bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of amino acid transport in Escherichia coli by transcription termination factor rho
Authors: Quay S C , Oxender D L
Published: January 2020

Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur.
Purification and Characterization of a Neutral Protease fromSaccharomycopsis lipolytica
Authors: Abdelal Ahmed T. H. , Kennedy Emily H. , Ahearn Donald G.
Published: January 2020

Saccharomycopsis lipolytica37-1 produced two inducible extracellular proteases, one under neutral or alkaline growth conditions and the second under acid conditions. Secretion of the neutral protease was repressed in the presence of glycerol or glucose, both of which supported rapid growth of the organism. Ammonium ions also repressed the secretion of the enzyme. The neutral protease activity copurified with esterase activity during ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose, and gel filtration on Sephadex G-150. The molecular weight of the enzyme was estimated to be 42,000 by sucrose density gradient centrifugation and 38,500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme had a pH optimum of 6.8. Phenylmethylsulfonylfluoride inhibited both protease and esterase activities, indicating the presence of a serine residue in the active center. Protease, but not esterase, activity was sensitive to ethylenediaminetetraacetate and was significantly activated by divalent ions. Dithiothreitol inhibited both protease and esterase activities, indicating the presence of a critical disulfide bridge. The enzyme hydrolyzed casein (Km= 25.6 μM) and hemoglobin as well as the nitrophenyl esters of tyrosine (Km= 2.4 mM), glycine, tryptophan, and phenylalanine.
Energy Requirements for the Transport of Methylthio-β-
d
-Galactoside by
Escherichia coli
: Measurement by Microcalorimetry and by Rates of Oxygen Consumption and Carbon Dioxide Production
Authors: Long Richard A. , Martin W. G. , Schneider Henry
Published: January 2020

The energy cost for maintenance of gradients of methylthio-β-
d
-galactoside in
Escherichia coli
was evaluated. Information was also obtained concerning the energy flow associated with gradient establishment under some circumstances. Energy flow was evaluated from transport-induced changes in the rate of heat evolution, oxygen consumption, and carbon dioxide production in metabolically active cells. Heats were measured with an isothermal calorimeter. Energy expenditure behavior was characterized by a transition that depended on the level of accumulation. The data for steady-state maintenance could be rationalized in terms of the Mitchell hypothesis, two models for influx and efflux, and a transition between them. At low levels of uptake, steady-state proton-methylthio-β-
d
-galactoside (TMG) symport for influx and efflux occurred via a nonenergy-requiring exchange process. The only energy requirement was that necessary to pump back in any TMG exiting via a leakage pathway (model I). Above the transition, all influx occurred with proton symport, but all exit, leak and carrier mediated, occurred without proton symport (model II). The H
+
/TMG stoichiometric ratio computed for the region of model II applicability (carbon source present, high level of uptake) approached 1. This value agreed with that of other workers for downhill β-galactoside flow, suggesting that the energy cost for both downhill and uphill flow was approximately the same. For low levels of uptake, initial establishment of the gradient was followed by a burst of metabolism that was much larger than that expected on the basis of the chemiosmotic hypothesis. In the absence of carbon source, the stimulation in respiration was sufficient to produce 13 times more protons than are apparently necessary to establish the gradient. The results indicate also that the nature of the biochemical process stimulated by TMG depends on its level of uptake. Insight into several aspects of the nature of these processes was provided through analysis of the heat, oxygen, and CO
2
data. The key factor controlling the transition in energy flow behavior is suggested to be rate of flux. The present data suggest that it occurs at a flux of ∼120 nmol/min per mg of protein.
Structural analysis of the surface polysaccharide of Staphylococcus aureus M
Authors: Liau D F , Hash J H
Published: January 2020

The chemical structure of the surface polysaccharide from Staphylococcus aureus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl-(1 leads to 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-D-aminogalacturonic acid via an amide bond.
Relationships among some R plasmids found in Haemophilus influenzae
Authors: Elwell L P , Saunders J R , Richmond M H , Falkow S
Published: January 2020

Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.
Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2
Authors: Glass T L , Bryant M P , Wolin M J
Published: January 2020

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.
Transfer of chromosomal genes mediated by plasmid r68.45 in Rhodopseudomonas sphaeroides
Authors: Sistrom W R
Published: January 2020

