Oligopeptide uptake by Bacteroides ruminicola

Bacteroides ruminicola did not take up (14)C from exogenous (14)C-labeled l-proline or (14)C-labeled l-glutamic acid and took up very little (14)C from exogenous (14)C-labeled l-valine. Growing cultures of B. ruminicola rapidly took up (14)C from (14)C-proline-labeled peptides of molecular weights up to 2,000 and incorporated it into trichloroacetic acid-insoluble cell material. Uptake and incorporation did not occur at 0 C and were reduced or eliminated in glucose-starved cells, depending upon the length of time the cells were starved. The initial rate of uptake of peptides seemed to exhibit saturation kinetics, but it was impossible to establish this conclusively. The initial uptake of (14)C from peptides was not affected by chloramphenicol but the incorporation of it into trichloroacetic acid-insoluble cell material was virtually eliminated. Only moderate amounts of trichloroacetic acid-extractable, labeled material were present in cells during peptide uptake, whether or not chloramphenicol was present. (14)C-proline was rapidly released from labeled peptides during uptake, whether or not chloramphenicol was present. The amount of (14)C fixed into trichloroacetic acid-insoluble cell material was directly related to the size of peptides originally supplied in the medium. It is concluded that B. ruminicola possesses a general system for the uptake of peptides, that peptides are rapidly hydrolyzed during or after uptake, and that oligopeptides function only to supply amino acids in a form available to the organism.     

Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis. Following an overnight uptake, 20-30% of the accumulated protein was subsequently regurgitated into the medium in a trichloroacetic acid/phosphotungstic acid-precipitable form. This material co-migrated with the authentic protein during molecular sieve chromatography on Sephadex G-50. The rates of appearance of trichloroacetic acid/phosphotungstic acid-insoluble products were greater than expected for cell death and leakage. The observed first order rate constants, kobs, for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products in the culture medium were identical, indicating that both products were released in parallel from MPM and BHK cells. The kobs for intracellular BSA degradation and regurgitation were independent of the initial BSA concentration in the uptake medium, but were decreased about 35% when degradation was allowed to proceed in the presence of high concentrations of serum. Degradation was also inhibited by chloroquine and pepstatin. Inhibition of degradation was accompanied by an increase in the total amount of regurgitated protein appearing in the medium. Remarkably, however, these inhibitors also decreased kobs for regurgitation, thereby preserving the similarity in the observed rate constants for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products. These and other results were inconsistent with desorption of proteins from the surface of the culture dish or the surface of cells as the source of the trichloroacetic acid/phosphotungstic acid-insoluble label appearing in the medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Testosterone and 6-N,2''-O-dibutyryladenosine 3'':5''-cyclic monophosphate stimulate protein and lysosomal enzyme secretion in rat seminal vesicle.

Rat seminal-vesicle secretion was studied in vitro in a slice-incubation system. Seminal-vesicle slices were preincubated with 32Pi for 15 min, rinsed, and incubated in an isotope-free 'chase' medium for up to 4h. Gland slices spontaneously discharged protein, three lysosomal hydrolases and trichloroacetic acid-insoluble 32P into the medium in a time- and temperature-dependent manner. Testosterone (10 muM) and dibutyryl cyclic AMP (1 mM) stimulated the discharge of protein, acid hydrolases and trichloroacetic acid-insoluble 32P, and also stimulated the incorporation of 32Pi into trichloroacetic acid-insoluble components. The acid phosphatase and beta-N-acetylhexosaminidase isoenzymes were separated by isoelectric focusing. These hydrolases were secreted into the medium as acidic isoenzymes, presumably contained within primary lysosomes, whereas they occurred largely as less acidic and basic isoenzymes in the glandular tissue.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.     

Conversion of Citrate to Extracellular Glutamate by Penicillin-treated Resting Cells of Corynebacterium glutamicum

Triggering of glutamate excretion by penicillin is thought to occur by increasing cell permeability. It seemed odd that glucose-grown resting cells, after penicillin treatment, would not convert citrate to extracellular glutamate especially since citrate had been reported to be a substrate for the glutamate fermentation. Citrate was not even taken up by such cells. Upon addition of at least 2 percent glucose, citrate was converted to extracellular glutamate. Both glucose and citrate were used simultaneously and citrate metabolism continued even after sugar was exhausted. It was suspected that glucose was required as energy source for induction of a citrate-transport system. Resting cells pregrown in glucose plus citrate, were indeed found to take up citrate and convert it to extracellular glutamate even in the absence of sugar. In line with the induction hypothesis, chloramphenicol inhibited the metabolism of citrate by glucose-grown resting cells but had no such effect on the citrate-adapted cells. The antibiotic did not inhibit glucose utilization by citrate-adapted or unadapted resting cells.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer.

