bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and activity of ribonucleic acid polymerase in Escherichia coli
Authors: Shepherd N S , Churchward G , Bremer H
Published: January 2020

The amounts of ribonucleic acid (RNA) polymerase (beta' subunits) and ribosomes (RNA), and the fraction of RNA polymerase actively engaged in transcription, were measured in Escherichia coli B/r as a function of growth rate. By an improved method of quantitating protein bands on electrophoresis gels, the systematic error and reproducibility of the RNA polymerase determination were estimated to be less than 15 and 6%, respectively. For a threefold increase in growth rate, the fractional synthesis of polymerase (relative to protein) increased 1.5-fold, whereas the fractional synthesis of ribosomal protein increased 2.2-fold. The decrease in the amount of RNA polymerase per ribosome with increasing growth rate is interpreted as an expression of the control of the transcriptional read-through from the genes for ribosomal protein, rplJ,L, to the adjacent genes for RNA polymerase subunits, rpoB,C. The number of active RNA polymerase molecules was determined from the synthesis rates of stable and messenger RNA and the known RNA chain growth rates. Comparison of active and total RNA polymerase indicates that the fraction of active enzyme increases from 20 to 30% in the range of growth rates between 0.6 and 2.0 doublings per hour. Possible causes for the inactive enzyme are discussed.
Polyol metabolism by Rhizobium trifolii
Authors: Primrose S B , Ronson C W
Published: January 2020

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.
Coordination between chromosome replication and cell division in Escherichia coli
Authors: Tang M S , Helmstetter C E
Published: January 2020

Cell division properties of Escherichia coli B/r containing either a dnaC or a dnaI mutation were examined. Incubation at nonpermissive temperature resulted in the eventual production of cells of approximately normal size, or slightly smaller, which lacked chromosomal DNA. The cell division patterns in cultures which were grown at permissive temperature and then shifted to nonpermissive temperature were consistent with: first, division and equipartition of chromosomes by cells which were in the C and D periods at the time of the shift; second, an apparent delay in cell division; and third, commencement of the formation of chromosomeless cells. In glucose-grown cultures of the dnaI mutant, production of chromosomeless cells continued for at least 120 min, whereas in the dnaC mutant chromosomeless cells were formed during a single interval between 110 and 130 min after the temperature shift. The results are discussed in light of the hypothesis that replication of a specific chromosomal region is not an obligatory requirement for the initiation and completion of the processes leading to division in a cell which contains at least one functioning chromosome.
Lysine decarboxylase mutants of Escherichia coli: evidence for two enzyme forms
Authors: Goldemberg S H
Published: January 2020

Mutants with reduced lysine decarboxylase activity were isolated from an Escherichia coli polyamine auxotroph. These mutants could not produce induced enzyme, showing only a very low level of an apparently constitutive form. Both enzyme forms could be demonstrated in the parental strain.
Creatinine metabolism in Cryptococcus neoformans and Cryptococcus bacillisporus
Authors: Polacheck I , Kwon-Chung K J
Published: January 2020

The pathogenic species of Cryptococcus, C. neoformans and C. bacillisporus, utilized creatinine as a source of nitrogen but not of carbon. Chromatographic and autoradiographic studies suggest that creatinine metabolism in both species involves a single step resulting in the production of methylhydantoin and ammonia. The enzyme responsible for this step, creatinine deiminase, was produced by the cells only in the presence of creatinine in both species. The synthesis of creatinine deiminase was repressed by ammonia in C. neoformans, but not in C. bacillisporus. A possible explanation for this variation, based on the ecological differences between the two species, is discussed. A novel method for measuring creatinine deiminase activity is also described.
Genetic and biomedical studies demonstrating a second gene coding for asparagine synthetase in Escherichia coli
Authors: Humbert R , Simoni R D
Published: January 2020