Plasmid R68.45 was transferred from Pseudomonas aeruginosa PAO25 to the photosynthetic species Rhodopseudomonas gelatinosa and Rhodopseudomonas sphaeroides by selection for resistance to antibiotics. R. sphaeroides strains carrying the plasmid could transfer the plasmid and also chromosomal genes to other strains of R. sphaeroides.
Fermentative metabolism of pyruvate by Rhodospirillum rubrum after anaerobic growth in darkness
Authors: Gorrell T E , Uffen R L
Published: January 2020

Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.
Source of Energy for Gliding Motility inFlexibacter polymorphus: Effects of Metabolic and Respiratory Inhibitors on Gliding Movement
Authors: Ridgway Harry F.
Published: January 2020

The effects of selected metabolic and respiratory inhibitors on the gliding motility ofFlexibacter polymorphuswere examined. Motility and oxygen consumption were quantitatively inhibited in a reversible manner by specific respiratory poisons, suggesting that gliding velocity was linked to electron transport activity. Arsenate had little influence on the number or rate of gliding filaments, despite a 95% decrease in the concentration of intracellular adenosine 5′-triphosphate (ATP). At concentrations of cyanide or azide that abolished gliding movement, cells possessed a level of ATP that should have been sufficient to allow motility. Proton-conducting uncouplers of oxidative phosphorylation, such as carbonylcyanidem-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide, strongly inhibited locomotion yet did not suppress respiratory activity or intracellular ATP sufficiently to account for their effect on movement. Inhibition of motility by CCCP (but not by tetrachlorosalicylanilide) was partially reversed by sulfhydryl compounds. However, unlike CCCP, inhibition of motility byp-chloromercuribenzoate, a known sulfhydryl-blocking reagent, was associated with a corresponding reduction in respiratory activity and ATP content of cells. Protein synthesis was not blocked by concentrations of CCCP inhibitory for motility, indicating that utilization of existing ATP in this energy-requiring process was not impaired. These data suggest (but do not unequivocally prove) that ATP may not function as the sole energy donor for the gliding mechanism, but that some additional product of electron transport is required (e.g., the intermediate of oxidative phosphorylation).
Ultrastructure of lipid bodies in Tilletia caries teliospores
Authors: Gardner J S , Hess W M
Published: January 2020

The ultrastructure of lipid bodies within developing, dormant, and germinating Tilletia caries (DC). Tul. (race T-16) teliospores was studied by freeze-etching and thin-sectioning techniques. When teliospores were prefixed in sodium cacodylate-buffered glutaraldehyde-acrolein for 24 h before further processing, most of the lipid bodies appeared to have a uniformly osmiophilic matrix. Some of these lipid bodies were surrounded by thin electron-dense lines that appeared to be half-unit membranes. Occasionally this membrane seemed to be absent, allowing for a direct interface between lipid and cytosol. Irregular electron-dense patterns were occasionally observed in lipid bodies of developing, dormant, and germinating teliospores. A lamellar substructure with 6- to 10-nm center-to-center spacing was visible in the electron-dense patterns at high magnifications. Irregular fracture patterns were visible in freeze-etch replicas.
Phosphoenolpyruvate:sugar phosphotransferase system in Ancalomicrobium adetum
Authors: Saier M H , Staley J T
Published: January 2020

Ancalomicrobium adetum possesses a membrane-associated phosphoenolpyruvate:sugar phosphotransferase system, the components of which exhibited enzymatic cross-reactivity with those from Salmonella typhimurium.
Fine-structure mapping of the firA gene, a locus involved in the phenotypic expression of rifampin resistance in Escherichia
Authors: Lathe R
Published: January 2020

The firA (Ts)200 mutation not only eliminates the resistance to rifampin of certain genetically resistant strains, but, moreover, renders ribonucleic acid synthesis thermolabile. The firA gene has been mapped by P1 tranduction and is located extremely close to the structural gene for deoxyribonucleic acid polymerase III at 4 min on the Escherichia coli linkage map.
Myxospore Coat Synthesis in
Myxococcus xanthus
: In Vivo Incorporation of Acetate and Glycine
Authors: Filer D. , White D. , Kindler S. H. , Rosenberg E.
Published: January 2020

