bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase
Authors: Adya S , Elbein A D
Published: January 2020

An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.
Isolation of acetyl esterase mutants of Bacillus subtilis 168
Authors: Higerd T B
Published: January 2020

Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified. By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity. All of the back-crossed esterase mutants were able to sporulate at wild-type frequency and produce exoprotease(s) and antibiotic(s). No difference in motility could be attributed to the esterase mutation. PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker.
Gluconeogenesis in Saccharomyces cerevisiae: determination of fructose-1,6-bisphosphatase activity in cells grown in the presence of glycolytic carbon sources
Authors: Foy J J , Bhattacharjee J K
Published: January 2020

The activity of fructose-1,6-bisphosphatase (FBP), a gluconeogenic enzyme, was determined in wild-type Saccharomyces cerevisiae X2180 grown in the presence of the glycolytic carbon sources, glucose, fructose, and galactose. The activities of phosphofructokinase (PFK), a glycolytic enzyme, and phosphoglucose isomerase (PGI), an enzyme functioning both in glycolysis and gluconeogenesis, were determined for purposes of comparison. A measurable amount of FBP activity was present in 20-h-old cells grown with moderate shaking in 1% glucose-nutrient or minimal medium. This activity increased significantly in 40 and 60-h-old cells. Similar levels of FBP activity were also present in 20-, 40-, and 60-h-old cells grown in 1% fructose-nutrient medium. A higher level of FBP activity was present in 20-h-old cells grown in 1% galactose-nutrient medium than in 20-h-old cells grown in 1% glucose- or fructose-nutrient medium. The FBP activity in glucose- or fructose-grown cells was higher than the corresponding activity in cells grown under similar conditions for 40 and 60 h in the presence of ethanol, a gluconeogenic carbon source. The PFK activity was significantly less in galactose- and ethanol-grown cells. The PGI activity was relatively constant in 20-, 40-, and 60-h-old cells grown in the presence of glucose, fructose, and galactose, but this activity was reduced approximately 50% in ethanol-grown cells. It is concluded from these results that, depending upon the concentration of carbon source and the time of incubation, FBP, a strictly gloconeogenic enzyme, is synthesized by S. cerevisiae grown in the presence of glycolytic carbon sources.
Two unlinked genes for the pyruvate dehydrogenase complex in Aspergillus nidulans
Authors: Payton M A , McCullough W , Roberts C F , Guest J R
Published: January 2020

The activity of the overall pyruvate dehydrogenase complex was found to be similar in extracts of Aspergillus nidulans after growth on either sucrose or acetate. Eight mutants lacking the activity of this complex were found among some 200 glycolytic mutants selected for their inability to grow on sucrose. The absence of pyruvate dehydrogenase complex activity was also confirmed for a mutant, g6 (pdhA1), isolated previously. Studies with the mutants supported the existence of two unlinked genes, pdhA and pdhB, controlling the function of the complex. In vivo and in vitro complementation between mutations at the two loci were shown by the ability of forced heterokaryons to grow on sucrose and by the restoration of overall pyruvate dehydrogenase complex activity in mixed cell-free extracts. The mutations were recessive to their wild-type alleles, and the pdhA and pdhB loci were assigned to linkage groups I and V, respectively.
Characterization of a lysine-specific active transport system in Rickettsia prowazeki
Authors: Smith D K , Winkler H H
Published: January 2020

Rickettsia prowazeki possesses an active transport system for lysine with a Kt of influx of 1 muM. Extraction and chromatographic analysis of the accumulated labeled material show the material to be lysine rather than a derivative. This intracellular lysine pool can be exchanged with external unlabeled substrates for at least 10 min; The lysine analogues L-aminoethyl cysteine, N-methyl lysine, hydroxylysine, and D-lysine competitively inhibit uptake of L-lysine, but cadaverine, diaminopimelate, arginine, ornithine, and epsilon-aminocaproate do not. Accumulation of lysine can be inhibited by the energy poisons potassium cyanide, triphenylmethyl phosphonium bromide, and 2,4-dinitrophenol. The effect of potassium cyanide, but not 2,4-dinitrophenol or triphenylmethyl phosphonium bromide, can be overcome by adenosine 5'-triphosphate. Both energy-dependent influx and energy-independent efflux are inhibited by the sulfhydryl reagents N-ethyl maleimide and p-chloromercuriphenyl sulfonic acid.
Interconversion of large packets and small groups of cells of Micrococcus rubens: dependence upon magnesium and phosphate
Authors: Yamada M , Koyama T , Matsuhashi M
Published: January 2020

