bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli
Mutants Tolerant to Beta-Lactam Antibiotics
Authors: Kitano Kazuaki , Tomasz Alexander
Published: January 2020

Two types of
Escherichia coli
mutants tolerant to beta-lactam antibiotics were isolated. One is
E. coli
χ2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42°C, and the others are the mutants C-80 and C-254, selected from mutagenized
E. coli
χ1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of χ1776 or in χ2452 grown at 32°C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of χ2452 grown at 42°C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of χ2452 cells grown at 42°C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of χ2452 grown either at 42 or 32°C. Possible mechanisms for the penicillin tolerance of
E. coli
are considered on the basis of these findings.
In vitro binding of cloacin DF13 to its purified outer membrane receptor protein and effect of peptidoglycan on bacteriocin-receptor interaction
Authors: Oudega B , van der Molen J , de Graaf F K
Published: January 2020

The in vitro neutralization of the killing activity of cloacin DF13 by incubation with its purified receptor protein was shown to be the result of the formation of a direct and specific equimolar complex of both proteins. The binding of cloacin DF13 to its receptor protein did not result in a fragmentation of the cloacin molecules nor in the expulsion of immunity protein from the bacteriocin. The rate of the cloacin DF13-receptor interaction in vitro was found to be enhanced significantly in the presence of peptidoglycan, but lysozyme-treated peptidoglycan did not affect this interaction. Incubation of the cloacin DF13 as well as its receptor protein with peptidoglycan showed that the receptor protein but not the cloacin DF13 was able to bind to the peptidoglycan.
Precursor in cotranslational secretion of diphtheria toxin
Authors: Smith W P , Tai P C , Murphy J R , Davis B D
Published: January 2020

By extracellular labeling of peptides of intact Corynebacterium diphtheriae, followed by fractionation of the cells and chain completion by isolated polysomes, it is shown that diphtheria toxin is formed and secreted cotranslationally by membrane-bound polysomes; free polysomes from none. Moreover, when the chains on these polysomes were completed in vitro, in the absence of membrane they were found to include not only diphtheria toxin of a molecular weight of 62,000, but also a larger precursor of a molecular weight of 68,000. The precursor was identified by several properties: immune precipitation; conversion into toxin fragments A and B; adenosine diphosphate ribosyl-transferase activity after activation with trypsin; and cleavage to 62,000 daltons by membrane enzymes. The precursor yields an N-terminal A fragment with a broadened molecular weight distribution, compared with that from authentic toxin, thus supporting the expectation that the extra segment of the precursor is N-terminal.
Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus
Authors: Mansour J D , Henry S , Shapiro L
Published: January 2020

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.
Amphotericin B-induced changes in K+ content, viability, and ultrastructure of yeast-phase Histoplasma capsulatum
Authors: Arnold W N , Pringle A T , Garrison R G
Published: January 2020

Yeast-phase cells of Histoplasma capsulatum were challenged with amphotericin B, and membrane perturbation was monitored by K+ efflux. Suspensions of washed cells readily absorbed about 1.12 microgram of amphotericin B per mg (dry weight) and further nonspecific sites were also apparent. The dose-response curve for initial rate of K+ efflux was sigmoidal within the range 0.1 to 1.0 microgram of amphotericin B per ml. A fungistatic concentration of amphotericin B (0.3 microgram/ml) evoked an efflux of 85 to 90% K+ from the cells within 15 min, but cell viability decreased only 13% (yeast phase) or 33% (transformed to mycelial units). Ultrastructural changes in treated cells were detected within 5 min, and the hallmark was expansion of vacuoles during the 1-h monitoring period. In contradistinction to a previous report, the appearance of the protoplasmic membrane was not altered by fungistatic concentration. When treated cells were returned to a fresh growth medium, there was a pronounced lag (20 h). During this apparent recovery phase, the large vacuoles fragmented and returned to normal size. It is proposed that vacuoles of H. capsulatum act as a spatial buffer of considerable survival value to stressed cells.
Iron storage in Mycoplasma capricolum
Authors: Bauminger E R , Cohen S G , Labenski de Kanter F , Levy A , Ofer S , Kessel M , Rottem S
Published: January 2020

Considerable quantities or iron were incorporated into the Mycoplasma capricolum cell membrane. Mossbauer studies showed that the iron is in a form which becomes magnetically ordered at low temperatures. The iron-enriched cells contained membrane-bound electron-dense particles of about 6.0 nm in diameter.
Polarity of amber mutations in ribosomal protein genes of Escherichia coli
Authors: Cabezón T , Delcuve G , Faelen M , Desmet L , Bollen A
Published: January 2020