Myxospore coat synthesis in
Myxococcus xanthus
was studied by incorporation of [
14
C]acetate into intermediates in the biosynthesis of coat polysaccharide and into acid-insoluble material during vegetative growth and after glycerol induction of myxospores. During short labeling periods at 27°C, the radioactivity was shown to be located primarily in
N
-acetyl groups rather than sugar moieties. Two hours after glycerol induction, the pools of
N
-acetylglucosamine 6-phosphate and uridine 5′-diphosphate-
N
-acetylgalactosamine (UDPGalNAc) plus uridine 5′-diphosphate-
N
-glucosamine increased about twofold and were labeled at twice the rate measured for vegetative cells. The increased rate of synthesis of UDPGalNAc and its precursors could be correlated with increased enzyme activities measured in vitro. Controlled acid hydrolysis revealed that the galactosamine portion of the myxospore coat was
N
-acetylated. After glycerol induction, the incorporation of acetate into acid-insoluble material increased threefold. This enhanced incorporation was sensitive to neither penicillin nor
d
-cycloserine. In contrast, bacitracin inhibited the incorporation of [
14
C]acetate into acid-insoluble material more effectively 2 h after myxospore induction than during vegetative growth. Chloramphenicol added to cells 90 min after induction blocked further increase in the rate of [
14
C]acetate incorporation. Since the myxospore coat contains glycine, polymer synthesis was also measured by chloramphenicol-insensitive [
14
C]glycine incorporation into acid-insoluble material. Although protein synthesis decreased after glycerol induction, glycine incorporation increased. Two hours after induction, glycine incorporation was only 75% inhibited by chloramphenicol and rifampin. The chloramphenicol-insensitive rate of incorporation of [
14
C]glycine increased during the first hour after myxospore induction and reached a peak rate after 2 to 3 h. The chloramphenicol-resistant incorporation of [
14
C]glycine was resistant to penicillin but sensitive to bacitracin.
Inhibition and facilitation of transfer among Pseudomonas aeruginos R plasmids
Authors: Sagai H , Uyobe S , Mitsuhashi S
Published: January 2020

Esamining 12 plasmids in Pseudomonas aeruginosa, we found two types of interaction in their transfer (inhibition and facilitation), using donor cells carrying two compatible plasmids. (i) Ten plasmids representing incompatibility groups P-1, P-2, P-5, P-6, and P-7 were all transmissible at a high frequency, 10-2 to 10-1, except for one with a lower frequency of about 10-3. The transfer of P-5 plasmids was inhibited by P-2 plasmids reciprocally or unilaterally, and the unilateral transfer inhibition was observed in other combinations between plasmids belonging to groups P-1, P-2, P-6, and P-7. It was characteristic of Pseudomonas plasmids that most plasmids with high transferability inhibited the transfer of other coexisting plasmids without distinct inhibition of their own transfer. (ii) Two plasmids, Rms149 of P-8 group and Rlb679, which was not classified, were transmissible at an exceptionally low frequency of 10-7 to 10-6, but their transfer was facilitated by plasmids with high transferability.
Myo-inositol transport system in Pseudomonas putida
Authors: Rber G , Belet M , Deshusses J
Published: January 2020

The kinetic features of the myo-inositol transport system in Pseudomonas putida are reported. The system is sensitive to osmotic shock, is not operative in membrane vesicles, and does not involved substrate phosphorylation. Line-weaver-Burk plots indicate the presence of two different systems, whose Kt are 5 micrometer and 0.43 mM and whose V max are 7.9 and 27 nml/mg per min, respectively. Transport activity of glucose-grown cells is very low. myo-Inositol-grown cells lose the high-affinity system upon osmotic shock; concentrated shock fluid possesses myo-inositol-binding activity. The system is very specific for the myo-configuration of the cyclitol.
Carbon Monoxide Oxidation by Methanogenic Bacteria
Authors: Daniels L. , Fuchs G. , Thauer R. K. , Zeikus J. G.
Published: January 2020

Different species of methanogenic bacteria growing on CO
2
and H
2
were shown to remove CO added to the gas phase. Rates up to 0.2 μmol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed.
Methanobacterium thermoautotrophicum
was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to CO
2
and CH
4
according to the following equation: 4CO + 2H
2
O → 1CH
4
+ 3CO
2
. However, growth was slight, and the growth rate on CO was only 1% of that observed on H
2
/CO
2
. Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of
M. thermoautotrophicum
contained both an active factor 420 (F
420
)-dependent hydrogenase (7.7 μmol/min per mg of protein at 35°C) and a CO-dehydrogenating enzyme (0.2 μmol/min per mg of protein at 35°C) that catalyzed the reduction of F
420
with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F
420
, viologen dyes were effective electron acceptors for the enzyme. The apparent