Genetic experiments have indicated that asparagine auxotrophs of Escherichias coli K-12 can be made asparagine prototrophs at either of two sites on the chromosome and that wild-type strains require both sites to be mutated to produce asparagine auxotrophy. The former asn locus is now called asnA, and the new gene is designated asnB. The asnB gene is located near gal.AsnA+ asnB and asnA asnB+ strains were constructed, and the asparagine synthetic reaction was characterized in extracts. These studies revealed that the asnA gene codes for the enzyme previously described (H. Cedar and J.H. Schwartz, J. Biol. Chem. 244: 4112-4121, 1969), whereas the asnB gene is involved in the production of an enzyme which differs from the one previously described in its specific activity in extracts, its stability at low and high temperatures, and its apparent ability to use either glutamine or ammonia as amide nitrogen donor. Physiological studies showed that either enzyme alone is sufficient to allow a maximal growth rate under conditions of asparagine limitation.
A mutation, adjacent to gene amdS, defining the site of action of positive-control gene amdR in Aspergillus nidulans
Authors: Hynes M J
Published: January 2020

In Aspergillus nidulans the acetamidase enzyme is inducible by omega-amino acids, sources of acetyl-coenzyme A, and benzoate. The amdR (or intA) gene is a positive-control gene involved in omega-amino acid induction only. A cis-acting mutation amdI93 located in a complex controlling region adjacent to the acetamidase structural gene was found to abolish induction by omega-amino acids but not induction by other sources of induction. As predicted, this mutation was epistatic to constitutive amdR alleles but did not affect the expression of mutations in other regulatory genes.
Influences of growth substrates and oxygen on the electron transport system in Acinetobacter sp. HO1-N
Authors: Ensley B D , Finnerty W R
Published: January 2020

The electron transport system of Acinetobacter sp. HO1-N was studied to determine the specific cytochromes and to measure changes in the composition of the respiratory system due to growth in various concentrations of oxygen or types of growth substrates. Spectrophotometric analysis revealed that the quantity and types of cytochromes changed in response to growth under various concentrations of oxygen. Growth on alkane and nonalkane substrates resulted in only minor differences in cytochrome composition or oxidase activities. Membranes prepared from cells grown under oxygen-limiting conditions contained at least one b-type cytochrome, cytochrome o, cytochrome d, and slight traces of cytochrome a1, whereas membranes prepared from cells grown in the presence of high oxygen concentrations contained only low levels of cytochromes b and o. Polarographic measurements, electron transport inhibitor studies, and photoaction spectrum analyses indicated that cytochromes o, a1, and d were potentially capable of functioning as terminal oxidases in this organism. These experiments also revealed that all three cytochromes may be involved in the oxidation of reduced nicotinamide adenine dinucleotide, succinate, or N,N,N',N'-tetramethyl-p-phenylenediamine.
Basic amino acids and inorganic polyphosphates in Neurospora crassa: independent regulation of vacuolar pools
Authors: Cramer C L , Vaughn L E , Davis R H
Published: January 2020

At least 78%, and perhaps all, of inorganic polyphosphate is shown to be contained within the vesicles (vacuoles) of Neurospora crassa, where over 97% of the soluble arginine, lysine, and ornithine pools are known to accumulate. Furthermore, synthetic polyphosphate can concentrate arginine up to 400-fold from dilute (0.01 mM) solutions in equilibrium dialysis. For these reasons and because the molar ratio of basic amino acids and polyphosphate phosphorus is approximately 1, we tested the hypothesis that there was an obligate physiological relationship between them. Experiments in which nitrogen starvation and arginine excess were imposed upon cells showed that polyphosphate content was insensitive to changes in the basic amino acid content. Experiments involving phosphate starvation and restoration showed that basic amino acid content was almost wholly independent of polyphosphate pools. Moreover, the normal high degree of compartmentation of arginine in vesicles was maintained despite polyphosphate depletion, and arginine was still exchanged across the vesicular membrane. We conclude that N. crassa, like yeasts, can regulate polyphosphates and basic amino acids independently, and that the accumulation of basic amino acids in vesicles may depend upon an energy-requiring mechanism in addition to the demonstrated charge interaction with polyphosphate.
Plasmids coding for heat-labile enterotoxin production isolated from Escherichia coli O78: comparison of properties
Authors: McConnell M M , Smith H R , Willshaw G A , Scotland S M , Rowe B
Published: January 2020