Two amber mutations have been mapped inside the spcA-strA region (now called rpsE-rpsL) on the bacterial genome. Derivatives of the transducing phage lambda fus3 carrying each mutation were constructed and assayed in ultraviolet-irradiated bacteria to identify the mutated genes and measure the polarity of the mutations. The data indicated that both mutations, 3162(Am) and 3161(Am), affect genes coding for ribosomal proteins: rplC (L3) and rpsN (S14), respectively. It was shown also that each mutation exerts, inside of its respective operon (S10 and spc units), a relatively strong polar effect on genes distal to the mutated locus.
Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation
Authors: Postma P W , Stock J B
Published: January 2020

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.
Extracellular and membrane-bound proteases from Bacillus subtilis
Authors: Mäntsälä P , Zalkin H
Published: January 2020

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.
Function of positive regulatory gene gal4 in the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae: evidence that the GAL81 region codes for part of the gal4 protein
Authors: Matsumoto K , Adachi Y , Toh-e A , Oshima Y
Published: January 2020

A meiotic fine structure map of the gal4 locus was constructed, which extended over 0.44 units on the chromosome (units in percent frequency of supposed recombination). Several nonsense gal4 mutations (four UAA and two supposed UGA [gal4-62 and gal4-69]) were placed at various sites on the map. In reversion experiments with 20 independently isolated gal4 mutants, only the gal4-62 and gal4-69 alleles, which are located at the same site on the map, could revert to overcome the superrepression of gal80s-1 spontaneously with a frequency of approximately 4 x 10(-7). Secondary mutations in the revertants occurred in the region of gal4-62 or were due to unlinked suppressors. A total of 15 GAL81 mutations in 19 isolates were found to be located in the same region as gal4-62 by three-point crosses with the aid of gal4 mutants; the other four could not be analyzed. The reverted gal4 gene and GAL81 mutations were semidominant over the wild-type GAL4+ allele and fully dominant over a nonsense gal4 mutation. Four suppressors (one dominant and three recessive) effective against gal4-62 and gal4-69 were isolated. The dominant suppressor was also effective against three independent, authentic auxotrophic UGA nonsense mutations, and one of the three recessive suppressors were effective against the authentic auxotrophic UAA and UAG mutations. These results strongly support the idea that the gal4 locus is expressed constitutively and codes for a regulatory protein. The GAL81 site mapped inside the locus codes for a part of the gal4 protein but does not work as an operator.
Protein expression in Escherichia coli minicells containing recombinant plasmids specifying trimethoprim-resistant dihydrofolate reductases
Authors: Fling M E , Elwell L P
Published: January 2020

Deoxyribonucleic acid fragments containing the structural genes for several trimethoprim-resistant dihydrofolate reductases from naturally occurring plasmids were inserted into small cloning vehicles. The genetic expression of these hybrid plasmids was studied in purified Escherichia coli minicells. The type I dihydrofolate reductase, encoded by plasmid R483 and residing within transposon 7 (Tn7), had a subunit molecular weight of 18,000. The type II dihydrofolate reductase, specified by plasmid R67, had a subunit molecular weight of 9,000. These two enzymes were antigenically distinct in that anti-type II dihydrofolate reductase (R67) antibody did not cross-react with the type I (R483) protein. The trimethoprim-resistant reductase specified by plasmid R388 had a subunit molecular weight of about 10,500 and was immunologically related to the type II (R67) enzyme. A 9,000 subunit of the dihydrofolate encoded by the transposition element Tn402 was also antigenically related to the R67 reductase.
Temporal dissociation of late events in Bacillus subtilis sporulation from expression of genes that determine them
Authors: Jenkinson H F , Kay D , Mandelstam J
Published: January 2020