K
m

for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (ΔH‡) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 μM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of
M. thermoautotrophicum
exhibited several characteristic properties found for the enzyme of
Clostridium pasteurianum
but differed mainly in that the clostridial enzyme did not utilize F
420
as the electron acceptor.
Functional modifications of the translational system in Bacillus subtilis during sporulation
Authors: Chambliss G H , Legault-Demare L
Published: January 2020

Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA. The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA. The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis. The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA. The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes. The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E. coli ribosomes. The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA.
Bisulfite reductase of Desulfovibrio vulgaris: explanation for product formation
Authors: Drake H L , Akagi J M
Published: January 2020

Bisulfite reductase, purified from Desulfovibrio vulgaris, was coupled with the pyruvate phosphoroclastic reaction. Moderate to low reducing conditions resulted in the formation of trithionate; however, when the concentration of reductant was high, a mixture of trithionate and thiosulfate was formed. Sulfide was also a detectable product, but only when the concentration of bisulfite was low. Flavodoxin mediated native coupling between bisulfite reductase and the phosphoroclastic reaction. A model for bisulfite reductase activity is proposed.
Immunological study of the regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes
Authors: Murooka Y , Yamada T , Tanabe S , Harada T
Published: January 2020

Regulation of cellular arylsulfatase synthesis in Klebsiella aerogenes was analyzed by immunological techniques. Antibody directed against the purified arylsulfatase from K. aerogenes W70 was obtained from rabbits and characterized by immunoelectrophoresis, double-diffusion, quantitative precipitation, and enzyme neutralization tests. Arylsulfatase was located in the periplasmic space when the wild-type strain was cultured with methionine or with inorganic sulfate plus tyramine, but not with inorganic sulfate without tyramine, as the sole sulfur source. Tyramine oxidase was retained in the membrane fraction prepared from cells grown in the presence of tyramine. Arylsulfatase protein was not synthesized in the presence of tyramine and inorganic sulfate by mutant K611, which is deficient in tyramine oxidase (tynA). We conclude that the expression of the arylsulfatase gene (atsA) is regulated by the expression of tynA and that inorganic sulfate serves as a corepressor. In addition, strains mutated in the atsA gene were analyzed by using antibody.
Properties of the Beta-Lactamase Specified by the
Pseudomonas
Plasmid RPL11
Authors: Matthew Margaret , Sykes R. B.
Published: January 2020

The
Pseudomonas
plasmid RPL11 specifies a β-lactamase that has properties distinguishing it from other plasmid-mediated β-lactamases so far described, although it closely resembles plasmid Rms139 β-lactamase.
Variations in the Adenylate Energy Charge During Phased Growth (Cell Cycle) of
Candida utilis
Under Energy Excess and Energy-Limiting Growth Conditions
Authors: Thomas K. Chandapillai , Dawson Peter S. S.
Published: January 2020

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast
Candida utilis
growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + ½ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5′-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.
Role of threonine dehydrogenase in Escherichia coli threonine degradation
Authors: Potter R , Kapoor V , Newman E B
Published: January 2020

Threonine was used as nitrogen source by Escherichia coli K-12 through a pathway beginning with the enzyme threonine dehydrogenase. The 2-amino-3-ketobutyrate formed was converted to glycine, and the glycine was converted to serine, which acted as the actual nitrogen donor. The enzyme formed under anaerobic conditions and known as threonine deaminase (biodegradative) is less widespread than threonine dehydrogenase and may be involved in energy metabolism rather than in threonine degradation per se.
Growth and cell division during nitrogen starvation of the yeast Saccharomyces cerevisiae
Authors: Johnston G C , Singer R A , McFarlane S
Published: January 2020

During nitrogen starvation, cells of the yeast Saccharomyces cerevisiae increased threefold in number, and little ribonucleic acid (RNA) and protein were accumulated. Both RNA and protein were extensivley degraded during starvation, suggesting that intracellular macromolecules could supply most of the growth requirements. The types and proportions of stable RNA synthesized during nitrogen deprivation were characteristic of exponentially growing cells; however, the complement of proteins synthesized was different. We conclude that, once events in the deoxyribonucleic acid division cycle are initiated, cells can complete division with little dependence on continued net cell growth.
Cell size and budding during starvation of the yeast Saccharomyces cerevisiae
Authors: Johnston G C
Published: January 2020

When starved for nitrogen, cells of the yeast Saccharomyces cerevisiae produced abnormally small cells. Nonetheless, during starvation, only cells of a size characteristic of growing cells were capable of initiating a bud. Even when growth was severely limited, some event(s) in G1 required growth to a critical size for completion.
Isolation and Preparation of Pretyrosine, Accumulated as a Dead-End Metabolite by
Neurospora crassa
Authors: Jensen R. A. , Zamir L. , St. Pierre M. , Patel N. , Pierson D. L.
Published: January 2020

