Steady-State Measurement of the Turnover of Amino Acid in the Cellular Proteins of Growing Escherichia coli: Existence of Two Kinetically Distinct Reactions

Turnover of cellular protein has been estimated in Escherichia coli during continuous exponential growth and in the absence of extensive experimental manipulation. Estimation is based upon the cumulative release into carrier pools of free leucine-1-(14)C over a number of time intervals after its pulsed incorporation into protein. Breakdown rates obtained with other labeled amino acids are similar to those obtained with leucine. Two kinetically separate processes have been shown. First, a very rapid turnover of 5% of the amino acid label occurs within 45 sec after its incorporation, most likely indicating maturative cleavages within the proteins after their assembly. A slower heterogeneous rate of true protein turnover follows, falling by 39% in the remaining proteins for each doubling of turnover time. At 36 C, the total breakdown rate of cellular protein is 2.5 and 3.0% per hr over a threefold range of growth rate in glucose and acetate medium, respectively. This relatively constant breakdown rate is maintained during slower growth by more extensive protein replacement, one fifth of the protein synthesized at any time in the acetate medium being replaced after 4.6 doubling times. Intracellular proteolysis thus appears to be a normal and integral reaction of the growing cell. The total rate equals minimal estimates obtained by others for arrested or decelerated growth but is kinetically more heterogeneous. Quantitatively proteolysis is not directly affected by growth arrestment per se as caused by alpha-methylhistidine, chloramphenicol, or uncouplers of oxidative phosphorylation, but qualitatively it can gradually become more homogeneous kinetically as a secondary event of starvation. Under more extreme conditions as with extensive washing, prolonged phosphorylative uncoupling, or acidification of the growth medium, the proteolytic rate can increase severalfold.     

Intracellular protein breakdown in growing cells of Escherichia coli

1. When Escherichia coli was grown exponentially in a defined medium at 35 degrees , the rate of protein breakdown was initially rapid, but decreased to 0.6%/hr. after about 30min. The latter rate was maintained for at least 3.5hr. 2. The initial rapid rate may have been due to the presence of a small protein fraction (about 1%) that was degraded with a half-life of 13min. 3. The rate of protein degradation was the same during balanced growth at low rates imposed in a bactogen. However, it increased during the period immediately after a decrease of the growth rate.     

Prostaglandin synthesis by murine mammary gland is modified by the state of the estrus cycle

Mammary glands were excised from female C₃H mice at various stages of their estrus cycle. Homogenates incubated at 37°C ± 10⁻⁵ M indomethacin synthesized prostaglandins (PG) E and F_2α at rates that varied as a function of the stage at estrus. The rate of PGF_2α synthesis was maximal at 1300 pg per mg protein per 2 hr in early diestrus and fell to undetectable levels in early estrus. PGE synthesis exhibited a similar pattern, being maximal at 83 pg per mg protein per 2 hr in early diestrus. These observations suggest that prostaglandins play a role in the cyclic changes observed within the mammary gland.     

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).     

Effect of Decreasing Growth Temperature on Cell Yield of Escherichia coli

Studies of the relationship between yield coefficient and growth rate, as affected by temperature of growth, in Escherichia coli have shown that, over a wide range of temperature, yield is relatively constant until the specific growth rate falls below about 0.2 hr(-1), at which point the yield begins to fall off precipitously. No intermediates of glucose metabolism in a form utilizable at higher temperatures could be found in the medium, and no toxic product was produced which limited growth. At 10 C, 37% of the carbon from glucose-UL-(14)C was assimilated into cellular material, whereas, at 30 C, 53% was assimilated. Cells grown at 10 C contained more carbohydrate than did cells grown at 37 C, and the glycogen-to-protein ratio of cells grown at 10 C was approximately three times higher than that of cells grown at 37 C. Adenosine triphosphatase activities of cells grown at 10 and 35 C were similar. Growth rates on glucose, glycerol, and succinate were quite similar at 10 C, but at 35 C growth was most rapid on glucose and slowest on succinate. The data suggest that the decrease in yield with decrease in temperature is a result of uncoupling of energy production from energy utilization.     