Micrococcus rubens, a gram-positive occus, usually forms large, cubic packets of more than 500 cells that are regularly arranged in three-dimensional cell groups. In medium with extremely low concentration of Mg2+ and phosphate, in which the cells can only grow on a agar surface, it formed small groups of 2 to 20 cells. Irregularly arraged cell groups of intermediated size were obtained in culture media containing intermediated concentrations of Mg2+ and phosphate. Mutants that formed irregular cell groups of intermediate size under normal culture conditions were also obtained.
relA gene control of the synthesis of lipid A fatty acyl moieties
Authors: Spencer A , Muller E , Cronan J E , Gross T A
Published: January 2020

The incorporation of [14C]acetate into the fatty acid moieties of lipid A was measured during amino acid starvation of rel+ and relA strains of Escherichia coli K-12. The synthesis of the beta-hydroxymyristate and other fatty acid moieties was inhibited two- to fourfold in rel+ strains, whereas no inhibition was observed in relA strains. The fatty acid compositions of the phospholipids synthesized after amino acid starvation or rel+ and relA strains were also determined.
Classification of Bacillus subtilis flagellins
Authors: Simon M I , Emerson S U , Shaper J H , Bernard P D , Glazer A N
Published: January 2020

Purified flagellins derived from 16 strains of Bacillus subtilis were classified into at least five distinct groups on the basis of their reaction with antiflagellar filament antibody and antiflagellin antibody. This classification was in good accord with that derived independently on the basis of amino acid analyses of the flagellins. Flagellar antigenicity appears to provide a useful typological character in classifying B. subtilis strains.
Extracellular phosphatases of Chlamydomonas reinhardi and their regulation
Authors: Patni N J , Dhawale S W , Aaronson S
Published: January 2020

Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.
Klebsiella serotype 25 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase
Authors: Niemann H , Kwiatkowski B , Westphal U , Stirm S
Published: January 2020

By partial acid hydrolysis, methylation and gas-liquid chromatography-mass spectrometry of the methylated monomers (as the alditol acetates), mass spectrometry of trimethylsilylated disaccharide alditols, as well as proton magnetic resonance, the primary structure of the Klebsiella serotype 25 capsular polysaccharide was elucidated. A glycanase activity, associated with the particles of newly isolated Klebsiella bacteriophage no. 25, was shown to catalyze the hydrolysis of the glycan.
Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium
Authors: Kier L D , Weppelman R , Ames B N
Published: January 2020

A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.
Properties of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium
Authors: Weppelman R , Kier L D , Ames B N
Published: January 2020

The properties of three phosphatases from Salmonella typhimurium have been examined. A cyclic 2',3'-nucleotide phosphodiesterase (EC 3.1.4.d) hydrolyzes cyclic 2',3'-purine and -pyrimidine nucleotides, as well as 3'-mononucleotides, and has a pH optimum of about 7.5. It requires divalent cations for activity and has a molecular weight of 67,000. Acid hexose phosphatase (EC 3.1.2.2) possesses activity towards hexose phosphates as well as other sugar phosphates. The enzyme is apparently a dimer of 37,000-dalton subunits. Nonspecific acid phosphatase (EC 3.1.3.2) hydrolyzes a variety of phosphate esters, including nucleotides and sugar phosphates. The enzyme also hydrolyzes the phosphoric anhydride bonds of pyrophosphate and nucleotides. Michaelis constants of the nonspecific acid phosphatase for several of its substrates are in the 1 to 2 mM range. Nonspecific acid phosphatase is a dimer of 27,000-dalton subunits.
Efficiency of oxidative phosphorylation in continous cultures of Candida parapsilosis
Authors: Rogers P J
Published: January 2020

By using a gas balance technique, the effective oxidative phosphorylation ratio in glucose-limited continuous cultures of Candida parapsilosis was calculated to be 2.4 +/- 0.2. When this value was used, the molar growth yield with respect to adenosine 5'-triphosphate was found to be 10 g of cells produced per mol of ATP.
New R plasmid-mediated restriction-modification system of deoxyribonucleic acid conferred by group E R plasmids
Authors: Arai T , Aoki T
Published: January 2020

A new R plasmid-mediated restriction-modification system of deoxyribonucleic acid was identified. This system is specific for group E plasmids which have been detected in unidentified marine Vibrio fish pathogens.
Differential binding of cyclic adenosine 3' ,5'-monophosphate to the cyclic adenosine 3' ,5'-monophosphate receptor protein in Escherichia coli
Authors: Danley D E , Drexler M , Botsford J L
Published: January 2020