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Methylamine metabolism in Hansenula polymorpha: an in vivo 13C and 31P nuclear magnetic resonance study.

Methylamine uptake, oxidation, and assimilation were studied in Hansenula polymorpha, a methylotrophic yeast. The constitutive ammonia transport system was shown to be effective at accumulating methylamine within cells cultured with methylamine or ammonia as a nitrogen source. [13C]methylamine oxidation rates were measured in vivo in methylamine-adapted cells by 13C nuclear magnetic resonance and were found to be lower than its uptake rate into the cells. The 13C label of methylamine was found exclusively in trehalose and glycerol, and [13C]formaldehyde was also extensively assimilated, indicating the presence of an assimilation pathway for the methylamine carbon. In vivo 31P nuclear magnetic resonance analysis showed major differences in the endogenous polyphosphate levels and mean chain length during adaptation of the cells from ammonia to methylamine, indicating that methylamine accumulated in the vacuole in the same manner as basic amino acids and purines. [13C]glucose metabolism was drastically altered during adaptation of the cells from ammonia to methylamine as a nitrogen source. The total rate of glucose utilization and the rate of ethanol production fell. Direct trehalose synthesis from glucose increased, indicating a switch from carbon utilization for growth to that for storage. The rate of methylamine oxidation was sufficient to support a much higher flow of carbon into central biosynthetic pathways. These results suggest that this reduction in biosynthetic carbon flow, rather than nitrogen availability, was the main factor responsible for reducing the growth rate of the yeast when ammonia was replaced by methylamine as the nitrogen source.     

Uptake and utilization of glutamic acid by Cryptococcus albidus

Cryptococcus albidus utilizes glutamate as a sole carbon source. The kinetics of uptake of this amino acid were studied. l-Glutamic acid was taken up by two saturable systems: a high affinity system with a Michaelis constant (K(m)) of 1.15 x 10(-5) M and a V(max) of 0.049 mumol per mg per h and a low affinity system with a K(m) of 2.5 x 10(-3) M and a V(max) of 3.61 mumol per mg per h. Both systems possessed characteristics of active transport which were dependent on temperature and pH and which required metabolic energy. Uptake was inhibited at 37 C but the temperature-sensitive step was reversible. Chemical fractionation of cells with 5% trichloroacetic acid showed that glutamic acid initially entered a soluble pool which decreased after 1 h as the amino acid was incorporated into the protein and nucleic acid fractions of the yeast. Some of the glutamate was completely oxidized and could be recovered as (14)CO(2). Therefore, the amino acid was also used as an energy source.     

Abolition of crypticity of Arthrobacter pyridinolis toward glucose and alpha-glucosides by tricarboxylic acid cycle intermediates

Arthrobacter pyridinolis cannot grow on glucose as sole carbon source, although the cells possess catabolic enzymes of the Embden-Meyerhof and pentose phosphate pathways as well as a complete tricarboxylic acid cycle. Crypticity toward glucose is abolished by a period of growth in a medium containing malate, succinate, citrate, or fumarate in addition to glucose. Other carbon sources, which support as rapid growth as does malate (e.g. asparagine), do not enable the cells to use glucose. Malate, succinate, citrate, and fumarate abolish crypticity toward glucose only in the second phase of diauxic growth after the tricarboxylic acid cycle intermediate has been depleted. This sequence of events, first observed in growth curves, has been verified by experiments in which the incorporation of radioactive substrates into trichloroacetic acid-insoluble cellular material was followed. The tricarboxylic acid cycle intermediates which confer the ability to utilize glucose also enhance the utilization of the alphaglucosides sucrose and maltose. The mechanism whereby growth on certain tricarboxylic acid cycle intermediates confers the subsequent ability to grow on glucose is related to a transport system for glucose and alpha-glucosides. This transport system has been assayed by measuring the uptake of [1-(14)C]-2-deoxyglucose. Cells grown for varying periods of time in asparagine, asparagine plus glucose, or malate do not transport 2-deoxyglucose. Cells from malate-glucose cultures that are in the exponential phase of growth on glucose can transport 2-deoxyglucose. Transport of 2-deoxyglucose shows Michaelis-Menten kinetics with a K(m) of 2.9 x 10(-4) M. It is competitively inhibited by glucose, alpha-methylglucopyranoside, and maltose. The transport of 2-deoxyglucose is inhibited by cyanide, dinitrophenol, azide, and N-ethylmaleimide, but not by malonate or fluoride. No phosphoenolpyruvate: d-glucose phosphotransferase activity has been detected, and the 2-deoxyglucose transported into the cell is not phosphorylated.     

