bluuebunny/crossref_metadata_2025 huggingface dataset:
Catabolism of aromatic acids in Trichosporon cutaneum
Authors: Anderson J J , Dagley S
Published: January 2020

Trichosporon cutaneum readily metabolized protocatechuate, homoprotocatechuate, and gentisate, but lacked ring fission dioxygenases for these compounds. Benzoic, salicylic, 2,3-dihydroxybenzoic, and gentisic acids were converted into beta-ketoadipic acid before entry into the Krebs cycle. Benzoic acid gave rise successively to 4-hydroxybenzoic acid, protocatechuic acid, and hydroxyquinol (1,3,4-trihydroxybenzene), which underwent ring fission to maleylacetic acid. Salicylate and 2,3-dihydroxybenzoate were both initially metabolized to give catechol. 2,3-Dihydroxybenzoate was the substrate for a specific nonoxidative decarboxylase induced by salicylate, although 2,3-dihydroxybenzoate was not a catabolite of salicylate. Gentisate was metabolized to maleylacetic acid and was also readily attacked by salicylate hydroxylase at each stage of a partial purification procedure. Phenylacetic acid was degraded through 3-hydroxyphenylacetic, homogentisic, and maleylacetoacetic acids to acetoacetic and fumaric acids. All the reactions of these catabolic sequences were catalyzed by cell extracts, supplemented with reduced pyridine nucleotide coenzymes where necessary, except for the hydroxylations of benzoic and phenylacetic acids which were demonstrated with cell suspensions and isotopically labeled substrates.
Stabilization and purification of ornithine transcarbamylase from Neisseria gonorrhoeae
Authors: Powers C N , Pierson D L
Published: January 2020

Ornithine transcarbamylase was stabilized in cell-free extracts by the presence of either carbamyl phosphate or glycerol. The enzyme was rapidly purified by a procedure consisting of ion-exchange chromatography and electrofocusing. The native molecular weight of the enzyme was determined by gel filtration to be 110,000. A subunit molecular weight of 36,000 was determined by polyacrylamide electrophoresis under dissociating conditions. These findings indicated a trimeric quaternary structure for the enzyme. The isoelectric point of the purified enzyme was 4.75, and no evidence of multiple forms of active enzyme was found in either crude or purified preparations. An inactive form of the enzyme appeared upon storage in the absence of stabilization buffer.
High-frequency heterokaryon formation by Mucor racemosus
Authors: Lasker B A , Borgia P T
Published: January 2020

Heterokaryons from Mucor racemosus were produced from two auxotrophic strains of the fungus. Germlings were converted to spheroplasts by using commercial chitinase and purified Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed and fused in solutions of polyethylene glycol and CaCl2. Under optimal conditions, prototrophic heterokaryons were formed at a frequency of about 5%.
Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli
Authors: Ray P H
Published: January 2020

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.
Ribosome assembly during recovery of heat-injured Staphylococcus aureus cells
Authors: Flowers R S , Martin S E
Published: January 2020

The process of ribosome formation during repair of sublethal heat injury was examined in Staphylococcus aureus. Sublethal heating of this organism results in the degradation of the 30S ribosomal subunit and alteration of the 50S subunit. Cells recovering from sublethal injury were examined for changes with time in the sedimentation and electrophoretic properties of ribonucleoprotein particles and ribonucleic acid, respectively. When cells were allowed to recover in [3H]uridine, the label could be followed into ribonucleic acid species that coelectrophoresed with 23S and 16S ribonucleic acid. Three ribonucleoprotein particles (49S, 36S, and 30S) were isolated from repairing cells by sedimentation through sucrose gradients. Polyacrylamide gel electrophoresis showed that the 49S particle contained 23S ribonucleic acid, the 36S particle contained both 23S ribonucleic acid and 16S precursor and mature ribonucleic acid, and the 30S particle contained 16S and precursor 16S ribonucleic acid. Particles with similar sedimentation properties were found in unheated cells.
Ferrisiderophore reductase activity in Bacillus megaterium
Authors: Arceneaux J E , Byers B R
Published: January 2020