Binding of cyclic adenosine 3' ,5'-monophosphate (cAMP) by the cAMP receptor protein in crude cell-free extracts of Escherichia coli was characterized. When cell were grown in glucose, binding was inhibited 50% relative to extracts from cells grown with succinate as carbon source . This inhibition could be relieved by dialysis.
Functional capacities and the adenylate energy charge in Escherichia coli under conditions of nutritional stress
Authors: Walker-Simmons M , Atkinson D E
Published: January 2020

Functional capacities in Escherichia coli cells starved for glucose were examined by comparing protein synthesis, utilization of new substrates, and maintenance of viability with the adenylate energy charge of the culture. When growth ceased because of glucose exhaustion in an E. coli culture, the energy charge dropped from 0.90 to about 0.80. During this time, the viable-cell count and the capacity for protein synthesis and for induction of new enzymes were maintained only if other substrates were available in the medium. The culture could be maintained for many hours without growth or death if glucose was added slowly; the energy charge in this case stabilized at about 0.80. A consistent transient decrease in the energy charge to around 0.80, accompanied by a decrease in protein synthesis, was also observed during the adaptation from glucose to other substrates during diauxic growth on glucose and glycerol or lactose.
Deoxyribonucleic acid synthesis after inhibition of initiation of rounds of replication in Escherichia coli B/r
Authors: Bremer H , Churchward G
Published: January 2020

The theory describing the effect of inhibition of initiation of rounds of deoxyribonucleic acid (DNA) replication on the accumulation of DNA is derived, and an analysis is presented which allows the determination of the time C taken to replicate the bacterial chromosome from the kinetic changes in the accumulation of DNA. This analysis is applied to experiments in which inhibition of initiation was achieved by inhibiting protein or protein and ribonucleic acid synthesis with chloramphenicol or rifampin. The results for both antibiotics are identical and indicate that there is a delay of 6 to 11 min in the effect of the antibiotics on initiation of rounds of replication. If this delay is taken into account, then the value of the C period estimated from such experiments agrees with values obtained by other methods, whereas by conventional data evaluation of such experiments the C period would be overestimated. In the low thymine-requiring derivative of Escherichia coli B/r ATCC 12407 used here, the C period was found to be between 38 and 41 min for cultures growing with a mass doubling time of 29 min in glucose-amino acids medium, supplemented with 20 micrograms of thymine/ml.
Escherichia coli K-12 tolF mutants: alterations in protein composition of the outer membrane
Authors: Chai T J , Foulds J
Published: January 2020

Outer membrane materials prepared from three independently isolated spontaneous Escherichia coli tolF mutants contained no detectable protein Ia. The loss of this protein was nearly completely compensated for by an increase in other major outer membrane proteins, Ib and II. Thus, the major outer membrane proteins accounted for 40% of the total cell envelope protein in both tol+ and tolF strains. No changes were found in the levels of inner membrane proteins prepared from tolF strains when compared with similar preparations from the tol+ strain. Phage-resistant mutants were selected starting with a tolF strain by using either phage TuIb or phage PA2. These phage-resistant tolF strains contained neither protein Ia nor protein Ib. The mutation leading to the loss of protein Ib in these strains is independent of the tolF mutation and is located near malP on the E. coli genetic map.
Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella
Authors: Israel D W , Gronostajski R M , Yeung A T , Schmidt R R
Published: January 2020

Earlier studies indicated that the gene of an ammonium-inducible glutamate dehydrogenase (GDH) was inducible throughout the cell cycle and was expressible shortly after replication early in the S-phase in synchronous Chlorella cells growing at a rate of 13% per h in the absence of inducer. In the present study, synchronous cells cultured at the same growth rate in the continuous presence of inducer accumulated this enzyme in a linear manner, with a positive rate change observed late instead of early in the S-phase. At a growth rate of 26% per h, the positive rate change appeared to be displaced to 1.5 h before the S-phase in the next cell cycle. With 2'-deoxyadenosine, an in vivo inhibitor of deoxyribonucleic acid (DNA) synthesis, the magnitude of the positive rate change was shown to be proportional to the relative increase in DNA in the previous cell cycle. Collectively, these data support the idea that expression of newly replicated genes of this enzyme can be delayed into the subsequent cell cycle in cells in the continuous presence of inducer. Studies with cycloheximide indicated that the inducible GDH and another GDH isozyme were stable in fully induced cells in the absence of protein synthesis. However, after ammonium was removed from the culture medium, the activity of the inducible GDH decreased rapidly in vivo, with a half-time of 5 to 10 min at 38.5 degrees C, whereas the rate of accumulation of the other GDH isozyme did not change. Addition of cycloheximide, at the time of inducer removal, prevented this loss in activity of the inducible GDH. The inability to rescue the activity of the inducible GDH, by readdition of ammonium during the deinduction period, indicates that this enzyme probably underwent irreversible inactivation and/or proteolytic degradation.
Genetic analysis of Azotobacter vinelandii mutant strains unable to fix nitrogen
Authors: Bishop P E , Brill W J
Published: January 2020