Nineteen enterotoxigenic Escherichia coli strains of serogroup O78, isolated in different geographical areas from humans with diarrheal diseases, were tested for their ability to transfer enterotoxin production. All of the strains originally produced heat-labile enterotoxin, and 16 also produced heat-stable enterotoxin and colonization factor antigen I. Plasmids coding for the production of heat-labile enterotoxin only were transferred from 13 strains. Some properties of these plasmids were compared. All were fi+, but they could be divided into three groups on the basis of their incompatibility reactions, ability to restrict E. coli K-12 phages, and size. The three heat-labile enterotoxin plasmids isolated from African strains all belonged to one enterotoxin plasmid group. The heat-labile enterotoxin plasmids from the Asian strains were divided into two groups, those from serotype O78.H11 differing from those from serotype O78.H12.
Heat stability of Bacillus cereus enzymes within spores and in extracts
Authors: Warth A D
Published: January 2020

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.
Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis
Authors: Hofstra H , Van Tol J D , Dankert J
Published: January 2020

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.
Chemical basis for selectivity of metal ions by the Bacillus subtilis cell wall
Authors: Doyle R J , Matthews T H , Streips U N
Published: January 2020

The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations. The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix. Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased. Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants. Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals. Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity. Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls. When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites.
Plasmid-mediated resistance to aminocyclitol antibiotics in group D streptococci
Authors: Courvalin P , Carlier C , Collatz E
Published: January 2020

Streptococcus faecalis BM4100 was resistant to high levels of gentamicin, kanamycin, and structurally related antibiotics. The genes conferring resistance to aminocyclitols in this strain were carried by a plasmid, pIP800, self-transferable to other S. faecalis strains. The aminocyclitol resistance was mediated by constitutively synthesized phosphotransferase and acetyltransferase activities. It was inferred that phosphorylation occurred at the 2"-hydroxyl group and that acetylation occurred at the 6'-hydroxyl group of the aminocyclitols. The enzyme activities were not separable by gel filtration or by isoelectric focusing. Their apparent molecular weight was 31,000, and their isoelectric point was 5.3. With respect to substrate profile, size, and charge, the transferases from strain BM4100 resembled closely those with identical site specificity described in staphylococci. These results suggest that plasmid gene transfer may occur between the two pathogenic bacterial genera.
Regulation of carbamylphosphate synthesis in Serratia marcescens
Authors: Crane C J , Abdelal A T
Published: January 2020

Serratia marcescens HY possessed a single carbamylphosphate synthase (CPSase) which was subject to cumulative repression by arginine and a pyrimidine. CPSase did not appear to be a part of a multifunctional enzyme complex as is the case for other enzymes of pyrimidine biosynthesis in this organism. CPSase was purified to homogeneity. The molecular weight of the enzyme was estimated to be 167,000 by sucrose density gradient ultracentrifugation. The double-reciprocal plot for magnesium adenosine triphosphate was linear, yielding a Km value of 2.5 mM. The enzyme utilized either glutamine (Km, 0.1 mM) or NH3 (Km, 10.5 mM) as a nitrogen donor in the reaction. CPSase activity was subject to activation by ornithine and feedback inhibition by uridine monophosphate, as is the case for other enteric bacteria. Carbamate kinase activity, detected in crude extracts of S. marcescens, was shown to be due to a constitutive acetate kinase. The absence of carbamate kinase from S. marcescens HY is consistent with the inability of this organism to utilize arginine as a source of energy under anaerobic conditions.
Induction of chondroitin sulfate lyase activity in Bacteroides thetaiotaomicron
Authors: Salyers A A , Kotarski S F
Published: January 2020

Chondroitin sulfate lyase (EC 4.2.2.4) was present constitutively at low levels (0.06 to 0.08 U/mg of protein) in cells of Bacteroides thetaiotaomicron which were growing on glucose or other monosaccharides. When these uninduced bacteria were incubated with chondroitin sulfate A (5 mg/ml), chondroitin sulfate lyase specific activity increased more than 10-fold within 90 min. Synthesis of ribonucleic acid and of protein was required for induction, and induction was sensitive to oxygen. The disaccharides which resulted from chondroitinase action did not act as inducers, nor did tetrasaccharides or hexasaccharides obtained by digestion of chondroitin sulfate with bovine testicular hyaluronidase. None of these substances was taken up by uninduced cells; they may not have been able to penetrate the outer membrane. The smallest oligomer capable of acting as an inducer was the outer membrane. The smallest oligomer capable of acting as an inducer was the octassacharide. Oligomers larger than the octassacharide induced chondroitin lyase activity nearly as well as intact chondroitin sulfate.
Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups
Authors: de la Cruz F , Müller D , Ortiz J M , Goebel W
Published: January 2020

