Barotolerant variant of Streptococcus faecalis with reduced sensitivity to glucose catabolite repression

Physiological characterization of the APR-11 variant of Streptococcus faecalis ATCC 9790 revealed that the variant has reduced sensitivity to glucose catabolite repression. This reduced sensitivity was indicated by the synthesis of enzymes for catabolism of lactose or arginine in cultures growing at 0.1, 40, or 70 MPa in media with levels of glucose highly repressive for the parent strain. Reduced catabolite repression appeared to be due to reduced activity of the glucose-specific, phosphotransferase system in APR-11 cells. Conversion of pyruvate to lactate or to acetate and ethanol did not appear to be altered in the variant. The APR-11 variant produced a greater final yield of biomass than the parent at all pressures tested, and its barotolerance was especially marked in media with low levels of glucose and high levels of lactose in which derepression of the lactose catabolic system was necessary for full growth. Overall, the greater barotolerance of the APR-11 strain appeared to be due to its enhanced capacity for catabolism related to its reduced sensitivity to catabolite repression by glucose.     

Magnesium and calcium ions enhance barotolerance of streptococci

Summary Magnesium and calcium ions were found to enhance barotolerance of Streptococcus faecalis ATCC strain 9790 growing in a complex, glucose-containing medium. Enhancement was indicated both by higher growth rates and yields at 408 atm, and also by an increase in the maximum pressure permitting growth from 550 to 700 atm. The optimum concentration of either ion was ca. 50 mM, and both ions appeared to be equipotent in affecting the same processes by chemically specific interactions. Sodium, potassium, strontium, manganous, chloride, bromide or sulfate ions were all ineffective or only marginally effective in enhancing barotolerance. Mg⁺⁺ and Ca⁺⁺ also enhanced growth of compressed, ribose-degrading cultures. Pressure increased the sensitivity of streptococcal growth to low pH, and there appeared to be two distinct effects of Mg⁺⁺ and Ca⁺⁺ on barotolerance. First, the rate of exponential growth was enhanced prior to the time at which culture acidity began to limit growth. Second, growth was possible in more acid conditions under pressure when the ions were present, and enhanced yields from compressed cultures were related to this partial reversal of the potentiating effect of high pressure on acid inhibition of growth.Barotolerance of Escherichia coli or Saccharomyces cerevisiae was not enhanced by these ions; while tolerance of two types of chain-forming cocci freshly isolated from a rotting mussel was enhanced.     

Energetics of streptococcal growth inhibition by hydrostatic pressure.

Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5' -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K(+) levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 mumol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 mumol/ml at 1 atm and 5.5 mumol/ml at 408 atm. N,N'-dicyclohexylcarbodiimide at a 10 muM concentration improved growth efficiency under pressure, as did Mg(2+) or Ca(2+) ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.     

Energetics of Streptococcal Growth Inhibition by Hydrostatic Pressure

Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5′ -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K⁺ levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 μmol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 μmol/ml at 1 atm and 5.5 μmol/ml at 408 atm. N,N′-dicyclohexylcarbodiimide at a 10 μM concentration improved growth efficiency under pressure, as did Mg²⁺ or Ca²⁺ ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.     

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.     

Growth of Streptococcus faecalis under High Hydrostatic Pressure and High Partial Pressures of Inert Gases

Growth of Streptococcus faecalis in a complex medium was inhibited by xenon, nitrous oxide, argon, and nitrogen at gas pressures of 41 atm or less. The order of inhibitory potency was: xenon and nitrous oxide > argon > nitrogen. Helium appeared to be impotent. Oxygen also inhibited streptococcal growth and it acted synergistically with narcotic gases. Growth was slowed somewhat by 41 atm hydrostatic pressure in the absence of narcotic gases, but the gas effects were greater than those due to pressure. In relation to the sensitivity of this bacterium to pressure, we found that the volume of cultures increased during growth in a volumeter or dilatometer, and that this dilatation was due mainly to glycolysis. A volume increase of 20.3 ± 3.6 ml/mole of lactic acid produced was measured, and this value was close to one of 24 ml/mole lactic acid given for muscle glycolysis, and interestingly, close to the theoretic volume increase of activation calculated from the depression of growth rate by pressure.     