The release of iron from ferrisiderophores (microbial ferric-chelating iron transport cofactors) by cell-free extracts of Bacillus megaterium was demonstrated. Reductive transfer of iron from ferrisiderophores to the ferrous-chelating agent ferrozine was measured spectrophotometrically. This ferrisiderophore reductase activity (reduced nicotinamide adenine dinucleotide phosphate:ferrisiderophore oxidoreductase) was associated primarily with the cell soluble rather than particulate (membrane) fraction. Ferrisiderophore reductase was inhibited by oxygen and required the addition of a reductant (reduced nicotinamide adenine dinucleotide phosphate was most effective) for maximal activity. The activity was destroyed by both heat and protease treatments and was inhibited by iodoacetamide treatment. Ferrisiderophore reductase activity for several microbial ferrisiderophores was measured; highest activity was displayed for ferrischizokinen, the ferrisiderophore produced by this organism. The Km and Vmax values of the reductase for ferrischizokinen were 2.5 x 10(-4) M and 35.7 nmol/min per mg of the ferrisiderophore reductase reaction. Preliminary fractionation of the cell soluble material by gel filtration chromatography resulted in the demonstration of ferrisiderophore reductase activity in three peaks of different molecular weight. Ferrisiderophore reductase probably mediates entrance of iron into cellular metabolism.
Involvement of the ftsA gene product in late stages of the Escherichia coli cell cycle
Authors: Tormo A , Martínez-Salas E , Vicente M
Published: January 2020

Strain D-3 was shown, by genetic complementation with lambda phage vectors carrying either the wild type or a temperature-sensitive allele, to contain an ftsA mutation. The presence of this mutation rendered the strain unable to divide at 42 degrees C. The action of the ftsA gene product in strain D-3 takes place at a late stage of the cycle, starting near the end of the replication cycle and extending until cell division is completed. The action of the ftsA gene product seemed to take a constant time, independent of the duration of the cell division cycle. Experiments in which all other requirements for division except a "termination protein" were completed show that cells of strain D-3 can divide under these conditions only at the permissive temperature. We conclude that a likely role for the ftsA gene product is that of a termination protein.
Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus
Authors: Orndorff P E , Dworkin M
Published: January 2020

We have developed methods for separating the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus. The total membrane fraction from ethylenediaminetetraacetic acid-lysozyme-treated cells was resolved into three major fractions by isopycnic density centrifugation. Between 85 and 90% of the succinate dehydrogenase and cyanide-sensitive reduced nicotinamide adenine dinucleotide oxidase activity was found in the first (I) fraction (rho = 1.221 g/ml) and 80% of the membrane-associated 2-keto-3-deoxyoctonate was found in the third (III) fraction (rho = 1.166 g/ml). The middle (II) fraction (rho = 1.185 g/ml) appeared to be a hybrid membrane fraction and contained roughly 10 to 20% of the activity of the enzyme markers and 2-keto-3-deoxyoctonate. No significant amounts of deoxyribonucleic acid or ribonucleic acid were present in the three isolated fractions, although 26% of the total cellular deoxyribonucleic acid and 3% of the total ribonucleic acid were recovered with the total membrane fraction. Phosphatidylethanolamine made up the bulk (60 to 70%) of the phospholipids in the membrane fractions. However, virtually all of the phosphatidylserine and cardiolipin were found in fraction I. Fraction III appeared to contain elevated amounts of lysophospholipids and contained almost three times the amount of total phospholipid as compared with fraction I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved approximately 40 polypeptides in the total membrane fraction. Two-thirds of these polypeptides were enriched in fraction I, and the remainder was enriched in fraction III. Fraction II contained a banding pattern similar to the total membrane fraction. Electron microscopy revealed that vegetative cells of M. xanthus possessed an envelope similar to that of other gram-negative bacteria; however, the vesicular appearance of the isolated membranes was somewhat different from those reported for Escherichia coli and Salmonella typhimurium. The atypically low bouyant density of the outer membrane of M. xanthus is discussed with regard to the high phospholipid content of the outer membrane.
Modification of aspartate before its condensation with dihydroxyacetone phosphate during quinolinic acid formation in Escherichia coli
Authors: Steiner B M , Heard J T , Tritz G J
Published: January 2020

A crude enzyme preparation from a nadA mutant of Escherichia coli was used to catalyze the conversion of [14C]aspartic acid into a precursor of quinolinic acid, a key intermediate in the biosynthesis of nicotinamide adenine dinucleotide.
Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae
Authors: Kay D G , Singer R A , Johnston G C
Published: January 2020

In the yeast Saccharomyces cerevisiae, the specific activity of the enzyme ornithine decarboxylase (ODC) was correlated with overall growth status. The activity of ODC was highest in actively growing cells, whereas the specific activity was lower in slow-growing cultures limited for nitrogen or inhibited by low concentrations of cycloheximide. Specific activities of ODC were also low in cultures arrested in the stationary phase (in the G1 portion of the cell cycle) by starvation for required nutrients. Although correlated with overall growth, ODC activity was not required for growth or cell cycle regulation. Cells continued to grow in the presence of the polyamine spermidine or spermine, which markedly reduced ODC specific activities. Thus, high levels of ODC activity were not necessary for growth, nor were decreased ODC specific activities sufficient to cause cells to arrest in G1. Conversely, one agent (o-phenanthroline) which causes growing cells to arrest in G1 did so with no effect on ODC specific activity. Therefore, ODC specific activity changes are not necessary for cell cycle regulation but simply reflect the normal growth status of cells.
Altered immunochemical reactivity of Saccharomyces cerevisiae a-cells after alpha-factor-induced morphogenesis
Authors: Lipke P N , Ballou C E
Published: January 2020