Pretyrosine is an amino acid intermediate of phenylalanine and/or tyrosine biosyntheses in a variety of organisms. A procedure for the isolation of high-quality pretyrosine as the barium salt is described. Stable solutions of ammonium pretyrosine that are suitable for use as substrate in enzyme assays can be prepared in good yield with relatively few purification steps. A triple mutant of
Neurospora crassa
, bearing genetic blocks corresponding to each initial enzyme step of the three pathway branchlets leading to the aromatic amino acids, accumulates prephenate and pretyrosine. Although the time courses of prephenate and pretyrosine accumulations were found to be parallel in any given experiment, the ratios of the two metabolites varied as much as 100-fold depending upon such variables as carbon source, temperature of growth, accumulation, and especially the presence of aromatic pathway metabolites. Under appropriate nutritional conditions of accumulation, pretyrosine concentrations in excess of 4 mM in culture supernatant fluids were obtained. Strains individually auxotrophic for phenylalanine or tyrosine accumulate lesser amounts of prephenate and pretyrosine. The metabolic blocks of the mutant result in high intracellular levels of prephenate, which is then partially transaminated to pretyrosine. In
N. crassa
, pretyrosine is a dead-end metabolite since it is not enzymatically converted to phenylalanine or tyrosine. At a mildly acidic pH, pretyrosine is quantitatively converted to phenylalanine in a nonenzymatic reaction.
Host-dependent, thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae
Authors: Yamamoto T , Yokota T
Published: January 2020

The thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae, was studied. pJY5 consisted of 61 million daltons of covalently closed circular (CCC) deoxyribonucleic acid (DNA) with a buoyant density of 1.714 g/cm3 (55 mol % guanine plus cytosine). In Escherichia coli, this plasmid replicated stringently at 32 degrees C, but ceased its CCC DNA replication after a short incubation at 42 degrees C, resulting in production of R- segregants. The thermosensitive replication of pJY5 was not overcome by the coexistence of non-thermosensitive R plasmids. The plasmid manifested an inhibitory effect on host bacterial cell growth at 42 degrees C, although the effect was less prominent than that of R plasmids belonging to the T-incompatibility group, Rts1, R401, and R402. When the pJY5 plasmid was transferred into E. cloacae, however, no R- segregants were detected at any culture temperature, even 42 degrees C. Alkaline sucrose gradient analysis revealed that a significant amount of pJY5 CCC DNA was synthesized in E. cloacae at the high temperature but not in E. coli. Furthermore, the growth-inhibitory effect of pJY5 on hosts at 42 degrees C was not observed in E. cloacae. On the other hand, Rts1 and R401 were found to be thermosensitive in E. cloacae as well as in E. coli.
Synthesis of mot and che gene products of Escherichia coli programmed by hybrid ColE1 plasmids in minicells
Authors: Matsumura P , Silverman M , Simon M
Published: January 2020

Hybrid Escherichia coli ColE1 plasmids carrying the genes for motility (mot) and chemotaxis (che) were transferred to a minicell-producing strain. The mot and che genes on the hybrid plasmid directed protein synthesis in minicells. Polypeptides synthesized in minicells were identical to the products of the motA, motB, cheA, cheW, cheM, cheX, cheB, cheY, and cheZ genes previously identified by using hybrid lambda and ultraviolet-irradiated host cells (Silverman and Simon, J. Bacteriol. 130:1317-1325, 1977), thus confirming these gene product assignments. The products of some che genes (cheA and cheM) appeared as more than one band on polyacrylamide gel electrophoresis, but analysis of partial peptide digests of these polypeptides suggested that the multiple forms were coded for by a single gene. Measurement of the physical length of the hybrid plasmids allowed an estimate of the amount of coding capacity of the cloned deoxyribonucleic acid, which was devoted to the synthesis of the mot and che gene products. These estimates were also consistent with the hypothesis that the multiple polypeptides corresponding to cheA and cheM were the products of single genes.
A colI-specified product, synthesized in newly infected recipients, limits the amount of DNA transferred during conjugation of Escherichia coli K-12
Authors: Boulnois G J , Wilkins B M
Published: January 2020