Transformation was used to perform ratio test crosses with mutant strains of Azotobacter vinelandii unable to fix N2. Mutations that simultaneously eliminated both components of nitrogenase (nif-1 and nif-2) were tightly linked. The nif-45 mutation that resulted in the absence of an active molybdenum cofactor was closer to nif-1 and nif-2 than to any of the other nif mutations. Strains that lacked component I carried mutations that were closely linked to each other. Mutations that probably were located in the structural genes for components I and II appeared to be relatively close to each other on the A. vinelandii genome.
Dominance of dnaA+ to dnaA in Escherichia coli
Authors: Gotfried F , Wechsler J A
Published: January 2020

The dominance of dnaA+ to the dnaA508 mutation was complete and was unaffected by the presence of a copy of the chromosomal replication origin on the episome. These results prove that the dnaA gene of Escherichia coli produces a diffusible product.
Initiation of Deoxyribonucleic Acid Replication in Germinating Spores of
Bacillus subtilis
168 Carrying the
dnaB
(Ts)
134
Mutation
Authors: Callister Heather , Mesurier Sue Le , Wake R. G.
Published: January 2020

The nature of the deoxyribonucleic acid synthesis reported by others to occur at 45°C in germinating spores of the temperature-sensitive deoxyribonucleic acid initiation mutant of
Bacillus subtilis
168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of
B. subtilis
168, one carrying and the other without the
dnaB
(Ts)
134
mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 45°C. At a slightly higher temperature, 49°C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts
+
strain. Thus, it is concluded that, after the germination of a spore, the action of the
dnaB134
gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 45°C is presumably due to incomplete inactivation of the
dnaB134
gene product under such conditions.
Pathways of Nucleotide Biosynthesis in
Mycoplasma mycoides
subsp.
mycoides
Authors: Mitchell Alana , Finch Lloyd R.
Published: January 2020

By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of
14
C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in
Mycoplasma mycoides
subsp.
mycoides. M. mycoides
did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of
M. mycoides
. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5′-monophosphate to adenosine 5′-monophosphate via the intermediate inosine 5′-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter.
Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida
Authors: Rogers J E , Gibson D T
Published: January 2020

The purification of (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase from cells of Pseudomonas putida grown with toluene as the sole source of carbon and energy is reported. The molecular weight of the enzyme is 104,000 at pH 9.7. The enzyme is composed of four apparently identical subunits with molecular weights of 27,000. The enzyme is specific for nicotinamide adenine dinucleotide and oxidizes a number of cis-dihydrodiols. Both enantiomers of a racemic mixture of cis-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol are oxidized by the enzyme. No enzymatic activity is observed with trans-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol.
Comparative lipid composition of heterotrophically and autotrophically grown Sulfolobus acidocaldarius
Authors: Langworthy T A
Published: January 2020

Complex lipids from the thermoacidophilic facultative autotroph Sulfolobus acidocaldarius, as well as a strictly autotrophic isolate, were compared between cells grown on yeast extract and elemental sulfur. Lipids from both organisms grown autotrophically were nearly identical. Each contained about 15% neutral lipids, 35% glycolipids, and 50% acidic lipids. Glycolipids and acidic lipids contained C40H82-76-derived glycerol ether residues. Major glycolipids included the glycerol ether analogues of glucosyl galactosyl diglyceride (5%) and glucosyl polyol diglyceride (75%). Acidic lipids were comprised mainly of the glycerol ether analogues of phosphatidyl inositol (7%), inositolphosphoryl glucosyl polyol diglyceride (72%), and a partially characterized sulfate- and phosphate-containing derivative of glucosyl polyol diglyceride (13%). The lipids from cells grown heterotrophically were similar to those from autotrophically grown cells, except that the partially characterized acidic lipid was absent. In addition, the two glycolipids as well as the respective inositolphosphoryl derivatives were each present in nearly equal proportions.
Inactivation and partial degradation of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase in nongrowing cultures of Escherichia coli
Authors: Mosteller R D , Nishimoto K R , Goldstein R V
Published: January 2020