Antibodies were raised against Saccharomyces cerevisiae a-cells that had been exposed to the sex pheromone, alpha-factor. After adsorption of the antiserum with diploid cells, antibodies remained that reacted specifically with the mannan from haploid cells. The characteristic determinant was observed in mannan from pheromone-treated a-cells, in mannan from untreated alpha-cells, and at a much lower concentration, in mannan from control a-cells. The antigens from these three mannans appeared to be identical. The determinant was destroyed by mild-acid hydrolysis or periodate oxidation, but not by proteolysis or digestion with exo-alpha-mannanase. Mutants with altered mannan were unable to express the antigen. Complete acid hydrolysates mannan from alpha-factor-treated a-cells contained mannose, glucose, and N-acetylglucosamine. Partial acid hydrolysis, under conditions that destroyed the antigenic determinant, released only mannose and mannobiose. The mannose fraction was labeled to high specific activity during response of a-cells to alpha-factor if radioactive glucose was the carbon source. Neither alpha- not beta-D-mannopyranosyl phosphate was a hapten. The results are consistent with the presence of a haploid-specific antigen containing an acid-labile mannose determinant and show that the amount of this antigen in a-cell mannan is increased in response to alpha-factor.
Polymethylpolysaccharide synthesis in an ethionine-resistant mutant of Mycobacterium smegmatis
Authors: Maloney D H , Ballou C E
Published: January 2020

Mutants of Mycobacterium smegmatis were selected for resistance to ethionine in an effort to obtain methylation-defective strains that were altered in their ability to make methylmannose polysaccharides (MMP) or methylglucose lipopolysaccharides. Two methods were developed for the detection of MMP in cell extracts to aid in the screening for potential mutants, one a qualitative procedure based on iodine binding by the sample after paper chromatography and the other a quantitative procedure based on fluorimetric titration of the MMP with parinaric acid. An ethionine-resistant mutant was obtained that contained only about 25% of the normal level of S-adenosylmethionine and 10% of the normal level of methionine adenosyltransferase (adenosine 5'-triphosphate:L-methionine S-adenosyltransferase, EC 2.5.1.6) activity. When grown in the presence of 0.1% ethionine, the mutant cells contained about 50% of the wild-type levels of methylglucose lipopolysaccharides but only about 7% of the normal level of MMP (wild-type cells contain about 0.14 mM MMP and the mutant contains about 0.01 mM MMP). The amount of fatty acid synthesis in the ethionine-resistant mutant grown in the presence of ethionine was not dramatically altered although the mutant accumulated more short-chain and less long-chain unsaturated fatty acids than the wild-type cells.
Deoxyribonucleic acid-deficient strains of Candida albicans
Authors: Olaiya A F , Steed J R , Sogin S J
Published: January 2020

We analyzed a series of germ tube-negative variants isolated from Candida albicans 3153A for deoxyribonucleic acid content. As analyzed by flow microfluorometry, the deoxyribonucleic acid level in these variant strains was 50% of that of the parental strain and equivalent to that of haploid Saccharomyces cerevisiae. This finding was confirmed by comparison of survival rates when exposed to the mutagens ultraviolet light, ethyl methane sulfonate, and methyl methane sulfonate. The diameter of the variant cells as compared to the diameter of the parental 3153A strain showed a relationship similar to that of the diameters of haploid versus diploid S. cerevisiae. These results indicate that those strains may be representative of the imperfect stage of C. albicans.
Inhibition of transformation in Streptococcus pneumoniae by lysogeny
Authors: Moynet D J , Tiraby G J
Published: January 2020

Streptococcus pneumoniae R6X was lysogenized with bacteriophage 304 isolated after mitomycin induction of an ungrouped alpha-hemolytic streptococcus. Lysogenized pneumococci lost their capacity to undergo genetic transformation: transformability was restored after cells were spontaneously cured of their prophage. Both lysogens and nonlysogens produced activator substance (competence factor), and both bound deoxyribonucleic acid in a deoxyribonuclease-resistant form. However, nonlysogens retained deoxyribonucleic acid after washing, whereas lysogens did not. The latter did not liberate phage nor (unlike nonlysogens) degrade transforming deoxyribonucleic acid and contained normal levels of endonuclease.
Protein identifications of O'Farrell two-dimensional gels: locations of 81 Escherichia coli proteins
Authors: Bloch P L , Phillips T A , Neidhardt F C
Published: January 2020