The amount of ColI DNA transferred between mating cells of Escherichia coli K-12 increased about fourfold when rifampin-resistant donors were mated with sensitive recipients in the presence of the drug. Conjugational synthesis of ColI in dnaB recipients, shown primarily to reflect conversion of the transferred DNA into double-stranded material, was also enhanced when the recipients were treated with either rifampin or streptomycin. It is suggested that the amount of ColI transfer is normally limited by the synthesis of one or more proteins in the newly infected recipients. The protein is thought to be plasmid-specified because rifampin also quadrupled transfer to UV-irradiated recipients which were deficient in the transcription of the resident DNA. Successive strands of ColI appear to be transferred discontinuously, because the transferred DNA accumulated in normal and rifampin-treated recipients in the form of circular and linear monomeric units. Although rifampin treatment of recipients also increased transfer of a second Ialpha plasmid, R144drd-3, by about four times, the drug failed to cause a substantial increase of Flac transfer in comparable matings.
Tween 80 effect on glucosyltransferase synthesis by Streptococcus salivarius
Authors: Wittenberger C L , Beaman A J , Lee L N
Published: January 2020

Streptococcus salivarius (ATCC 25975) produced very low or nondetectable amounts of the extracellular enzyme glucosyltransferase (GTase) when grown in a chemically defined medium. The addition of Tween 80 to this medium resulted in the production of markedly enhanced levels of the enzyme. Oleic acid, the methyl ester of oleic acid, and sucrose each could not substitute for Tween 80 in this regard. The surfactant had no direct activating effect on performed enzyme activity. Tween 80 also stimulated the production of GTase by concentrated cells suspended in defined medium during a time when no measurable growth occurred. Under these conditions, the stimulatory effect of Tween 80 was blocked by chloramphenicol. It was further found that the surfactant dramatically stimulated the differential rate of GTase synthesis. These and other data strongly suggest that Tween 80 stimulates the production of extracellular GTase by acting either directly or indirectly at the level of enzyme synthesis.
Temperature-sensitive nature of the rodB maturation in Bacillus subtilis
Authors: Rogers H J , Thurman P F
Published: January 2020

The Mg2+ requirement of a morphological mutant of Bacillus subtlis, rodB strain 104 was highly temperature sensitive in the presence of halide or nitrate anions. Likewise the morphological change from rod shapes to spheres was dependent upon temperature, the same anions, and the Mg2+ concentration. The three factors interacted. Other rodB mutants behaved similarly. If the rodB strain 104 in its rod form was treated at high temperatures in the absence of either protein or peptidoglycan synthesis and restored to lower temperatures with the syntheses restarted, a partial temporary change toward cocci occurred. In the absence of halides or in the presence of Cl- but not Br-, the cells increased in volume when they changed from rods to cocci.
Regulation of gamma-aminobutyric acid degradation in Escherichia coli by nitrogen metabolism enzymes
Authors: Zaboura M , Halpern Y S
Published: January 2020

The possible role of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase in the regulation of enzyme formation in the gamma-aminobutyric acid (GABA) catabolic pathway of Escherichia coli K-12 was investigated. Evidence is presented indicating that glutamine synthetase acts as a positive regulator in the E. coli GABA control system. Mutations impairing glutamate synthase activity prevent the depression of the enzymes of the GABA pathway in ammonia-limited glucose media. However, mutations resulting in constitutive synthesis of glutamine synthetase (GlnC) restore the ability of the glutamate synthase-less mutants to grow in glucose-GABA media and result in depressed synthesis of the GABA enzymes. It is suggested that the loss of glutamate synthesis activity affects the GABA control system indirectly by lowering glutamine synthetase levels.
Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides
Authors: Markwell J P , Lascelles J
Published: January 2020

Rhodopseudomonas sphaeroides has a pyridine nucleotide-independent L-lactate dehydrogenase associated with the membrane fraction of cells grown either aerobically or phototrophically. The dehydrogenase is present in cells grown on a variety of carbon sources, but at levels less than 20% of that found in cells grown with DL-lactate. The dehydrogenase has been purified 45-fold from membranes of strain L-57, a non-photosynthetic mutant, by steps involving solubilization with lauryl dimethylamine oxide and three anion-exchange chromatography steps. The purified enzyme was specific for the L-isomer of lactate. The Km of the purified enzyme for L-lactate is 1.4 mM, whereas that of the membrane-associated enzyme is 0.5 mM. The enzyme activity was inhibited competitively by D-lactate and non-competitively by oxalate and oxamate. Quinacrine, a flavin analog, also inhibited the activity. The inducible enzyme may serve as a marker of membrane protein in studies of membrane development.
Stimulation of Lipase Production During Bacterial Growth on Alkanes
Authors: Breuil Colette , Shindler D. B. , Sijher J. S. , Kushner D. J.
Published: January 2020