During sporulation in replacement medium, resistance to toluene to heating at 65 degrees C, to lysozyme, and to heating at 80 degrees C appeared in sequence between 4 and 8 h after the induction of sporulation (i.e., between t4 and t8). The addition of sufficient chloramphenicol at t4.5 to prevent protein synthesis nevertheless allowed the emergence of all of these types of resistance except lysozyme resistance. The numbers of spores with these types of resistance (lysozyme resistance again excepted) increased about fourfold when phenylmethylsulfonyl fluoride (an inhibitor of serine protease activity) was also present. Thus, the observed increases in resistance in the 2 h after the addition of chloramphenicol resulted from the utilization of preformed protein elements. Dipicolinate did not seem to be a determining factor in the development of any of these forms of resistance. Electron micrographs showed that inhibition of protein synthesis did not prevent deposition of the outer layers of the spores. Lysozyme resistance developed differently; synthesis of the relevant proteins began later (t5), and continued synthesis was necessary up to t8. Some processing of proteins made earlier was a prerequisite for lysozyme resistance. Therefore, it appears that from the viewpoint of regulation, the expression of the genes and the production of the proteins for resistance to toluene, heating at 65 degrees C, and heating at 80 degrees C are all stage IV sporulation events, although the resistance properties themselves appear only during stages V and VI. Lysozyme resistance is the only real late event among those examined. The germination characteristics of the spores, which are also late events, are discussed in this context, as they too are dependent on proteins that are synthesized much earlier.
Effect of macromolecular synthesis and lytic capacity on surface growth of Streptococcus faecalis
Authors: Higgins M L , Daneo-Moore L
Published: January 2020

Exposure of exponential-phase cultures of Streptococcus faecalis to any of three inhibitors of protein synthesis was accompanied by an increase in the average distance that the cross wall extended into the cytoplasm. This resulted in: (i) an increase in the average surface area of the cross wall (Sa) and (ii) septation occurring in the envelope growth sites that were much smaller than the controls. However, although at the concentrations used, all three antibiotics inhibited protein synthesis and autolytic capacity to the same extent and with the same kinetics, cells treated with these agents showed large differences in the rate at which Sa values increased above those of the untreated cells. The largest increases in Sa were observed in cells that synthesized the least amount of cytoplasmic macromolecules (deoxyribonucleic acid, plus ribonucleic acid, plus protein). The observations were interpreted in terms of a model in which a decreased lytic capacity reduces the rate of splitting of the nascent cross wall into two layers of peripheral wall, preferentially using wall precursors to close open cross walls. However, the extent to which centripetal growth occurs would be inversely related to the rate at which cytoplasmic macromolecules are synthesized. In contrast, inhibition of deoxyribonucleic acid synthesis was accompanied by decreased extension of the leading edge of the cross wall into the cytoplasm, thus antagonizing septation. These findings are discussed in relation to the normal cell division cycle of S. faecalis.
Identification of two glutamine synthetases in Agrobacterium
Authors: Fuchs R L , Keister D L
Published: January 2020

Two distinct glutamine synthetases have been identified in Agrobacterium and in the fast-growing rhizobia. A limited survey indicates that GSII may be found only in the Rhizobiaceae family.
Caffeine resistance of Saccharomyces cerevisiae
Authors: Bard M , Neuhauser J L , Lees N D
Published: January 2020

Four caffeine-resistant haploid isolates, two resistant to 50 mM caffeine and two resistant to 100 mM caffeine, were genetically analyzed. Complementation and tetrad analysis indicated that all four mutations are alleles of the same locus. All four isolates demonstrated incomplete dominance when hybridized to the wild-type strain and dominance of high to low resistance when hybridized to one another. Differences in caffeine resistance were found between wild-type grande cells and its petite derivative.
Biosynthesis and catabolism of 6-deoxy L-talitol by Klebsiella aerogenes mutants
Authors: St Martin E J , Mortlock R P
Published: January 2020

A mutant strain of Klebsiella aerogenes was constructed and, when incubated anaerobically with L-fucose and glycerol, synthesized and excreted a novel methyl pentitol, 6-deoxy L-talitol. The mutant was constitutive for the synthesis of L-fucose isomerase but unable to synthesize L-fuculokinase activity. Thus, it could convert the L-fucose to L-fuculose but was incapable of phosphorylating L-fuculose to L-fuculose 1-phosphate. The mutant was also constitutive for the synthesis of ribitol dehydrogenase, and in the presence of sufficient reducing power this latter enzyme catalyzed the reduction of the L-fuculose to 6-deoxy L-talitol. The reducing equivalents required for this reaction were generated by the oxidation of glycerol to dihydroxyacetone with an anaerobic glycerol dehydrogenase. The parent strain of K. aerogenes was unable to utilize the purified 6-deoxy L-talitol as a sole source of carbon and energy for growth; however, mutant could be isolated which had gained this ability. Such mutants were found to be constitutive for the synthesis of ribitol dehydrogenase and were thus capable of oxidizing 6-deoxy L-talitol to L-fuculose. Further metabolism of L-fuculose was shown by mutant analysis to be mediated by the enzymes of the L-fucose catabolic pathway.
Stimulation of deoxyribonucleic acid replication fork movement by spermidine analogs in polyamine-deficient Escherichia coli
Authors: Geiger L E , Morris D R
Published: January 2020