The two-dimensional gel electrophoresis method of P. H. O'Farrell readily resolves approximately 1,000 proteins from whole-cell homogenates. We have found that the location of most individual proteins is sufficiently reproducible and precise to permit different laboratories to exchange information about them. We present the location of 81 Escherichia coli structural proteins, binding proteins, enzymes, and factors, identified with the aid of purified proteins supplied to us by many investigators.
Is cyclic guanosine 3',5'-monophosphate a cell cycle regulator?
Authors: Cook W R , Kalb V F , Peace A A , Bernlohr R W
Published: January 2020

Transient increases in the intracellular level of cyclic guanosine 3',5'-monophosphate have been observed at a periodicity of one generation time in two spoT strains of Escherichia coli and in Bacillus licheniformis.
Effect of Growth Conditions on Yield and Heme Content of
Vitreoscilla
Authors: Lamba Parveen , Webster Dale A.
Published: January 2020

Vitreoscilla
, a gliding bacterium in the
Beggiatoaceae
, is an obligate aerobe in which cytochrome
o
functions as the terminal oxidase. Protoheme IX is the only heme type present in this organism. The yield and heme content of
Vitreoscilla
cells grown in yeast extract, peptone, and acetate were dependent on growth conditions. Cells harvested in early stationary phase contained roughly three times as much heme as cells in early log phase. There was an optimal shaking rate for maximum heme content of cells harvested in stationary phase at fixed initial nutrient concentration. The heme content of cells grown at a fixed shaking rate increased from 5 nmol/g (wet weight) in media which had low nutrient concentration to a maximum of 45 nmol/g (wet weight) in media which had high nutrient concentration, and there was a corresponding sixfold increase in cytochrome
o
content and an eightfold increase in respiratory rate, evidence that some of the additional heme was incorporated into respiratory pigments. Heme content may be controlled jointly by competition for oxygen and availability of nutrients. Temperature and initial pH affected the growth rate but not the final yield or heme content. Growth rate was optimal at pH 8.0 to 8.5. A defined medium for
Vitreoscilla
, which is based on glutamate as the carbon source, is described; the other organic components of this medium are acetate, tryptophan, thiamine, biotin, and riboflavin.
Adenosine 5'-triphosphate formation in Thiobacillus ferrooxidans vesicles by H+ ion gradients comparable to those of environmental conditions
Authors: Apel W A , Dugan P R , Tuttle J H
Published: January 2020

Vesicles prepared from iron-grown Thiobacillus ferrooxidans, and subsequently loaded with adenosine 5'-diphosphate and inorganic phosphate, produced adenosine 5'-triphosphate when subjected to H+ gradients comparable to those in the cells' normal environment (i.e., an internal pH in the range of 6.0 to 8.0 with an optimum of 7.0 to 7.8 and an external pH in the range of 2.1 to 4.1 with an optimum of 2.8). Nigericin, dicyclohexylcarbodiimide, and pentachlorophenol decreased adenosine 5'-triphosphate synthesis. Valinomycin at concentrations of 2.5 and 5.0 micrograms/ml increased adenosine 5'-triphosphate formation by 25 and 30%, respectively.
Polyethylene glycol-assisted transfection of Streptomyces protoplasts
Authors: Suarez J E , Chater K F
Published: January 2020

In the presence of polyethylene glycol (concentration optimum 20%), protoplasts of appropriate Streptomyces strains could be transfected by deoxyribonucleic acid (DNA) of five temperate phages (phi C31, VP5, R4, phi 448, and S14) belonging to four different immunity groups. Quantitation of transfection was made possible by plating the transfection mixture with excess uninfected protoplasts in soft agar overlays on protoplast regeneration medium so that plaques were easily detected. Optimum frequencies of transfection in the ranges of 10(-6)/DNA molecule and 10(-5)/viable protoplast were invariably obtained. It appeared that single DNA molecules initiated transfection events, and that the conformation of the DNA (i.e., circular or linear) was not important. Inhibition of transfection by ethylenediaminetetraacetic acid suggested that divalent cations were also observed. A minor subpopulation of protoplasts appeared to be particularly sensitive to transfection (i.e., "competent") in some DNA-host combinations. In such cases the size of this subpopulation was the major limiting factor in obtaining high transfection frequencies. The same protoplast
Organization of genes in the ftsA-envA region of the Escherichia coli genetic map and identification of a new fts locus (ftsZ)
Authors: Lutkenhaus J F , Wolf-Watz H , Donachie W D
Published: January 2020

Complementation tests have revealed that the mutation in the filamenting mutant PAT84 is distinct from ftsA and has been designated ftsZ. By isolating transducing phages carrying various amounts of the bacterial deoxyribonucleic acid in this region, it was possible to locate the ftsZ gene between ftsA and envA. It is concluded that these cell division genes are expressed independently of the neighboring murein genes.
Multilamellar surface layer of the cell wall of Albugo candida and Phycomyces blakesleeanus
Authors: Tewari J P , Skoropad W P , Malhotra S K
Published: January 2020