The stability of tryptophan biosynthetic enzyme activities was examined in cultures of repressor-negative (trpR) strains of Escherichia coli K-12 incubated under conditions of nutrient starvation of chloramphenicol inhibition. The results show that four of the five activities examined are stable under most nongrowing conditions, whereas one activity, indoleglycerol phosphate (InGP) synthetase, carried by the trpC protein, is unstable under most conditions tested. Phosphoribosylanthranilate (PRA) isomerase activity, which is also carried by the trpC protein, is unstable during starvation for ammonium, cysteine, or sulfate but is stable under other nongrowing conditions where InGP synthetase is not. InGP synthetase activity but not PRA isomerase activity is also diminished about twofold in cultures using glycerol as a carbon-energy source. These results indicate that one or both activities of the trpC protein is specifically inactivated under several culture conditions. Experiments with antibodies to the trpC protein show that sulfate-starved and ammonium-starved cultures contain 20 to 40% less immunologically reactive trpC protein than unstarved cultures. This indicates that the trpC protein is probably partially degraded under these conditions. During recovery from sulfate starvation or ammonium starvation, cultures slowly regain normal levels of InGP synthetase and PRA isomerase activities, suggesting that inactivation may be reversible.
Identification of lipopolysaccharides and phospholipids of Escherichia coli in polyacrylamide gels
Authors: Bailey S C , Apirion D
Published: January 2020

Polyacrylamide gel electrophoresis of unfractionated lysates of radioactively labeled cells resolves not only proteins and polynucleotides into discrete bands but also cellular lipopolysaccharides and phospholipids. This allows a determination of the intracellular amounts of all of these macromolecules. In addition, this technique is sensitive enough to detect mutational alterations in lipopolysaccharide structure. Polyacrylamide gel electrophoresis is herein shown to be a useful tool for investigations into the structure of lipopolysaccharides and the synthesis of lipopolysaccharides and phospholipids.
Gamma-ray sensitivity during synchronous cell differentiation in Caulobacter crescentus
Authors: Iba H , Fukuda A , Okada Y
Published: January 2020

Gamma-ray sensitivity of Caulobacter crescentus during its cell cycle was examined. Survival curves of the swarmer and stalked cells were similar and exponential in shape, whereas that of the predivisional cell was sigmoidal, with an extrapolation number of 1.8.
Characterization of lysocardiolipin from Acinetobacter sp. HO1-N
Authors: Torregrossa R E , Makula R A , Finnerty W R
Published: January 2020

Triacyl-lysocardiolipin (triacyl-LCL) and diacyl-LCL were isolated from Acinetobacter sp. HO1-N, and their structures were determined by chemical, physical, and enzymatic procedures. Deacylation of triacyl-LCL and diacyl-LCL yielded bis-glycerylphosphorylglycerol. Periodate oxidation of both lysolipids was negative. Diglyceride and 2-monoglyceride resulted from the acetic acid hydrolysis of triacyl-LCL, whereas 2-monoglyceride was the sole product obtained from diacyl-LCL. Cardiolipin (CL)-specific phospholipase D treatment of triacyl-LCL yielded lysophosphatidylglycerol and phosphatidic acid. Pancreatic lipase treatment of CL yielded triacyl-LCL and diacyl-LCL. 31P nuclear magnetic resonance spectrometry showed two resonance peaks separated by 40 HZ for CL, two overlapping peaks separated by 14 HZ for triacyl-LCL, and one peak for diacyl-LCL. The proportion of lysocardiolipin increased as a function of cell age, representing 2 to 3% of the total phospholipids in early- and mid-exponential growth, 5 to 7% in late-exponential growth, and 12% in the stationary growth phase.
Purification of the extracellular protease of Bacillus licheniformis and its inhibition by bacitracin
Authors: Vitković L , Sadoff H L
Published: January 2020

Sporulating cells of Bacillus licheniformis excrete three seryl proteases that are of similar size, 28,000 daltons, but of different charge at pH 6. The peptide antibiotic bactracin is released from the cells at the same time and exists, in part, as a bacitracin-protease complex that is stable throughout chromatographic procedures employed in enzyme purification. However, preextraction of crude protease with CHCl3 and subsequent gel filtration effect separation of the antibiotic and the enzyme. Three purified, bacitracin-free proteases, designated CMC I, CMC II, and CMC III and whose ratios of total activity are 1:3.7:10.3, respectively, are obtained by chromatography on carboxymethyl cellulose. The major component, CMC III, is inhibited by commercial bacitracin at near-physiological concentrations of the antibiotic.
Peptide transport in yeast: utilization of leucine- and lysine-containing peptides by Saccharomyces cerevisiae
Authors: Marder R , Becker J M , Naider F
Published: January 2020