By using cloned deoxyribonucleic acid fragments from the hemolysis determinant of the hemolytic plasmid pHly152 as hybridization probes, a deoxyribonucleic acid segment of about 3.8 megadaltons was identified as a common sequence in several hemolytic (Hly) plasmids of Escherichia coli belonging in four different incompatibility groups. This segment contained the genetic information for the synthesis and secretion of the extracellular toxin alpha-hemolysin of E. coli. With the exception of pSU5, representing a composite plasmid, one part of which seems to be very similar to pHly152, the overall sequence homology of these Hly plasmids with pHly152 seems to be rather restricted. However, the Hly plasmid pSU316 showed sequence homology with pHly152 that did not extend beyond the hemolysis determinant. The two other plasmids, pSU233 and pSU105, also shared homology with pHly152 in the hemolysis determinant as well as in various other parts of this plasmid which did not seem to be directly linked to the hemolysis determinant. This suggests that the hemolysis determinant has spread to presumably unrelated plasmids of E. coli.
Major heat-modifiable outer membrane protein in gram-negative bacteria: comparison with the ompA protein of Escherichia coli
Authors: Beher M G , Schnaitman C A , Pugsley A P
Published: January 2020

The outer membranes of several strains of Escherichia coli, other enteric bacteria, and a variety of nonenteric gram-negative bacteria all contain a major heat-modifiable protein similar to the OmpA protein of E. coli K-12. The heat-modifiable proteins from these bacteria resemble the K-12 protein in molecular weight, in preferential release from the outer membrane by sodium dodecyl sulfate in the presence of Mg2+, and in characteristic cleavage by proteases to yield a smaller fragment which remains membrane bound. Antiserum directed against the K-12 protein precipitated the heat-modifiable protein from all strains of Enterobacteriaceae, and chemical comparison by isoelectric focusing, cyanogen bromide cleavage profiles, and proteolytic peptide analysis indicated that the proteins from the various enteric bacteria were nearly identical in primary structure. The heat-modifiable proteins from bacteria phylogenically distant from E. coli shared many of the properties of the E. coli protein but were chemically distinct. Thus, it appears that the structure (and, presumably, the function) of the heat-modifiable protein of gram-negative bacteria is strongly conserved during evolution.
Reproduction and morphogenesis of Rickettsiella chironomi, an unusual intracellular procaryotic parasite of midge larvae
Authors: Federici B A
Published: January 2020

The reproduction and morphogenesis of Rickettsiella chironomi, an unusual procaryotic parasite of midge larvae, was studied with electron microscopy. The morphogenic cycle was similar to that found in chlamydia and consisted of four major cell types: (i) medium-sized spherical initial bodies (ca. 1 micrometers in diameter), (ii) large spherical initial bodies (1.5-2 micrometers), (iii) spherical intermediate bodies (600 to 700 nm), and (iv) disk-shaped elementary bodies (60 X 600 nm). The primary mode of reproduction involved binary fission of initial bodies to form other initial bodies or intermediate bodies. Each intermediate body condensed, forming an elementary body. The morphogenesis of R. chironomi is compared with that of several other organisms to which it is possibly related, including vertebrate chlamydia and invertebrate pathogens of the genera Rickettsiella and Porochlamydia, and its taxonomic position in regard to these is discussed. Additionally, a brief description of the pathology caused by the development of R. chironomi in larvae of Chironomus decorus and Chironomus frommeri is given.
Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12
Authors: Grundström T , Jaurin B , Edlund T , Normark S
Published: January 2020

Hybrid plasmids carrying the ampC gene of Escherichia coli K-12 that codes for the chromosomal beta-lactamase were physically studied. The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1,370 base pairs. The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (gamma delta) close to ampC. The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1,370-base pair deoxyribonucleic acid segment that contains the ampC gene. Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC, or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal beta-lactamase. In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater. In addition, a protein with a slightly higher molecular weight (38,000) was expressed by both ampA+ ampC and ampA1 ampC plasmids in this protein labeling system. This protein might represent a precursor form of chromosomal beta-lactamasee. From E. coli K-12 strains carrying the ampA1 allele, second-step mutants were isolated that hyperproduced chromosomal beta-lactamase. By reciprocal recombination, plasmid derivatives were isolated that carried these mutations. Two second-step regulatory mutations mapped within the same 370-base pair region as ampA1. This piece of deoxyribonucleic acid therefore contains ampA, a control sequence region for ampC.
Insertion element IS102 resides in plasmid pSC101
Authors: Ohtsubo H , Zenilman M , Ohtsubo E
Published: January 2020