The surface layer of the cell wall of the sporangia of Albugo candida and of the sporangiophores of Phycomyces blakesleeanus was composed of a series of lamellae. The evidence from freeze-fracture, freeze-etch, and single-stage replicas indicated that the lamellae are organized as bilayers, an organization associated with the presence of lipids. The role of these lamellae in dispersibility and resistance is discussed.
Changes in cyclic nucleotide levels and dimorphic transition in Candida albicans
Authors: Niimi M , Niimi K , Tokunaga J , Nakayama H
Published: January 2020

The relationship between the levels of cyclic nucleotides and dimorphic transition in Candida albicans was examined. The results showed that cells of this pathogenic fungus contained both cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP), the concentration of the latter being about one-tenth that of the former in stationary-phase cells of the yeast form. Our results further indicated that germ tube formation induced by incubation at 40 degrees C followed a rise in cAMP concentration in the cell with no accompanying change in cGMP content. Cysteine, which suppressed germination, also reversed the increase in intracellular cAMP concentration. Dibutyryl cAMP (1 MM) significantly promoted germination in proline medium at temperatures of 32 to 34 degrees C. These results suggested that cAMP was one of the controlling factors in the morphological transition in Candida albicans.
Pyridine nucleotide cycle of Salmonella typhimurium: in vivo recycling of nicotinamide adenine dinucleotide
Authors: Foster J W , Baskowsky-Foster A M
Published: January 2020

The relative contribution of the two known pyridine nucleotide cycles of Salmonella typhimurium towards the intracellular recycling of nicotinamide adenine dinucleotide was determined. The results indicate that intracellular nicotinamide adenine dinucleotide is recycled by both the four-membered pyridine nucleotide cycle (PNC IV) and the six-membered pyridine nucleotide cycle (PNC VI) with a relative contribution of 60 to 69% and 31 to 40%, respectively. These studies also revealed a nicotinic acid mononucleotide-degradative activity which converts nicotinic acid mononucleotide to nicotinic acid. This represents the first demonstration of a functional PNC IV pathway in S. typhimurium.
Permeability properties of Escherichia coli outer membrane containing, pore-forming proteins: comparison between lambda receptor protein and porin for saccharide permeation
Authors: Nakae T , Ishii J
Published: January 2020

Outer membrane permeability conferred by lambda receptor protein and porins to maltose-maltodextrins and other oligosaccharides was studied in vitro with reconstituted vesicle membranes and in vivo with mutant strains lacking either one of these proteins. The vesicle membranes reconstituted from phospholipids, lipopolysaccharide, and purified lambda receptor allowed rapid diffusion of maltose and maltose-maltodextrins of up to six glucose residues, but the membranes acted essentially as a molecular sieve for sucrose, raffinose, stachyose, and inulins of molecular weights 800, 920, and 1,380. The vesicle membranes containing porins allowed rapid diffusion of maltose but not of maltose-maltodextrins larger than maltose. The apparent transport Km values for maltose-maltodextrins of up to six glucose residues from the strain carrying lamB+ ompB (lambda receptor+, porin-) were similar (about 5 X 10(-6) M), whereas the transport Km values for maltose- and maltotriose of the strain carrying lamB ompB+ (lambda receptor-, porin+) alleles appeared to be 300 and about 20,000 X 10(-6) M. These results suggest that lambda receptor protein forms permeability pores that facilitate the diffusion of maltose-maltodextrins and function as a molecular sieve for other saccharides.
Energization of Glucose Transport by
Pseudomonas fluorescens
Authors: Romano Antonio H. , Voytek Anna , Bruskin Arthur M.
Published: January 2020

We have measured the capacity of
Pseudomonas fluorescens
to transport the glucose analog 2-deoxy-
d
-glucose and the amino acids
l
-alanine and α-aminoisobutyric acid under conditions in which the cells could generate (i) both a membrane proton motive force and high-energy phosphate compounds, (ii) a proton motive force but not high-energy phosphate compounds, and (iii) neither a proton motive force nor high-energy phosphate compounds. This was done by depleting cells of adenosine triphosphate stores by treatment with sodium arsenate and then suspending them in a phosphate-free medium, where they could generate a proton motive force but not phosphate bond energy, or in a phosphate-containing medium, where they could generate both a proton motive force and phosphate bond energy. Inclusion of the proton-conducting ionophore carbonyl cyanide-
m
-chlorophenyl hydrazone under either condition precluded the generation of both a proton motive force and phosphate bond energy. The amino acids
l
-alanine and α-aminoisobutyric acid were transported independently of phosphate bond energy and required only a proton motive force. 2-Deoxy-
d
-glucose was transported only under conditions in which phosphate bond energy could be generated. These results are consistent with the findings of others that
Pseudomonas aeruginosa
produces an inducible shock-sensitive glucose-binding protein and conform to the generalization that binding protein-associated transport systems are energized by phosphate bond energy.
Interaction of Escherichia coli adenosine triphosphatase with aurovertin and citreoviridin: inhibition and fluorescence studies
Authors: Satre M , Bof M , Vignais P V
Published: January 2020