Growth energetics of Clostridium sporogenes NCIB 8053: modulation by CO2

The effects of the partial pressure of carbon dioxide on the growth energetics of Clostridium sporogenes NCIB 8053 grown in chemostat culture were investigated in defined minimal media. Both the 'maintenance' requirements and the growth yield coefficients were dependent upon the partial pressure of carbon dioxide in otherwise glucose-limited cultures. Since growth yield coefficients decreased along with the apparent 'maintenance' requirements in essential amino acid/fatty acid medium when the partial pressure of carbon dioxide was increased above 0.5 atm, the occurrence of some type of metabolic uncoupling seemed likely. By contrast, when the organism was grown in amino acid complete medium both the maintenance requirements and the growth yield coefficients were increased when the partial pressure of carbon dioxide was raised above 0.5 atm partial pressure of carbon dioxide, suggesting an increased efficiency of growth. A futile cycle involving carbon dioxide is proposed as a factor contributing to the variable extent of free energy dissipation within this organism.     

Production and Catabolite Repression of the Constitutive Polygalacturonic Acid trans-Eliminase of Aeromonas liquefaciens

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.     

Catabolite Repression Gene of Escherichia coli

A catabolite repression gene (cat) which alters the sensitivity of Escherichia coli to catabolite repression has been mapped by transduction and shown to be located between the pyrC and purB genes. When the cat-1 mutation was studied in a number of genetic backgrounds, the results showed that this mutation affects the synthesis of more than one catabolic enzyme but does not completely eliminate catabolic repression under all conditions. It is suggested that this mutation may cause a block in the accumulation of the catabolite effector. Our experiments show that this effector is not glucose-6-phosphate.     

Production of biomass and amylases by the yeast Lipomyces kononenkoae in starch-limited continous culture

Summary In strain IGC 4052 of the amylolytic yeast Lipomyces kononenkoae growing in starch-limited chemostat cultures the critical dilution rate was reduced to about half of its theoretical value due to severe catabolite repression of amylase formation while its value in a repression-resistant mutant was near its theoretical value. The enzyme yield coefficients and the specific production rates of α-amylase and glucoamylase passed through maxima at intermediate dilution rates. The shapes of the respective curves were partly determined by catabolite repression (parent strain) or its absence (mutant strain) while induction did not seem to play to role. An additional growth-linked regulatory mechanism seemed to be involved. The use of continuous culture as compared with batch culture, increased the maximum biomass productivity by a factor of 2.2 in the mutant strain and by a factor of 1.4 in the parent strain.     

Barotolerance is inducible by preincubation under hydrostatic pressure, cold-, osmotic- and acid-stress conditions in Lactobacillus sanfranciscensis DSM 20451T

AIMS: This study addresses the inducibility of barotolerance by preincubation of Lactobacillus sanfranciscensis DSM 20451T under various sublethal stress conditions. METHODS AND RESULTS: Stress conditions which reduce the growth rate of L. sanfranciscensis DSM 20451T to 10% of its maximum were determined. These conditions were met at 43, 12.5 degrees C, a pH value of 3.7, 1.9% NaCl, or 80 MPa respectively. In contrast to heat preincubation, other prestresses, including salt, cold and pressure led to an increase of barotolerance by hydrostatic pressure of 300 MPa for 30 min. Stationary-phase cells also showed an increased barotolerance. Sublethal pressure leads to enhanced heat tolerance. CONCLUSIONS: Stress response to salt, low temperature and acidic pH as well as starvation overlap with that one to high pressure by inducing barotolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivation of bacteria by high pressure treatment is influenced by their history which modulates barotolerance. Mechanisms of barotolerance appear different from heat shock defence.     

Catabolite repression of tryptophanase in Escherichia coli

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.     

High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth.

We describe a high-pressure reactor system suitable for simultaneous hyperbaric and hydrostatic pressurization of bacterial cultures at elevated temperatures. For the deep-sea thermophile ES4, the growth rate at 500 atm (1 atm = 101.29 kPa) and 95 degrees C under hydrostatic pressure was ca. three times the growth rate under hyperbaric pressure and ca. 40% higher than the growth rate at 35 atm.     