We examined the rate of deoxyribonucleic acid (DNA) replication fork movement in polyamine-deficient cells of Escherichia coli by two independent techniques. DNA autoradiography was used to directly visualize the length of DNA produced during a given time interval, and replication rates were calculated. The amount of DNA synthesized after blocking protein synthesis also allowed calculation of replication rates. We found that the DNA chain elongation rate in polyamine-deficient cells was about half that of putrescine- or spermidine-supplemented cells. We also found that spermidine homologs of increasing chain length, when present at equal intracellular concentrations, exhibited a decreasing ability to support growth and the rate of DNA replication fork movement. The kinetics of recovery of DNA synthesis from the polyamine-deficient state were also investigated. A new rate of DNA synthesis was reached about 20 min after addition of spermidine to polyamine-limited cells. The rise in the rate of DNA synthesis was preceded by a rise in the intracellular concentration of spermidine.
Differential synthesis of polypeptides during morphogenesis of Mucor
Authors: Hiatt W R , Inderlied C B , Sypherd P S
Published: January 2020

The extent of differential gene expression during morphogenesis of Mucor racemosus was investigated by two-dimensional polyacrylamide gel electrophoresis of neutral and acidic polypeptides. Cellular proteins were labeled with [35S]methionine in cells growing in either the yeast or hyphal form, or in yeast cells undergoing the transition of hyphae. The results showed that of the 400 to 500 polypeptides resolved by electrophoresis, relatively few were specific to one or the other morphological form. The major change in the patterns of proteins synthesized during morphogenesis was a change in rates of synthesis of individual polypeptides. Experiments in which morphogenesis was affected under aerobic or anaerobic conditions showed that the majority of changes in the protein patterns were associated with morphogenesis and were not a specific response to O2.
Inhibition of canavanyl-protein proteolysis in Escherichia coli by rifampin
Authors: George G N , Worthington V C , Hipkiss A R
Published: January 2020

Rifampin inhibited the intracellular proteolysis of canavanine-induced, rapidly sedimenting protein complexes in Escherichia coli.
Glucose catabolism during germination of Bacillus megaterium spores
Authors: Maruyama T , Otani M , Sano K , Umezawa C
Published: January 2020

When heat-activated spores of Bacillus megaterium germinated in glucose-containing medium, 10 to 30% of the glucose was found to be oxidized to gluconate.
Catabolite repression of enzyme synthesis does not prevent sporulation
Authors: Lopez J M , Uratani-Wong B , Freese E
Published: January 2020

In the presence of excess glucose, a decrease of guanine nucleotides in Bacillus subtilis initiated sporulation but did not prevent catabolite repression of three enzymes. Therefore, the ultimate mechanism(s) repressing enzyme synthesis differs from that suppressing sporulation.
Thermal modulation of fatty acid synthesis in Escherichia coli does not involve de novo enzyme synthesis
Authors: Garwin J L , Cronan J E
Published: January 2020

An increased ratio of unsaturated to saturated fatty acids was synthesized within 30 s after shift of Escherichia coli K-12 from 42 degrees C to 24 degrees C. This was more than 10-fold faster than the induction of beta-galactosidase. Inhibition of ribonucleic acid or protein synthesis had no effect on the response of fatty acid synthesis to temperature shift.
Position effect on expression of dsd genes cloned onto multicopy plasmids
Authors: Carothers A M , Heincz M C , McFall E
Published: January 2020

In the D-serine deaminase system of Escherichia coli, which is regulated by positive control, we have fouand a complete lack of trans activation in vivo with multicopy dsd hybrid plasmids. A PLASmid carrying the regulatory gene, dsdC+, did not promote expression of chromosomal dsdCO+A+ loci, nor did a chromosomal dsdC+ gene promote expression of plasmid-borne dsdC delta O+A+ (dsd regulatory gene negative) restriction fragments. However, hybrid plasmids that comprise the entire dsd system (dsdC+O+A+) are highly inducible for the enzyme. These dsd hybrid plasmid deoxyribonucleic acids functioned well as templates in the in vitro coupled transcription-translation system. In vitro-synthesized dsdC+ protein promoted expression of the dsdA+ operation efficiently. Exogenously purified dsdC+ protein also activated expression of several dsdC delta O+A+ plasmid deoxyribonucleic acid templates in vitro. An explanation that reconciles these results with previous dominance studies is presented.
Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens
Authors: Carman G M , Wieczorek D S
Published: January 2020