Aurovertins B and D inhibited the adenosine triphosphatase (ATPase) activity of soluble Escherichia coli coupling factor ATPase (BF1) isolated from wile-type E. coli K-12. Half inhibition was obtained with 2 microns aurovertin B and 0.9 microns aurovertin D. Aurovertins B and D had no inhibitory effect on BF1 isolated from the aurovertin-resistant E. coli mutant MA12. Acetylation or saponification of aurovertin D yielded a derivative which was devoid of inhibitory effect on BF1. Citreoviridin also inhibited wild-type BF1 but with much less efficiency (half inhibition at 60 microns) than aurovertin. Citreoviridin had no effect on the aurovertin-resistant BF1. The fluorescence intensity of aurovertins B and D was markedly enhanced upon addition to purified BF1. There was no enhancement of fluorescence when the aurovertins were added to BF1 isolated from the aurovertin-resistant mutant. The fluorescence of the aurovertin-BF1 complex was enhanced by adenosine 5'-diphosphate and by low concentrations of adenosine 5'-triphosphate. The adenosine 5'-diphosphate-enhanced fluorescence of the aurovertin-BF1 complex was quenched by high concentrations of adenosine 5'-triphosphate or by MG2+. Aurovertin bound selectively to the beta subunit of BF1 isolated from wile-type cells. By complementation assays in vitro, using a reconstituted system made of subunits isolated from wild-type and aurovertin-resistant BF1, it was shown that the altered peptide in aurovertin-resistant BF1 was the beta subunit.
Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase
Authors: Tommassen J , Lugtenberg B
Published: January 2020

Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.
Physiological and regulatory properties of the general amino acid transport system of Neurospora crassa
Authors: DeBusk R M , DeBusk A G
Published: January 2020

The fundamental properties of the general amino acid transport system of Neurospora crassa were investigated in the conidial stage of the life cycle. The transport activity was found to be under genetic control, and an isogenic set of mutants deficient for the neutral, basic, or general amino acid transport systems and combinations thereof was constructed and used for analyzing the properties specific to the general permease. Amino acid transport by this system was found to be a carrier-mediated active process with broad specificity for the neutral and basic amino acids. Kinetic analysis revealed that a common binding site functioned to transport both neutral and basic amino acids and that the permease had a high affinity for its substrates. The kinetic parameters Km, Vmax, and Ki were defined for several substrates. Two modes of regulation were detected: substrate inhibition and ammonium repression. Activity of the general system was enhanced by the removal of ammonium ions from the incubation medium with a concomitant decline in either neutral or basic permease activity, suggesting that a common component exists between the neutral and the general systems and between the basic and the general systems.
Cytoplasmic membrane proteins of spectinomycin-susceptible and -resistant strains of Neisseria gonorrhoeae
Authors: Miller M A , Schärli C S , Mills J
Published: January 2020

Cytoplasmic membranes were isolated and examined from two spectinomycin-susceptible and three spectinomycin-resistant clinical strains of Neisseria gonorrhoeae. A laboratory-derived spectinomycin-resistant mutant, obtained by serial passage on gradually increasing concentrations of the antibiotic, and a susceptible revertant, spontaneously arising from one of the resistant clinical strains, were also studied. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis revealed that a major protein, comprising about 7% of total cytoplasmic membrane protein (molecular weight 24,000), was absent in the three clinically isolated spectinomycin-resistant strains. In a revertant, this protein reappeared. During treatment of one of the susceptible strains with spectinomycin, the protein disappeared. However, this correlation was not maintained in the laboratory-derived spectinomycin-resistant mutant. This mutant was of comparable resistant to the clinical isolates, but the 24,000-molecular-weight protein was present in normal quantities. In addition, spectinomycin resistant in clinical isolates was variable compared with stable resistance exhibited by the laboratory-derived mutant. These findings suggested that differences in laboratory-derived versus clinical spectinomycin resistance may be due to different types of resistance mutations.
Induction of Normal Ascosporogenesis in Two-Spored
Saccharomyces cerevisiae
by Glucose, Acetate, and Zinc
Authors: Bilinski Carl A. , Miller John J.
Published: January 2020