Thermophilic mixed culture of bacteria utilizing methanol for growth

A thermophilic mixed population of bacteria, capable of utilizing methanol as its sole carbon-energy source at temperatures up to 65 C, was selected by enrichment and studied. A maximal cellular yield of 0.42 g per g of methanol was observed at 50 to 56 C. The maximal specific growth rate of the mixed population in continuous culture at 56 C was greater than 0.32 per h. The amino acid profile of the mixed culture indicated that a high quality protein was produced and the protein content was 71%. The properties of this culture and its ability to grow at elevated temperatures are discussed in terms of single-cell protein production and the treatment of industrial waste.     

Acquired Thermotolerance and Temperature-Induced Protein Accumulation in the Extremely Thermophilic Bacterium Rhodothermus obamensis

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium Rhodothermus obamensis. The survival at temperatures superoptimal for growth (90 and 95°C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.     

Effect of reduced temperatures on protein synthesis in mouse L cells.

The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.     

A thermodynamic comparison of mesophilic and thermophilic ribonucleases H

The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.     

Selective utilization of 2-thioribothymidine- and ribothymidine-containing tRNAs by the protein synthetic systems of Thermus thermophilus HB 8 depending on the environmental temperature

An extreme thermophile, Thermus thermophilus HB 8, contains two types of tRNAs, T- and S2T-containing tRNAs. Their relative content changes depend on the growth temperature of the bacterial cells (1-3). To elucidate the reason why the extreme thermophile possesses the two types of tRNAs, an attempt was made to clarify how these tRNAs are utilized in in vivo protein synthetic systems of the bacteria cultured at different temperatures. First, a method was developed to isolate active polysomes from the thermophile cells cultured at 55 degrees C, 65 degrees C, and 77 degrees C. Then, tRNAs were separated from the polysomes and the T- and S2T-contents of the tRNAs were determined by HPLC. The relative content of S2T-tRNAs in the polysomes from 77 degrees C cells was much higher than that in bulk tRNAs from whole cells cultured at the same temperature, but the situation was reversed in 50 degrees C cells. These results clearly show that the protein synthetic systems of the thermophile have some selection mechanism to utilize either T- or S2T-containing tRNAs preferentially depending on the environmental temperature.     

The first thermophilic alpha-oxoamine synthase family enzyme that has activities of 2-amino-3-ketobutyrate CoA ligase and 7-keto-8-aminopelargonic acid synthase: cloning and overexpression of the gene from an extreme thermophile, Thermus thermophilus, and characterization of its gene product

The first thermophilic alpha-oxoamine synthase family enzyme was identified. The gene (ORF TTHA1582), which is annotated to code putative alpha-oxoamine synthase family enzymes, 7-keto-8-aminopelargonic acid (KAPA) synthase (BioF, 8-amino-7-oxononanoate synthase, EC 2.3.1.47) and 2-amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29), in a genomic database, was cloned from an extreme thermophile, Thermus thermophilus, and overexpressed in Escherichia coli. The recombinant TTHA1582 protein was purified and characterized. It exhibited activity of BioF, which catalyzes the condensation of pimeloyl-CoA and L-alanine to produce a biotin intermediate KAPA, CoASH, and CO(2) with pyridoxal 5'-phosphate as a cofactor. The protein is a dimer with a subunit of 43 kDa that shows an amino acid sequence identity of 35% with E. coli BioF. The optimum temperature and pH were about 70 degrees C and about 6.0. The enzyme showed high thermostability at temperatures of up to 70 degrees C for 1 h, and a half-life of 1 h at 80 degrees C. Thus the TTHA1582 protein was found to have the highest optimum temperature and thermostablility of the alpha-oxoamine synthase family enzymes so far reported. Substrate specificity experiments revealed that it was also able to catalyze the KBL reaction, which used acetyl-CoA and glycine as substrates, and that enzyme activity was seen with the following combinations of substrates: acetyl-CoA and glycine, L-alanine, or L-serine; pimeloyl-CoA and L-alanine, glycine, or L-serine; palmitoyl-CoA and L-alanine. This suggests that the recombinant TTHA1582 protein has broad substrate specificity, unlike the reported mesophilic enzymes of the alpha-oxoamine synthase family.     

Energetics of Bacillus stearothermophilus growth: molar growth yield and temperature effects on growth efficiency.