A variety of leucine-containing di- and tripeptides and two lysine-containing dipeptides supported the growth of strain Z1-2D, a leucine, lysine auxotroph of Saccharomyces cerevisiae. However, (Lys)2, (Lys)3, (Lys)4, and (Lys)5 as well as Gly-Leu-Gly, three tetra- and one pentapeptide containing leucine were not utilized by the mutant. Cellular peptidases released leucine or lysine from all of these non-growth-supporting peptides, suggesting that the failure of strain Z1-2D to utilize these compounds reflects their failure to enter the yeast. Competition studies employing phenylalanine or non-leucine-containing peptides showed that the uptake of peptides into S. cerevisiae Z1-2D is distinct from that of amino acids and that di- and oligopeptides may share a common transport system. The failure of strain Z1-2D to utilize any peptide larger than (Leu)3 may indicate a transport size limit. Such a size limit would influence the construction of models that explain the action of yeast mating factors.
Xylitol and
d
-Arabitol Toxicities Due to Derepressed Fructose, Galactitol, and Sorbitol Phosphotransferases of
Escherichia coli
Authors: Reiner Albey M.
Published: January 2020

d
-Arabitol was observed to be toxic to many laboratory strains of
Escherichia coli
K-12, and xylitol was found to be toxic to an existing
E. coli
C mutant strain. Fructose-specific components of the phosphoenolpyruvate:sugar phosphotransferase system are required for xylitol toxicity. Selection for xylitol resistance results in Fru
−
strains blocked in fructose phosphotransferase. Introduction of the
ptsF
or
ptsI
mutation into a xylitol-sensitive strain eliminates sensitivity. [
14
C]fructose uptake experiments imply that the mutation to xylitol sensitivity, which is co-transducible with
ara
and
leu
, results in derepression of normally inducible fructose phosphotransferase. Wild-type strains also become xylitol sensitive if induced by (and then removed from) fructose. Xylitol toxicity is prevented by fructose in both wild-type and mutant strains. Circumstances causing xylitol, a new food additive, to become toxic to an otherwise insensitive wild-type organism have not been reported previously. The
d
-arabitol-sensitive laboratory strains are galactitol (dulcitol) utilizers, although most other strains are not. Selection for
d
-arabitol resistance results in Gat
−
strains blocked in a constitutive galactitol-specific component of the phosphotransferase system. A mutation causing
d
-arabitol sensitivity occurred many years ago in AB284, the parent of AB311, AB312, AB313, and many other strains.
d
-Arabitol sensitivity also occurs in sorbitol-constitutive strains and is shown, like the previous two instances of pentitol toxicities, to result from a constitutive phosphotransferase, which is blocked in mutants selected for resistance.
Sulfate Uptake in
Saccharomyces cerevisiae
: Biochemical and Genetic Study
Authors: Breton Annick , Surdin-Kerjan Yolande
Published: January 2020

Sulfate uptake is the first step of the sulfate assimilation pathway, which has been shown in our laboratory to be part of the methionine biosynthetic pathway. Kinetic study of sulfate uptake has shown a biphasic curve in a Lineweaver-Burk plot. The analysis of this plot indicates that two enzymes participate in sulfate uptake. One (permease I) has a high affinity for the substrate (

K
m

= 0.005 mM); the other (permease II) shows a much lower affinity for sulfate (

K
m

= 0.35 mM). Regulation of the synthesis of both permeases is under the control of exogenous methionine or
S
-adenosylmethionine. It was shown, moreover, that synthesis of sulfate permeases is coordinated with the synthesis of the other methionine biosynthetic enzymes thus far studied in our laboratory. An additional specific regulation of sulfate permeases by inhibition of their activity by endogenous sulfate and adenosyl phosphosulfate (an intermediate metabolite in sulfate assimilation) has been shown. A mutant unable to concentrate sulfate has been selected. This strain carried mutations in two independent genes. These two mutations, separated in two different strains, lead to modified kinetics of sulfate uptake. The study of these strains leads us to postulate that there is an interaction in situ between the products of these two genes.
Deoxyribonucleic Acid Synthesis in Permeabilized Spheroplasts of
Saccharomyces cerevisiae
Authors: Oertel Wolfgang , Goulian Mehran
Published: January 2020

Osmotically shocked spheroplasts from
Saccharomyces cerevisiae
incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro.
32
P-label transfer (“nearest-neighbor”) experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5′-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.
Metal ion dependence of a heat-modifiable protein from the outer membrane of Escherichia coli upon sodium dodecyl sulfate-gel electrophoresis
Authors: McMichael J C , Ou J T
Published: January 2020

