; on the other hand, this ion has inhibitory action on the sporulation of B. thuringiensis in its higher concentration range. We also found that the effects of Sm³⁺ ion on B. thuringiensis during the sporulation phase were far greater than that during the bacterial phase. It is concluded that the application of B. thruringiensis of controlling insecticides is not affected by the presence of the rare-earth elements in the environmental ecosystem.