In an apomictic strain of
Saccharomyces cerevisiae
that normally produces two-spored asci, production of three and four-spored asci was restored by adjusting zinc and carbon source levels in presporulation and sporulation media. Sporogenic capability was compared after growth in defined and undefined presporulation environments. Cultivation in defined presporulation medium containing 6% glucose followed by sporulation in 3.5% acetate yielded 16 ± 3% three- and four-spored asci. Inclusion of 25 μg of zinc sulfate per ml in presporulation medium or 25 μg of zinc acetate per ml in sporulation medium gave a further increase in yield of three- and four-spored asci; it reached 48 ± 1% when zinc was added to both media. Cells grown in undefined presporulation medium were incapable of three- and four-spored ascus development in acetate sporulation medium unless zinc was included in the latter medium. Cultivation in defined presporulation medium amended with yeast extract, peptone, or casein hydrolysate greatly reduced three- and four-spored ascus yields. Inclusion of a meiotic inhibitor (glucose) in sporulation medium inhibited three- and four-spored ascus production but not two-spored ascus production. Nuclear staining demonstrated two classes of asci: binucleate (one and two spored) and tetranucleate (three and four spored). Hence, some control over apomictic versus normal ascus development has been gained by nutritional manipulation.
Proton-linked D-xylose transport in Escherichia coli
Authors: Lam V M , Daruwalla K R , Henderson P J , Jones-Mortimer M C
Published: January 2020

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.
Correlation Between the Expression of an
Escherichia coli
Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide
Authors: Beher Michael , Pugsley Anthony , Schnaitman Carl
Published: January 2020

The
ompA
gene of
Escherichia coli
codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (
E. coli
K-12 and two clinical isolates of
E. coli
) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an
E. coli
K-12 recombinant carrying an unexpressed foreign
ompA
allele was subjected to two cycles of selection for an OmpA
+
phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign
ompA
gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the
ompA
gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.
Selective synthesis of plasmid-coded proteins by Escherichia coli during recovery from chloramphenicol treatment
Authors: Neidhardt F C , Wirth R , Smith M W , Van Bogelen R
Published: January 2020

Protein products of recombinant ColE1 plasmids are preferentially synthesized and can easily be identified in Escherichia coli cells recovering from prolonged treatment with chloramphenicol.
Effect of adenosine 5'-monophosphate on adenosine 5'-triphosphate activation of methyl coenzyme M methylreductase in cell extracts of Methanosarcina barkeri
Authors: Mountfort D O
Published: January 2020

In cell extracts of Methanosarcina barkeri, adenosine 5'-triphosphate (ATP)-activated methyl coenzyme M methylreductase was inhibited by adenosine 5'-monophosphate (AMP) but not by cyclic AMP. AMP (2 and 4 mM) shifted the saturation curve for ATP activation from hyperbolic (Hill coefficient [n] = 1.0) to sigmoidal (n = 1.5), decreased Vmax, and increased the apparent KmATP.
Sex pheromone cAD1 in Streptococcus faecalis: induction of a function related to plasmid transfer
Authors: Clewell D B , Brown B L
Published: January 2020

In short matings between two donor strains with distinguishable isogenic conjugative plasmids (derivatives of pAD1), only the strain preexposed to the sex pheromone cAD1 behaved as a donor.
Restriction map of the Escherichia coli malA region and identification of the malT product
Authors: Raibaud O , Schwartz M
Published: January 2020

A series of plaque-forming lambda h80 transducing phages carrying various portions of the malA region were isolated. A 5,800-base pair HindIII-EcoRI DNA fragment from one of these phages was cloned into pBR322 and shown to contain malT, which is the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase. A restriction map of the HindIII-EcoRI fragment was established, and it was correlated with the genetic map of the malA region (i) by mapping deletions which had been generated in vitro on the plasmid and (ii) by locating on the restriction map a DNA insertion of known genetic position. A 600-base pair HincII-HaeII segment was shown to contain all or part of the promoters for malT and malP, which are known to be transcribed in opposite directions. Strains carrying gene malT on a plasmid synthesized a 94,000-dalton polypeptide which was not produced by identical strains carrying similar plasmids in which malT was partially deleted. Estimates of the size of the malT gene support the conclusion that the 94,000-dalton polypeptide is the malT product.
Cloning of Gene
lon
(
capR
) of
Escherichia coli
K-12 and Identification of Polypeptides Specified by the Cloned Deoxyribonucleic Acid Fragment
Authors: Zehnbauer Barbara A. , Markovitz Alvin
Published: January 2020