One heat-modifiable protein of Escherichia coli outer membrane does not completely change to the high-temperature form in the presence of magnesium ion in sodium dodecyl sulfate solution. When the metal ion complexing reagents ethylenediaminetetraacetic acid, phosphate ion, hydroxyl ion, or the competitive cations Zn2+ or Ca2+ are added to the sodium dodecyl sulfate-solubilized sample of outer membrane, and then the sample is heated to 100 degrees C and recooled to room temperature, the protein is almost completely converted to the high-temperature form. In control samples, or if sodium chloride, magnesium chloride, or manganous chloride are added to these samples and treated the same way, a large amount of the low-temperature form of the protein is preserved. beta-Mercaptoethanol additions gave the same results as the metal ion complexing reagents and may owe its activity in these solutions to metal-binding activity and not to its role as a reducing reagent. We concluded that magnesium ion may be involved with stabilization of the low-temperature form of the protein either by directly binding the magnesium or by mediating interaction with other components of the membrane.
Isolation and genetic analysis of deletion mutants of colicin E1 plasmids carrying a TnA insertion
Authors: Inselburg J , Ware P
Published: January 2020

Deletions of colicin E1 (colE1) plasmid deoxyribonucleic acid (DNA) carrying the TnA transposon have been isolated. All except two were generated by nuclease digestion of plasmid DNA from its EcoRI-sensitive site. A plasmid containing about 16% of the ColE1 DNA (6.5 X 10(5) daltons) was generated that also contained the part of the TnA transposon conferring ampicillin resistance. The extents of different deletions were determined by analysis of restriction endonuclease fragments generated by the restriction endonucleases HaeII, BamHI, and HincII.
Thymidine 5′-Monophosphate-Requiring Mutants of
Saccharomyces cerevisiae
Are Deficient in Thymidylate Synthetase
Authors: Bisson Linda , Thorner Jeremy
Published: January 2020

Thymidylate synthetase activity was measured in crude extracts of the yeast
Saccharomyces cerevisiae
by a sensitive radiochemical assay. Spontaneous non-conditional mutants auxotrophic for thymidine 5′-monophosphate (
tmp1
) lacked detectable thymidylate synthetase activity in cell-free extracts. In contrast, the parent strains (
tup1, -2
, or
-4
), which were permeable to thymidine 5′-monophosphate, contained levels of activity similar to those found in wild-type cells. Specific activity of thymidylate synthetase in crude extracts of normal cells or of cells carrying
tup
mutations was essentially unaffected by the ploidy or mating type of the cells, by the medium used for growth, by the respiratory capacity of the cells, by concentrations of exogenous thymidine 5′-monophosphate as high as 50 μg/ml, or by subsequent removal of thymidine 5′-monophosphate from the medium. Extracts of a strain bearing the temperature-sensitive cell division cycle mutation
cdc21
lacked detectable thymidylate synthetase activity under all conditions tested. Its parent and another mutant (
cdc8
), which arrests with the same terminal phenotype under restrictive conditions, had normal levels of the enzyme. Cells of a temperature-sensitive thymidine 5′-monophosphate auxotroph arrested with a morphology identical to the
cdc21
strain at the nonpermissive temperature and contained demonstrably thermolabile thymidylate synthetase activity. Tetrad analysis and the properties of revertants showed that the thymidylate synthetase defects were a consequence of the same mutation causing, in the auxotrophs, a requirement for thymidine 5′-monophosphate and, in the conditional mutants, temperature sensitivity. Complementation tests indicated that
tmp1
and
cdc21
are the same locus. These results identify
tmp1
as the structural gene for yeast thymidylate synthetase.
Isolation and Properties of Two Classes of Low-Density Vesicles from
Saccharomyces cerevisiae
Authors: Cartledge T. G. , Rose A. H. , Belk Diane M. , Goodall Adelina A.
Published: January 2020