High-pressure, high-temperature bioreactor for comparing effects of hyperbaric and hydrostatic pressure on bacterial growth.

We describe a high-pressure reactor system suitable for simultaneous hyperbaric and hydrostatic pressurization of bacterial cultures at elevated temperatures. For the deep-sea thermophile ES4, the growth rate at 500 atm (1 atm = 101.29 kPa) and 95 degrees C under hydrostatic pressure was ca. three times the growth rate under hyperbaric pressure and ca. 40% higher than the growth rate at 35 atm.     

Control of mixed-substrate utilization in continuous cultures of Escherichia coli

The chemostat culture technique was used to study the control mechanisms which operate during utilization of mixtures of glucose and lactose and glucose and l-aspartic acid by populations of Escherichia coli B6. Constitutive mutants were rapidly selected during continuous culture on a mixture of glucose and lactose, and the beta-galactosidase level of the culture increased greatly. After mutant selection, the specific beta-galactosidase level of the culture was a decreasing function of growth rate. In cultures of both the inducible wild type and the constitutive mutant, glucose and lactose were simultaneously utilized at moderate growth rates, whereas only glucose was used in the inducible cultures at high growth rates. Catabolite repression was shown to be the primary mechanism of control of beta-galactosidase level and lactose utilization in continuous culture on mixed substrates. In batch culture, as in the chemostat, catabolite repression acting by itself on the lac enzymes was insufficient to prevent lactose utilization or cause diauxie. Interference with induction of the lac operon, as well as catabolite repression, was necessary to produce diauxic growth. Continuous cultures fed mixtures of glucose and l-aspartic acid utilized both substrates at moderate growth rates, even though the catabolic enzyme aspartase was linearly repressed with increasing growth rate. Although the repression of aspartase paralleled the catabolite repression of beta-galactosidase, l-aspartic acid could be utilized even at very low levels of the catabolic enzyme because of direct anabolic incorporation into protein.     

Utilization of d-Ribose by Veillonella

Three strains of Veillonella, representing two species, were unable to utilize carbohydrates as energy sources for growth. Ribose, however, was utilized biosynthetically by all three strains. Exponentially growing cultures removed (14)C-ribose from the growth medium and retained radioactivity throughout the growth cycle. The kinetics of removal of ribose from the growth medium was found to depend on the initial ribose concentration. Uptake by resting cells was found to require active metabolism, was greatly stimulated by the presence of an energy source, and was insensitive to the presence of other pentoses. Fractionation of cells showed that the ribose was used for synthesis of acid-precipitable material, with as much as 92% of the radioactivity being found in the nucleic acid fraction. The intracellular distribution of ribose radioactivity did not change during growth after uptake was completed.     

Effect of Escherichia coli biomass composition on central metabolic fluxes predicted by a stoichiometric model

The amino acid composition of proteins and the fatty acid composition of the cell membranes were measured in Escherichia coli growing exponentially in batch culture on glucose, succinate, glycerol, pyruvate, and acetate, and growing under continuous culture conditions on glucose at dilutions rates equivalent to the growth rates of the batch cultures. Although the fatty acid composition of the membranes did change significantly with carbon source and dilution rate, the amino acid content of proteins did not change significantly under either condition. A previously developed stoichiometric model of metabolism was used to calculate the fluxes through the metabolic reactions and to determine their sensitivity to changes in fatty acid and amino acid composition.     

Barotolerance is dependent on both trehalose and heat shock protein 104 but is essentially different from thermotolerance in Saccharomyces cerevisiae

The contribution of trehalose and hsp104 to barotolerance in Saccharomyces cerevisiae has been investigated. Mutant strains, which lacked the ability to accumulate trehalose and/or hsp104, were examined for barotolerance and thermotolerance. All the mutants showed lower barotolerance and thermotolerance than their control strains. Trehalose had a greater protective effect towards high pressure than high temperature. Thus, trehalose and hsp104 are important factors for barotolerance and thermotolerance, but trehalose is more important for barotolerance than for thermotolerance.