Acinetobacter lwoffi
strain O
16
, a facultative psychrophile, can grow on crude oil, hexadecane, octadecane, and most alkanes when tested at 20 but not at 30°C. Growth occurred on a few alkanes at 30°C but after a longer lag than at 20°C. Cells grown on alkanes as sole carbon sources had high levels of cell-bound lipase. In contrast, previous work has shown that those grown on complex medium produced cell-free lipase and those grown on defined medium without alkanes produced little or no lipase. Low concentrations of the detergent Triton X-100 caused the liberation of most of the lipase activity of alkane-grown cells and increased total lipase activity. When ethanol and hexadecane were both present in a mineral medium, diauxic growth occurred; until the ethanol was completely used up, hexadecane was not utilized, and the lipase activity was very low. When growth on hexadecane began, lipase activity increased, reaching a level 50- to 100-fold higher than that of cells growing on ethanol. A similar pattern of lipase formation and hexadecane utilization was observed with
Pseudomonas aeruginosa
. Whenever
A. lwoffi
and other bacteria degraded alkanes they exhibited substantial lipase activity. Not all bacteria that produced lipase, however, could attack alkanes. Bacteria that could not produce lipase did not attack alkanes. The results suggest that a correlation may exist between lipase formation and alkane utilization.
Effect of glucose starvation on the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of yeast
Authors: Mazón M J
Published: January 2020

Yeast cells growing on mineral medium plus ammonia and glucose contained high levels of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase activity, as measured in crude extracts. After suspension of cells in fresh medium lacking glucose, there was a loss of the glutamate dehydrogenase activity. Loss of activity was inhibited by 2,4-dinitrophenol, sodium azide, iodoacetic acid, and cycloheximide. The enzyme activity was restored when glucose was added back to the medium, and this recovery was fully prevented in the presence of cycloheximide.
Deg phenotype of Escherichia coli lon mutants
Authors: Gottesman S , Zipser D
Published: January 2020

Deg. one of the Escherichia coli systems for degrading abnormal polypeptides (e.g., nonsense fragments), is also involved in the degradation of some classes of missense proteins. Both missense proteins of beta-galactosidase and temperature-sensitive phage products appear to be degraded by the Deg system. Mutations in the Deg system are indistinguishable from mutations classically called lon or capR; all map near proC, all are mucoid, defective in protein degradation, sensitive to radiomimetic agents, and defective in P1 lysogenization. All are able to propagate temperature-sensitive phage better than lon+ parental strains. Mutations that suppress the radiation sensitivity of these strains (sul) also suppress the P1 lysogenization defect, but do not affect mucoidy or the degradation defect.
Near-UV mutagenesis: photoreactivation of 365-nm-induced mutational lesions in Escherichia coli WP2s
Authors: Webb R B
Published: January 2020

Reversion to tryptophan independence induced by 365-nm and 254-nm radiation was studied in Escherichia coli WP2s (B/r trp uvrA). Under aerobic conditions, the mutant frequency responses was of the fluence-square or "two-hit" type at both 365 and 254 nm when revertants were assayed on minimal agar supplemented with 2% nutrient broth (SEM plates). In contrast, when mutants were assayed on minimal agar supplemented with tryptophan only, the revertant yield was reduced to very low values at 365 nm, whereas values substantially greater than with SEM plates were obtained at 254 nm. Premutational lesions induced by both 365-nm and 254-nm radiation were photoreactivated more than 10-fold when assayed on SEM plates, implicating pyrimidine dimers as premutational lesions at both wavelengths. The strong photoreactivation of 365-nm-induced mutagenesis contrasted strikingly with the complete absence of photoreactivation of 365-nm-induced lethality in this strain.
Evolution of ribosomal proteins in Enterobacteriaceae
Authors: Hori H , Osawa S
Published: January 2020

The evolution of ribosomal proteins of about 70 bacterial strains belonging to the family Enterobacteriaceae has been studied by use of previously reported data (S. Osawa, T. Itoh, and E. Otaka, J. Bacteriol. 107:168-178, 1971) and those obtained in this paper. The proximity of the bacteria was quantified by co-chromatographing the differentially labeled ribosomal proteins from two strains on a column of carboxymethyl cellulose in various combinations. The were then classified into 12 groups (=species?) according to their ribosomal protein compositions and were placed in a phylogenic tree.
Binding and transport of thiamine by Lactobacillus casei
Authors: Henderson G B , Zevely E M
Published: January 2020