Cytidine 5'-diphospho (CDP)-1,2-diacyl-sn-glycerol (CDPdiacylglycerol):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycero-P synthase) and CDPdiacylglycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) activities were identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of phosphatidylglycero-P synthase and phosphatidylserine synthase with the cell envelope fraction of cell-free extracts was demonstrated by sucrose density gradient centrifugation, by both activities sedimenting with the 100,000 x g pellet and solubilization of both activities from the 100,000 x g pellet with Triton X-100. The pH optimum for both enzyme activities was 8.0 with tris(hydroxy-methyl)aminomethane-hydrochloride buffer. Phosphatidylglycero-P synthase activity was dependent on magnesium ions (100 mM). Phosphatidylserine synthase activity was dependent on manganese (0.1 mM) or magnesium ions (50 mM). Both enzyme activities were dependent on the addition of the nonionic detergent Triton X-100. Maximum phosphatidylglycero-P synthase and phosphatidylserine synthase activities were obtained when the molar ratio of Triton X-100 to CDP-diacylglycerol was 50:1 and 12:1, respectively. The Km for sn-glycero-3-P in the phosphatidylglycero-P synthase reaction was 0.1 mM. The Km for L-serine in the phosphatidylserine synthase reaction was 0.15 mM. Both enzyme activities were 100% stable for at least 20 min at 60 degrees C.
New cryptic plasmid of Bacillus subtilis and restriction analysis of other plasmids found by general screening
Authors: Uozumi T , Ozaki A , Beppu T , Arima K
Published: January 2020

A new cryptic plasmid, pTA1030 (4.5 megadaltons, copy number 16), was characterized by restriction analysis, together with some other plasmids of Bacillus subtilis.
Altered bacteriophage T4 ribonucleic acid metabolism in a ribonuclease II-deficient mutant of Escherichia coli
Authors: Birenbaum M , Schlessinger D , Ohnishi Y
Published: January 2020

Some early T4 ribonucleic acids were not found in an infected ribonuclease II-deficient strain but were formed in ribonuclease II+ transductants as well as in wild-type Escherichia coli.
Membrane association of a Staphylococcus aureus plasmid in Bacillus subtilis
Authors: Winston S , Sueoka N
Published: January 2020

The Staphylococcus aureus plasmid pUB110 was found to be enriched in deoxyribonucleic acid-membrane complexes isolated from Bacillus subtilis containing pUB110.
Absence of acetohydroxy acid synthetase in a clinical isolate of Neisseria gonorrhoeae requiring isoleucine and valine
Authors: Lerner S A , Friedman E L , Dudek E J , Kominski G , Bohnhoff M , Morello J A
Published: January 2020

A clinical isolate of Neisseria gonorrhoeae with an unusual growth requirement for isoleucine and valine lacked the activity of acetohydroxy acid synthetase, one of the enzymes required for the biosynthesis of these amino acids. A spontaneous mutant which no longer required isoleucine and valine had acquired this enzymatic activity.
Properties of beta-galactosidase III: implications for entry of galactosides into Klebsiella
Authors: Hall B G
Published: January 2020

Klebsiella sp. strain CT-200 lacks both its plasmid-borne lac operon, which specifies beta-galactosidase I, and its chromosomal lac operon, which specifies beta-galactosidase II, but it expresses a gene for a third beta-galactosidase, beta-galactosidase III, constitutively. CT-200 was examined to determine whether there was a beta-galactoside permease associated with the beta-galactosidase III gene. The failure of CT-200 to transport thiomethyl-beta-galactoside, o-nitrophenyl-beta-D-galactopyranoside, phenyl-beta-galactoside, lactulose, or galactosyl-arabinose was taken as evidence that beta-galactoside permease is not part of a beta-galactosidase III operon. Optimal assay conditions for beta-galactosidase II, whose activity was used as a measure of beta-galactoside transport, are reported here, as are an improved purification method and some physical and catalytic properties of the enzyme not previously reported.
Growth and physiology of Thiobacillus novellus under nutrient-limited mixotrophic conditions
Authors: Leefeldt R H , Matin A
Published: January 2020