A mixture of small (0.43-μm diameter) and large (0.62-μm diameter) low-density vesicles from spheroplasts of
Saccharomyces cerevisiae
was fractionated by rate centrifugation in a gradient of 0 to 8% (wt/vol) Ficoll to yield fractions rich (90 to 95%) in small or large vesicles. The large, but not small, vesicles swelled when diluted into mannitol solutions containing less than 0.4 M mannitol. The pH-electrophoretic mobility curve of the large vesicles showed that they are probably enclosed in a phospholipid-protein membrane. The dyes neutral red and toluidine blue, accumulated into large vesicles by intact cells and spheroplasts, were largely lost from large vesicles when these were separated from stained spheroplasts. Sudan black III stained small and large vesicles, both classes of vesicle retaining the stain on separation. Fractions rich in large vesicles contained proportionately more phospholipid and less free sterols, diacylglycerols, and free fatty acids compared with those enriched in small vesicles. The two classes of vesicles contained about the same proportions of esterified sterols and triacylglycerols. The free fatty acids in both small and large vesicles were free from unsaturated fatty-acyl residues; diacylglycerols and triacylglycerols contained appreciable proportions of unsaturated fatty-acyl residues. Small vesicles were richer in lipase activity, whereas the larger vesicles contained greater β-glucanase and α-mannosidase activities. Phospholipase activity could not be detected in any of the fractions.
Levels of acetyl coenzyme A, reduced and oxidized coenzyme A, and coenzyme A in disulfide linkage to protein in dormant and germinated spores and growing and sporulating cells of Bacillus megaterium
Authors: Setlow B , Setlow P
Published: January 2020

Dormant spores of Bacillus megaterium were found to contain approximately 850 pmol of coenzyme A (CoA) per milligram of dry weight. Of this total, less than 1.5% was acetyl-CoA, 25% was CoA-disulfide, 43% was in disulfide linkage to protein, and the remainder was the free thiol. Dormand spores of Bacillus cereus and Clostridium bifermentans contained 700 and 600 pmol of CoA per milligram of dry weight, respectively; in both species approximately 45% of the CoA 45% of the CoA was in disulfide linkage to protein. During germination of spores of all three species, greater than 75% of the CoA-protein disulfides were cleaved. In B. megaterium, cleavage of these disulfides during spore germination did not require exogenous metabolites and occurred at about the same time as the initiation of germination. Much of the CoA was converted to acetyl-CoA at this time. Dormant spores also contained reduced nicotinamide adenine dinucleotide-dependent CoA-disulfide reductase at levels higher than those in other stages of growth. The level of total CoA in the growing cells was two- to three-fold higher than in spores. This level remained constant throughout growth and sporulation, but less than 2% of the total cellular CoA was in disulfide linkage to protein until late in sporulation. The CoA-protein disulfides accumulated exclusively within the developing spore at about the time when dipicolinic acid was accumulated.
Membrane lipid biosynthesis in Acholeplasma laidlawii B: de novo biosynthesis of saturated fatty acids by growing cells
Authors: Saito Y , Silvius J R , McElhaney N
Published: January 2020

The de novo biosynthesis of fatty acids of 12 to 18 carbons from precursors of 5 carbons or fewer has been demonstrated in Acholeplasma laidlawii B. Radiolabeling experiments indicated that the normal primers for the synthesis of the even- and odd-chain fatty acids are acetate and propionate or valerate, respectively. Saturated straight-chain monomethyl-branched fatty acids of up to five carbons were readily utilized as primers, wheras more highly branched species and those possessing halogen substituents or unsaturation were not utilized. At primer concentrations of 1 to 3 mM, up to 80% of the total cellular lipid fatty acids were derived from exogenous primer. The mean chain length of the exogenous primer-derived fatty acids rose with increasing primer incorporation for methyl-branched short-chain fatty acids but was invariant for propionate. The products of de novo biosynthesis varied only slightly with temperature or cholesterol supplementation, suggesting that de novo biosynthesis is not directly influenced by membrane fluidity. Cerulenin inhibited de novo biosynthesis in a fashion that suggests the presence of two beta-ketoacyl thioester synthetases, which differ in substrate chain length specificity and in susceptibility to inhibition by the antibiotic.
Regulatory mutations of the Pseudomonas plasmid alk regulon
Authors: Fennewald M , Shapiro J
Published: January 2020

Pseudomonas putida strains with plasmids carrying pleiotropic alk mutations gave rise to alkane-positive "revertants," which differ from wild type. Some had restricted ability to utilize alkane and primary alcohol growth substrates, and others could grow on undecane and dodecanol, which are not utilized by alk+ strains. These revertants showed altered responses to normal inducers of alkA+, alkB+, and alkC+ activities. Some revertants were constitutive for these activities. Constitutive mutants could also be isolated directly from wild type, but they appeared spontaneously at a frequency of less than 2 X 10(-8). Regulatory mutations of all three types, pleiotropic negative, altered inducer specificity, and constitutive, were tightly linked in transduction crosses with a polar alkB mutation. These results demonstrate that the IncP-2 plasmid alk gene cluster constitutes a regulon. They also permit the identification of at least one cistron whose gene product participates in inducer recognition and suggest that the alkABC regulon is not under simple repressor control.