The relationship between thiamine transport and a membrane-associated thiamine-binding activity has been investigated in Lactobacillus casei. Thiamine transport proceeds via a system whose general properties are typical of active uptake processes; entry of the vitamin into the cells requires energy, is temperature dependent, exhibits saturation kinetics, and is inhibited by substrate analogs. A considerable concentration gradient of unchanged thiamine can be achieved by the system, although the vitamin is slowly metabolized to thiamine pyrophosphate. Consistent with these results, L. casei also contains a high-affinity, thiamine-binding component which could be measured by incubation of intact cells with labeled substrate at 4 degrees C (conditions under which transport is negligible). Binding was insensitive to iodoacetate, occurred at a level (0.5 nmol per 10(10) cells) nearly 20-fold higher than could be accounted for by facilitated diffusion, and was found to reside in a component of the cell membrane. Participation of this binder in thiamine transport is supported by the observations that the processes of binding and transport showed similarities in their (i) regulation by the concentration of thiamine in the growth medium, (ii) binding affinities for thiamine, and (iii) susceptibility to inhibition by thiamine analogs.
Glucose metabolism and dimorphism in Mucor
Authors: Inderlied C B , Sypherd P S
Published: January 2020

Mucor racemosus fermented glucose to ethanol, carbon dioxide, and glycerol. When this fungus was grown anaerobically in either the yeast or mycelial form, the catabolism of glucose was very similar. Yeast cells shifted to aerobic conditions maintained a high flux of glucose carbon through the glycolytic and pentose phosphate pathways. Mycelial cells grown aerobically catabolized glucose in a manner consistent with a respiratory metabolism. Although there was no consistent pattern of glucose metabolism in the mycelial form of Mucor, growth in the yeast form consistently was correlated with a high flux of glucose carbon through the catabolic pathways.
Involvement of Rhizobium japonicum O antigen in soybean nodulation
Authors: Maier R J , Brill W J
Published: January 2020

Non-nodulating mutant strains of Rhizobium japonicum lacked a surface antigen that was present on the wild type. This surface antigen is associated with the O antigen portion of the lipopolysaccharide. Paper chromatography of hydrolyzed lipopolysaccharide and O antigen revealed three major component differences between the non-nodulating strains and the wild type.
Positive detection of mycoplasma contamination by the whole-mount preparation of cell cultures for transmission electron microscopy
Authors: Maul G
Published: January 2020

Low-level mycoplasma contamination of cell cultures is difficult to recognize with presently available techniques. This report describes the adaptation of the whole-mount technique, usually used for scanning microscopy, for transmission electron microscopy. The differentiation between microvilli and the equal-sized filamentous mycoplasma is based on the differential density obtained by the use of the method described. This method allows positive identification of mycoplasma and reduces the preparation time and the time necessary for scanning the preparation.
Alkaline phosphatase possessing alkaline phosphodiesterase activity and other phosphodiesterases in Bacillus subtilis
Authors: Yamane K , Maruo B
Published: January 2020

In Bacillus subtilis Marburg strain, single-point mutations in the phoP locus brought about simultaneous losses of the major activities of alkaline phosphatase (APase) and alkaline phosphodiesterase (APDase). Revertants recovered the two activities. APases with APDase activity were purified from the membrane fraction of B. subtilis 6160-BC6 and from the culture fluid of an APase-secreting B. subtilis mutant strain, RAN 1. In addition to these major APases with APDase activity, at least two kinds of phosphodiesterase (PDase) without phosphatase activity were found in the cytoplasmic supernatants of RAN 1 and an APase-less B. subtilis mutant strain, SP25. Another minor APase with a molecular weight of about 80,000, which had almost no PDase activity, was isolated from the membrane fraction of strain 6160-BC6. Enzyme distribution in subcellular fractions from various strains cultured in high- and low-phosphate media was analyzed. The PDases did not cross-react with rabbit antiserum against the RAN 1 APase with APDase activity. The main component of the PDases had a molecular weight of about 80,000 and was most active at pH 8.0. These results suggest that APase with APDase activity is different from PDases detected in cytoplasmic supernatants and that phoP is the structural gene for the phosphate-repressible APase with APDase activity.