The major growth yield of a prototrophic strain of Bacillus stearothermophilus under aerobic conditions on salts medium containing ammonium nitrate as the nitrogen source and glucose or succinate as the carbon source was maximal at the lowest growth temperature employed and decreased steadily as the temperature was raised. The temperature optima for growth yield and for growth rate were thus different. The molar growth yield values of the thermophile, especially at the lower growth temperatures, were similar to those reported for aerobically grown mesophilic bacteria, both on glucose and on succinate. At the higher growth temperatures, a lower proportion of glucose carbon was incorporated into cells and a correspondingly greater proportion was left incompletely utilized in the medium, mostly as acetate. This suggests a greater inefficiency in the coordination of the nonoxidative and oxidative phases of glucose metabolism at the gigher temperatures. Another factor causing a decreased cell yield at higher temperatures was possibly an uncoupling of energy production from respiration. The rates of respiration by intact cells of the thermophile on glucose and on succinate followed the Arrhenius relationship from 55 C to 20 C, which is some 20 C below the minimal growth temperature of the organism. The Arrhenius constant was 17.1 kcal/mol for glucose oxidation and 13.5 kcal/mol for succinate oxidation. These results are comparable to those reported for some mesophiles, and they suggest that the inability of the thermophile to grow at temperatures below about 41 C is not due to an abnormally high temperature coefficient for the uptake and oxidation of the carbon source.     

Molecular breakdown of corn starch by thermal and mechanical effects

The molecular weight reduction of corn starch at 30–43% moisture during thermal treatment at temperatures 90–160 °C and during well-defined thermomechanical treatment at temperatures 90–140 °C was investigated. Thermal treatment resulted, during the first 5 min in a decrease in molecular weight as measured by intrinsic viscosity, after which longer heating had no significant effect. Higher moisture contents and temperatures generally resulted in more breakdown, although the effect diminished at higher temperatures. The decrease in intrinsic viscosity during thermomechanical treatment at relatively low temperatures and moisture contents was shown to be only dependent on the maximal shear stress. At higher temperatures, thermomechanical breakdown could be split into a mechanical part depending on maximal shear stress and a thermal breakdown part, which was again time-dependent on the shorter time-scales only. Higher moisture content during thermomechanical treatment resulted in more thermal breakdown and lowered the shear stresses required for mechanical breakdown. Consequences for process design are discussed briefly.     

An inserted Gly residue fine tunes dynamics between mesophilic and thermophilic ribonucleases H

Dynamic processes are inherent properties of proteins and are crucial for a wide range of biological functions. To address how changes in protein sequence and structure affect dynamic processes, a quantitative comparison of microsecond-to-microsecond time scale conformational changes, measured by solution NMR spectroscopy, within homologous mesophilic and thermophilic ribonuclease H (RNase H) enzymes is presented. Kinetic transitions between the observed major state (high population) and alternate (low population) conformational state(s) of the substrate-binding handle region in RNase H from the mesophile Escherichia coli (ecRNH) and thermophile Thermus thermophilus (ttRNH) occur with similar kinetic exchange rate constants, but the difference in stability between exchanging conformers is smaller in ttRNH compared to ecRNH. The altered thermodynamic equilibrium between kinetically exchanging conformers in the thermophile is recapitulated in ecRNH by the insertion of a Gly residue within a putative hinge between alpha-helices B and C. This Gly insertion is conserved among thermophilic RNases H, and allows the formation of additional intrahelical hydrogen bonds. A Gly residue inserted between alpha-helices B and C appears to relieve unfavorable interactions in the transition state and alternate conformer(s) and represents an important adaptation to adjust conformational changes within RNase H for activity at high temperatures.     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

Ribosomes, Polyribosomes, and Deoxyribonucleic Acid from Thermophilic, Mesophilic, and Psychrophilic Clostridia