Thiobacillus novellus was cultivated in a chemostate under the individual limitations of thiosulfate, glucose, and thiosulfate plus glucose. At dilution rate (D) of 0.05 h-1 or lower, the steady-state biomass concentration in mixotrophic medium was additive of the heterotrophic and autotrophic biomass at corresponding D values. The ambient concentrations of thiosulfate, glucose, or both in the various cultures were low and were very similar in mixotrophic, heterotrophic, and autotrophic environments at a given D value. At D = 0.05 h-1, mixotrophic cells possessed higher activities of sulfite oxidase and thiosulfate oxidation compared to autotrophic cells, as well as higher activities of glucose enzymes and glucose oxidation than heterotrophic cells. Thus, in contrast to nutrient-excess conditions, in nutrient-limited mixotrophic environments at these D values, T. novellus did not exhibit characteristics of uncoupled substrate oxidation, inhibition of substrate utilization, and repression of enzymes of energy metabolism. It is concluded that T. novellus responds to mixotrophic growth conditions differently in environments of different nutritional status, and the ecological and physiological significance of this finding is discussed.
Regulatory mutations affecting ornithine decarboxylase activity in Saccharomyces cerevisiae
Authors: Cohn M S , Tabor C W , Tabor H
Published: January 2020

We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.
Oligoglucuronide production in Mucor rouxii: evidence for a role for endohydrolases in hyphal extension
Authors: Dow J M , Villa V D
Published: January 2020

Culture filtrates of Mucor rouxii contained oligomers of glucuronic acid which were labeled rapidly during pulses with D-[U-14C]glucose. These oligomers were probably derived by enzymatic lysis of acidic polymers in the cell wall. The kinetics of the incorporation of label into oligouronides and cell wall polymers suggested that lysis of the wall was required for active hyphal extension. Experiments with cycloheximide, which inhibited hyphal extension, suggested that wall lysis was also required for the subapical cell wall synthesis which probably occurred under these conditions.
Morphology and ultrastructure of helical and nonhelical strains of Spiroplasma citri
Authors: Townsend R , Burgess J , Plaskitt K A
Published: January 2020

Cells of the nonhelical strain of Spiroplasma citri underwent changes of morphology comparable to those which occurred in the normal helical strain. Cells of the nonhelical strain had the same ultrastructural features as helical cells and released long flexible fibrils similar to those seen in other spiroplasmas. Nonhelical organisms showed an increased tendency to aggregate, forming cell clusters of an unusual annular form. The cytoplasmic membrane of the nonhelical strain lacked a single protein present in all helical strains. Loss of helicity associated with the senescence of spiroplasma cells was not accompanied by the disappearance of this protein. Differences in colony morphology were shown to be a consequence of motility, and a technique was developed which facilitated the identification of nonmotile organisms.
Co-Regulation in
Escherichia coli
of a Novel Transport System for
sn
-Glycerol-3-Phosphate and Outer Membrane Protein Ic (e, E) with Alkaline Phosphatase and Phosphate-Binding Protein
Authors: Argast Manfred , Boos Winfried
Published: January 2020

Mutants constitutive for the novel outer membrane protein Ic (e or E) contained a recently discovered binding protein for
sn
-glycerol-3-phosphate. The corresponding parental strains missing the outer membrane protein Ic (e, E) were negative or strongly reduced in the synthesis of the binding protein. In addition, strains that were previously isolated as mutants constitutive for the
sn
-glycerol-3-phosphate transport system (
ugp
+
mutants) and that produced the novel periplasmic proteins GP1 to GP4 also synthesized a new outer membrane protein with the same electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as protein Ic. Screening of different
ugp
+
mutants revealed the existence of three types in respect to the four novel periplasmic proteins GP1, -2, -3, and -4: (i) one containing all four proteins; (ii) one containing only proteins GP1, -2, and -3; (iii) one containing only proteins GP1, -2, and -4. In confirmation of the data presented in the accompanying paper by Tommassen and Lugtenberg (J. Bacteriol. 143:151–157, 1980), we found that purified GP1 is identical to alkaline phosphatase, whereas purified GP3 has binding activity of inorganic phosphate and is identical to the phosphate-binding protein. Moreover, growth conditions that lead in a wild-type strain to the derepression of alkaline phosphatase synthesis also derepressed the synthesis of the
sn
-glycerol-3-phosphate-binding protein as well as the corresponding transport system. Thus, the new
sn
-glycerol-3-phosphate transport system is part of the alkaline phosphatase regulatory system.
Choline transport in Saccharomyces cerevisiae
Authors: Hosaka K , Yamashita S
Published: January 2020

Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and various amino acids examined had no effect. Choline transport required metabolic energy; removal of glucose resulted in a great loss of transport activity, and the remaining activity was abolished by 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, arsenate, and cyanide. External Na+ was not required, and the transport was not effected by ionophores, valinomycin, and gramicidin D. These results indicate that S. cerevisiae possess an active choline transport system mediated by a specific carrier. This view is further supported by the isolation and characterization of a choline transport mutant. The choline transport activity in this mutant was very low, whereas the transport of L-leucine, L-methionine, D-glucose, and myo-inositol was normal. Together with the choline transport mutant, mutants defective in choline kinase were also isolated.
Protein synthesis elongation factors Tu and Tu.Ts from Caulobacter crescentus: sensitivity to kirromycin and activity in Q beta replicase
Authors: Stringfellow L A , Douglass J , Blumenthal T
Published: January 2020

The protein synthesis elongation factors Tu and Ts are responsible for binding aminoacyl-transfer ribonucleic acid (RNA) to the ribosome. In addition, they perform an undefined function, as the EF-Tu.Ts complex, in the RNA phage RNA replicases. In an effort to obtain insight into these two apparently unrelated roles, we purified the elongation factors from Caulobacter crescentus and compared them to the analogous Escherichia coli polypeptides. Although most physical and functional characteristics were found to be similar, significant differences were found in the molecular weight of EF-Ts and relative affinities of guanine nucleotides, sensitivity to trypsin cleavage, and rate of heat denaturation of EF-Tu. The antibiotic kirromycin was active with EF-Tu from both bacterial species. When C. crescentus EF-Tu.Ts was substituted for the E. coli elongation factors in Q beta phage RNA replicase, an enzyme capable of apparently normal RNA synthetic activity was formed.
Repression of nitrate reductase in Neurospora studied by using L-methionine-DL-sulfoximine and glutamine auxotroph gln-1b
Authors: Premakumar R , Sorger G J , Gooden D
Published: January 2020

The effect of L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, on the formation of nitrate reductase in the wild-type strain of Neurospora in the presence of ammonium ions and of glutamine was studied. Under conditions in which glutamine synthetase was inactivated, it was found that only glutamine could repress nitrate reductase. In a mutant of Neurospora, gln-1b, which requires glutamine for growth, only glutamine could repress nitrate reductase. These results suggest a direct role for glutamine as corepressor of nitrate reductase in Neurospora.
Purification and Some Properties of Maleylpyruvate Hydrolase and Fumarylpyruvate Hydrolase from
Pseudomonas alcaligenes
Authors: Bayly Ronald C. , Chapman Peter J. , Dagley Stanley , Berardino Dario Di
Published: January 2020

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (
cis
-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in
Pseudomonas alcaligenes
(P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn
2+
ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.
Positive correlation between size at initiation of chromosome replication in Escherichia coli and size at initiation of cell constriction
Authors: Koppes L J , Nanninga N
Published: January 2020

The variability of (i) the length (size) at which cells initiate chromosome replication, (ii) the length at which they initiate cell constriction, and (iii) the time interval between these events has been estimated for Escherichia coli B/r K at two different slow growth rates. Steady-state cultures were pulse-labeled with [3H]thymidine and, after fixation, analyzed by electron microscopic radioautography. The coefficient of variation of length at initiation of chromosome replication was found to be 15 to 22%, the coefficient of variation of length at initiation of cell constriction was 10%, and the coefficient of variation of the time interval between both events was 25%. With the help of these values we calculated a high positive coefficient of correlation (rho) between the length at which a round of chromosome replication is initiated and that at which the onset of cell constriction occurs. At both growth rates rho has a value of 0.6 to 1.0. This correlation excludes a model in which chromosome initiation and cell constriction are independently triggered by some aspects of cell growth. It favors a model in which an event before or at chromosome initiation triggers both.
Specific labeling of the Bacillus subtilis chromosome terminus
Authors: Sargent M G
Published: January 2020

The deoxyribonucleic acid labeled by a procedure described previously for labeling the chromosomal terminus of B. subtilis 168 was substantially enriched for sequences homologous to bacteriophages SP beta and phi 3T, which integrate in the terminal region.
Alterations in translatable ribonucleic acid after heat shock of Saccharomyces cerevisiae
Authors: McAlister L , Finkelstein D B
Published: January 2020