Effects of inositol starvation on phospholipid and glycan syntheses in Saccharomyces cerevisiae.

The early biochemical consequences of inositol starvation in an inositol auxotroph of Saccharomyces cerevisiae were examined as a means of determining the cellular role of inositol. Upon withdrawal of inositol, the rate of incorporation of 32P-labeled inorganic phosphate into phosphatidylinositol and into the phosphoinositol-containing sphingolipids immediately dropped by 80 and 50%, respectively; however, synthesis of the other major phospholipids continued for 2 to 3 h at control rates. The incorporation of [U-14C]glucose into cell wall glycans began to decline immediately poststarvation and decreased to 50% of the initial rate by 80 min for mannan and by 140 min for alkali- and acid-insoluble glucan. These changes in the rates of synthesis of cell wall glycan and phosphatidylinositol were the earliest effects of inositol starvation, preceding inhibition of the synthesis of protein and ribonucleic acid as measured by incorporation of radioactive precursors into trichloroacetic acid-insoluble cell material. These results suggest that phosphatidylinositol may play a direct role in the synthesis or secretion of yeast glycans.     

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.     

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.     

Drug-induced suppression of phospholipid synthesis in HeLa cells by inhibition of choline uptake.

2-(4-Anisidino)-4,6-bis (2-(diethylmethylammonium)ethylamino)-1,3,5-triazine diiodide was found to inhibit specifically the incorporation of [14 C]-choline into cold 5 percent trichloroacetic acid-insoluble materials in HeLa cells without affecting other vital areas of cellular metabolism. Further studies indicated that this drug did not affect any of the intracellular reactions leading to phosphatidylcholine formation; instead, the inhibition of lecithin synthesis was due primarily to the suppression of choline transport through the cytoplasmic membrane. The level of inhibition of the latter process was comparable to that observed for the inhibition of choline incorporation into cold 5 percent trichloroacetic acid-insoluble precipitates in whole cells.     

Filamentous fungal growth assay: correlation between [U-14C] glucose uptake and dry weight determinations

The filamentous fungus Trichophyton mentagrophytes was grown in [U-14C] glucose supplemented nutrient broth. Growth was monitored in 200 microliter microcultures and 5 ml macrocultures by measuring the incorporation of 14C into trichloroacetic acid-insoluble macromolecules and in macrocultures by dry weight determination. Adjustment of the specific activity of the [U-14C] glucose medium supplement from 1080 muCi/mmole to 108 muCi/mmole decreased the rate of isotope uptake and prolonged availability so that growth beyond 36 h could be monitored radiometrically. The dry mycelial weight of the fungus was directly proportional (r = 0.995) to the amount of isotope incorporated. Isotope uptake in microcultures and macrocultures was kinetically identical. These data clearly indicate that the uptake of 14C by T. mentagrophytes can be used to monitor fungal growth accurately in both macrocultures and microcultures.     

Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response

A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.     

Tunicamycin--an inhibitor of yeast glycoprotein synthesis

Tunicamycin, a glucosamine-containing antibiotic, halted synthesis of the external glycoproteins invertase, acid phosphatase and mannan by yeast protoplasts within 30 min; formation of two intracellular proteins, alpha-glucosidase and alkaline phosphatase, and of glucan continued at the control rate for at least 60–80 min. No accumulation of mannan-free acid phosphatase or invertase was evident in treated cells. Utilization of hexoses and incorporation of ¹⁴C-amino acids into protein were not affected. Incorporation of ³H-glucosamine into trichloroacetic acid-insoluble products was only partially reduced. In yeast tunicamycin acts primarily as an inhibitor of glycoprotein synthesis and not of general glucosamine metabolism.     

Concurrent Production and Consumption of Ethanol by Cultures of Pachysolen tannophilus Growing on d-Xylose

Growing cultures of Pachysolen tannophilus concurrently consumed and produced ethanol in the presence of substantial concentrations of d-xylose. Ethanol was also assimilated in the presence of other sugars, the amount depending on the sugar. Less ethanol assimilation occurred with d-glucose than with d-xylose. The rate of ethanol consumption decreased as the concentration of glucose was increased, but some consumption still occurred when 2% glucose was present. The rate increased with the amount of oxygen available to the culture when d-xylose or ethanol was the carbon source. In most instances, estimates of consumption were based on the extent of incorporation of C from [1-C]ethanol into trichloroacetic acid-insoluble material. The results are pertinent to the use of P. tannophilus for the production of ethanol from d-xylose.