Analysis of deoxyribonucleic acid (DNA) from four species of Clostridium, including two thermophiles, a mesophile, and a psychrophile, revealed no obvious relationship between growth temperature and DNA base composition. The melting temperatures (T(m)) of the DNA from the four species varied no more among the thermophilic, mesophilic, and psychrophilic species than among many related mesophilic species. Characterization of ribosomes from the clostridia by means of optical rotatory dispersion yielded similar spectra in common with other unrelated organisms. Only small differences were noted in the base composition of ribosomal ribonucleic acid (RNA) and in the amino acid composition of ribosomal proteins, including half-cystine content, as determined by cysteic acid analysis, and accessible sulfhydryl groups, as determined by titration with dithiobis (2-nitrobenzoic acid). Except for the two thermophiles, the ribosomal protein electrophoretic patterns were dissimilar. No unusual thermal stability was manifested in the T(m) values of thermophile ribosomal RNA. However, thermophile ribosome T(m) values (69 C) were higher than were mesophile and psychrophile T(m) values (64 C). Ribosomes from the four clostridial species were also examined in regard to the effect of heat on their functional integrity, measured by their activity in poly U-directed (14)C-phenylaline incorporation, and their gross physical integrity, measured by sucrose gradient analysis. The T(d, 5) values (temperature which produces 50% inactivation after 5 min) was found to be 70 and 72 C for the two thermophiles C. tartarivorum and C. thermosaccharolyticum, respectively; 57 C for a mesophile, C. pasteurianum; and 53 C for a psychrophile, Clostridium sp. strain 69. At 55 C, little effect was seen on the thermophile ribosomes, but the mesophile ribosomes lost 90% of their activity in 1 hr, and psychrophile ribosomes lost 100% of their activity within 10 min. According to sucrose gradient profiles, heating at 55 C results in dissociation of mesophile ribosomes and aggregation of psychrophile ribosomes. Thermophile S-100 fractions were also more thermostable than were mesophile or psychrophile S-100 fractions. The T(d, 5) values were 69 C for C. tartarivorum and C. thermosaccharolyticum S-100 and 41 C for C. pasteurianum and Clostridium sp. strain 69 S-100. The effect of heat on the endogenous incorporation of (14)C-valine by polysomes was also examined. In the case of thermophile polysomes, the extent of incorporation at 55 and 37 C was about equal. In the case of mesophile and psychrophile polysomes, the extent at 55 C was 44 and 39%, respectively, of the value at 37 C. The initial rates of incorporation in all four cases were greater at 55 C than at 37 C.     

Isolation and Characterization of a Thermotolerant Methanol-Utilizing Yeast

A yeast capable of growth on methanol as its sole carbon-energy source was isoalted from soil samples and identified as a strain of Hansenula polymorpha. A continuous enrichment culture at 37 C with a simple mineral salts medium was used to select this organism. The isolate, designated DL-1, has a maximal specific growth rate of 0.22 per h, at pH 4.5 to 5.5 and temperatures of 37 to 42 C, in simple mineral salts medium with methanol (0.5%), biotin, and thiamine. Growth occurred in a chemostat at temperatures up to 50 C, with strong growth at 45 C. The maximal growth yield of the yeast on methanol was 0.36 g of dry cell weight per g of methanol, and the yield on oxygen was 0.37 g of dry cell weight per g of O(2). Protein content of the isolate is 46%, and total nucleic acid content varies from 5.0 to 7.0% with increasing growth rate from 0.08 to 0.20 per h. The amino acid profile of this yeast protein indicates that it could serve as a good source of food protein. Feeding studies with rats show the yeast to have no toxic effects.     

Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and characterized, we have cloned two additional genes, amyB and amyC, coding for amylases of this thermophile, into Escherichia coli and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coli promoters. Almost all activity was detected in the intracellular fraction in the E. coli cells. The molecular mass and NH2-terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coli transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67,000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59,000). The optimal temperatures for AmyB and AmyC activities on soluble starch were 80 degrees C and 70 degrees C, respectively. Both AmyB and AmyC showed a pH optimum of 5.5. AmyB and AmyC showed a different pattern of starch hydrolysis when examined by thin-layer chromatography. Some homology in the amino acid sequences with the functional regions of Taka-amylase A was found in both AmyB and AmyC. The codon usage in the amyA, amyB and amyC genes was highly biased, which reflects the fact that the guanine-plus-cytosine (G + C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the codons was non-random; the G + C content of the first position of codons is significantly high, whereas that of the third position is somewhat low. In addition, codons consisting only of A and T were preferentially used in this thermophile.     

Temperature sensitive R plasmids isolated from Proteus strains.

Out of 32 R plasmids isolated from Proteus strains, 17 were found to be temperature sensitive with respect to inheritance in E. coli cells. They were fi- and classified into incompatibility group T or V. Cells carrying T group Rms273 plasmid were temperature sensitive with respect to growth and conjugal transfer in both E. coli and Proteus. The V group YOR-10 plasmid was stable in Proteus even at 42 C. However, the loss frequency of YOR-10 plasmid in E. coli reached 100% after 4 hr of incubation at 42 C, in spite of stable inheritance at 25 C. Conjugal transfer of the YOR-10 plasmid in E. coli was also strongly inhibited at 42 C. It has been concluded that instability of V group R plasmids in E. coli is due to their thermosensitive inheritance in the progeny cells at high temperatures.     

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.