A mutation in the
lon
(
capR
) gene of
Escherichia coli
K-12 results in overproduction of capsular polysaccharide and increased sensitivity to ultraviolet and ionizing radiations. The
lon
(
capR
) gene deoxyribonucleic acid was cloned from a new F′ factor. The new plasmids, designated pBZ201 and pBZ203, (i) contained an additional 8.2-megadalton (Md)
Eco
RI fragment that had the same mobility as one of the
Eco
RI fragments of the F′, and (ii) conferred repression of capsular polysaccharide synthesis and repression of sensitivity to ultraviolet radiation in a bacterial transformation experiment with
capR
mutant recipient strains. A
capR9
mutant plasmid, pBZ201M9, was also isolated and conferred expression of mucoidy and ultraviolet sensitivity to a
capR
+
(
lon
+
) strain, indicating that the
capR9
allele was dominant. Plasmids pBZ201M80, pBZ201M9-INSA, and pBZ201M9-INSB were characterized by transformation as containing recessive
capR
mutant alleles. Heteroduplex analyses and agarose gel electrophoresis of restriction endonuclease digests of plasmid DNA preparations revealed that (i) pBZ201M9-INSA and pBZ201M9-INSB each contains a 0.5-Md insertion (probably IS
1
) in the cloned DNA fragment at the same site, and (ii) pBZ201 and pBZ203, both
capR
+
plasmids, contain the same 8.2-Md fragment cloned in opposite orientations with respect to the cloning vehicle, pSC101. Plasmid-specified polypeptides were determined by using strain CSR603 maxicells containing each plasmid. Two new polypeptides were coded by the
lon
+
(
capR
+
) 8.2-Md DNA fragment: Z1, 94 kilodaltons (94K), and Z2, 67K. The maxicells containing recessive
capR
mutant plasmids were deficient only in synthesis of the 94K polypeptide, and the dominant (
capR9
) mutant plasmid specified 5 to 10 times more of the 94K polypeptide than the maxicells containing the
capR
+
plasmid. Other data indicated that the
capR9
-specified “94K polypeptide” was not identical to the
capR
+
-specified “94K polypeptide.” Thus the altered mutant polypeptide was synthesized in increased quantities, suggesting a defective mode of autogenous regulation for the
capR9
polypeptide and effective autogenous regulation of the
capR
+
polypeptide.
Cycloheximide resistance of Physarum polycephalum
Authors: Evans T E , Evans H H
Published: January 2020

In the presence of cycloheximide, wild-type plasmodia of Physarum polycephalum exhibit an immediate decrease in deoxyribonucleic acid synthesis, a reduction in the incorporation of [3H]thymidine into thymidine triphosphate, and an increase in the level of thymidine triphosphate, as well as a decrease in protein synthesis. In this study, we have utilized a cycloheximide-resistant (Cycr) amoebic strain selected from a population of cells mutagenized with nitrosoguanidine. Segregation data indicate that the resistance is due to a single mutation. We have used this Cycr mutant to construct Cycr plasmodial strains. Ribosomes isolated from such Cycr plasmodia showed resistance to cycloheximide in vitro, in contrast to ribosomes isolated from wild-type plasmodia. The Cycr plasmodia showed none of the cycloheximide-induced biochemical effects. Plasmodia heterozygous for the resistance marker were sensitive to cycloheximide with regard to growth but showed an intermediate response in the biochemical parameters. Heterokaryons formed by fusion of various proportions of the sensitive and resistant plasmodia showed a resistance with regard to both growth and biochemical parameters which was directly related to the fraction of Cycr plasmodia present in the heterokaryons. The data are consistent with the hypothesis that the effects of cycloheximide on deoxyribonucleic acid synthesis and nucleoside metabolism are secondary to the effect of the drug on protein synthesis in this organism.
Use of gene cloning to determine polarity of an operon: genes carAB of Escherichia coli
Authors: Crabeel M , Charlier D , Weyens G , Feller A , Piérard A , Glansdorff N
Published: January 2020

A gene-cloning approach was used to determine the transcription polarity of the carbamoylphosphate operon (carAB) of Escherichia coli. In agreement with the accompanying paper (J. Bacteriol. 143:914-920, 1980), our results lead to the conclusion that carA is the proximal gene of the carAB operon.
Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures
Authors: Darveau R P , Charnetzky W T , Hurlbert R E
Published: January 2020

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.
Structure of the Escherichia coli K2 capsular antigen, a teichoic acid-like polymer
Authors: Jann K , Jann B , Schmidt M A , Vann W F
Published: January 2020

The primary structure of the K2 antigen of Escherichia coli was elucidated by composition, alkaline fragmentation, dephosphorylation with hydrofluoric acid, periodate oxidation, and methylation analysis. The polymer contains galactose in the pyranosidic and furanosidic ring form. It consists of phosphogaolactopyranosyl glycerol and phosphagalactofuranosyl glycerol units in a molar ratio of 2:1. The sequence of these units is not known. The structure of the K2 antigen is reminiscent of that of certain teichoic acids of gram-positive bacteria. Using microprecipitation, it was shown that in the polymer galactoside is immunodominant.