Changes in populations of translatable messenger ribonucleic acids (mRNA's) after heat shock of Saccharomyces cerevisiae were examined and found to correlate very closely with transient alterations in patterns of in vivo protein synthesis. Initial changes included an increase in translatable species coding for polypeptides synthesized during heat shock; this increase was found to be dependent on transcription but did not require ongoing protein synthesis. A decrease was observed in the level of translatable mRNA's coding for polypeptides whose synthesis was repressed after heat shock. This decrease was much more rapid than can be explained solely by termination of transcription. Requirements for this rapid loss of RNA from the translatable pool included both transcription and an active rna1 gene product but not protein synthesis. After the initial changes in translatable RNA induced by heat shock, the patterns of both in vivo and in vitro translation products began to revert to the preshock levels. This recovery period, unlike the earlier changes, was dependent upon a requisite period of protein synthesis.
Deletion mapping and heterogenote analysis of a mutation responsible for osmosis-sensitive growth, spectinomycin resistance, and alteration of cytoplasmic membrane in Escherichia coli
Authors: Yamagata H , Dombou M , Sato T , Mizushima S , Uchida H
Published: January 2020

Lambda transducing phages carrying Escherichia coli deoxyribonucleic acid of various lengths from the aroE-rpsL region were lysogenized into the F'3 plasmid and were used for heterogenote analysis of YM101, a sucrose-dependent, spectinomycin-resistant mutant of E. coli. Three characteristics of the mutant strain, resistance to spectinomycin, sucrose dependence of growth, and lack of I-19 protein in the cytoplasmic membrane, were shown to be the result of a mutation in a region designated delta 53-spcl. This region extends over 3.6-kilobase pairs and is located within a cluster of ribosomal genes. The mutation is recessive to the wild-type allele.
Biochemical correlations among the thermophilic enteric yeasts Torulopsis bovina, Torulopsis pintolopesii, Saccharomyces telluris, and Candida slooffii
Authors: Watson K , Arthur H , Blakey M
Published: January 2020

Spontaneous and drug-induced respiration-deficient mutants were isolated from the thermophilic enteric yeasts Torulopsis bovina and Saccharomyces telluris. The biochemical properties of these yeasts were compared with those of the two naturally occurring respiration-deficient thermophilic yeasts T. pintolopesii and Candida slooffii. Succinate dehydrogenase was not detected in mitochondrial fractions from C. slooffii, but was present in all other species. Cytochrome c oxidase, succinate oxidase, and reduced nicotinamide adenine dinucleotide oxidase were not detected in C. slooffii, T. pintolopesii, and the respiration-deficient mutants. Low-temperature cytochrome spectra revealed the presence of cytochromes aa3, b, c1, and c in T. bovina and S. telluris; cytochromes b, c1, and c in C. slooffii and T. pintolopesii; and cytochromes c1 and c in the spontaneous respiration-deficient mutants. Palmitoleic and oleic acids were the major fatty acids in all the species. It was noteworthy that T. pintolopesii was rich in lauric and myristic acids. CsCl equilibrium centrifugation experiments showed the presence in all the yeasts of a light-buoyant-density (1.6785 to 1.6837-g/cm3) deoxyribonucleic acid band which was identified as mitochondrial deoxyribonucleic acid by its selective elimination on treatment of cells with ethidium bromide. The latter result indicated that the spontaneous respiration-deficient mutants were similar to cytoplasmic petite mutants of S. cerevisiae. Although classical assimilation and fermentation tests indicated that the spontaneous respiration-deficient mutants were strains of T. pintolopesii, it was concluded, on the basis of marked physiological and biochemical differences, that this was not the case.
Homologous structural genes and similar induction patterns in Azotobacter spp. and Pseudomonas spp
Authors: Durham D R , Ornston L N
Published: January 2020

Intergeneric comparison of the three enzymes that initiate metabolism of protocatechuate in Azotobacter and Pseudomonas species revealed close immunological relatedness of isofunctional proteins. Furthermore, beta-ketoadipate induces all of the enzymes of the protocatechuate pathway (except protocatechuate oxygenase) in Azotobacter and in Pseudomonas species of the "fluorescent" and "cepacia" groups. This regulatory property sets the organisms apart from other bacteria. Protocatechuate oxygenase from Pseudomonas cepacia, like the enzyme from fluorescent Pseudomonas species, cross-reacts strongly with antiserum prepared against protocatechuate oxygenase from Azotobacter vinelandii. Double-diffusion experiments conducted with the antiserum revealed relatedness of Azotobacter spp. Protocatechuate oxygenases in the following order: A. vinelandii = Azotobacter miscellum greater than Azotobacter chroococcum greater than Azotobacter beijerinkii. The antiserum also revealed serological heterogeneity among Pseudomonas spp. protocatechuate oxygenases which were serologically indistinguishable in earlier studies using Pseudomonas aeruginosa protocatechuate oxygenase as reference protein.