In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.
Synchronization of cell division in microorganisms by percoll gradients
Authors: Dwek R D , Kobrin L H , Grossman N , Ron E Z
Published: January 2020

We describe a method for obtaining synchronously dividing cells of bacteria (Escherichia coli B and K-12 and Bacillus subtilis 168) and fission yeasts (Schizosaccharomyces pombe) by the use of Percoll density gradients.
Molecular characterization of "plasmid-free" antibiotic-resistant Haemophilus influenzae
Authors: Roberts M C , Smith A L
Published: January 2020

We examined 14 multiresistant and 8 ampicillin- or tetracycline-resistant Haemophilus influenzae isolates and 4 ampicillin-resistant H. parainfluenzae isolates for plasmid deoxyribonucleic acid. Sixteen strains carried plasmids. Both "plasmid-free" and plasmid-carrying isolates transferred the antibiotic resistance by conjugation. All transconjugants carried plasmid deoxyribonucleic acid, suggesting that the apparent plasmid-free strains contained R plasmids encoding for antibiotic resistance.
Vacuoles: main compartments of potassium, magnesium, and phosphate ions in Saccharomyces carlsbergenis cells
Authors: Okorokov L A , Lichko L P , Kulaev I S
Published: January 2020

The uneven distribution of Mg2+, K+, and phosphate in Saccharomyces carlsbergensis was demonstrated by the differential extraction of ions. Their concentrations were 5, 60, and 1 mM in the cytoplasm and 73, 470, and 110 mM in vacuoles, respectively. The intracellular gradients of these ions were 1:15, 1:8, and 1:110, respectively, across the tonoplast. The determination of free Mg2+ (1.35 mM in the cytosol and 20 mM in vacuoles) showed that the ion accumulation in vacuoles could not be explained by the higher degree of ion complexing in these organelles.
Role of vacuolar ion pool in Saccharomyces carlsbergensis: potassium efflux from vacuoles is coupled with manganese or magnesium influx
Authors: Lichko L P , Okorokov L A , Kulaev I S
Published: January 2020

Saccharomyces carlsbergensis cells accumulated Mn2+ (or Mg2+) ions in the presence of glucose, fructose, or mannose, but not of deoxyglucose, 3-O-methylglucose, and sorbose. Accumulation of one equivalent of Mn/2+ was coupled with the efflux of two equivalents of K+ from the cells. Mg/2+ did not exit during Mn2+ uptake. Preliminary treatment of cells with various proton conductors or glucose led to the loss of K+ and to the proportional inhibition of Mn2+ uptake. Polyene antibiotic candicidin together with glucose elicited rapid efflux of K+ and completely inhibited Mn2+ accumulation. Exogenous K+ (more than 1 mM), 100 microM N,N'-dicyclohexylcarbodiimide, and 30 mM sodium arsenate inhibited both K+ efflux and Mn2+ influx. K+ efflux from S. carlsbergensis cells affected the vacuolar pool of K+ both during the accumulation of Mn2+ or Mg2+ and during glucose uptake.
Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation
Authors: Thomas T D , Turner K W , Crow V L
Published: January 2020

All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively, in galactose-grown cells compared with glucose- or lactose-grown cells. Reduced intracellular levels of both the lactate dehydrogenase activator (fructose, 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appeared to be the main factors involved in the diversion of lactate to the other products. S. lactis ML8 produced only lactate from galactose, apparently due to the maintenance of high intracellular levels of fructose 1,6-diphosphate and triose phosphates. The growth rates of all 10 Streptococcus cremoris strains examined decreased markedly with galactose concentrations below about 30 mM. This effect appeared to be correlated with uptake predominantly by the low-affinity galactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined, galactose uptake and initial metabolism involved more extensive use of the high-affinity galactose permease and Leloir pathway. With these strains the relative flux of galactose through the alternate pathways would depend on the exogenous galactose concentration.
Genetic and biochemical properties of Escherichia coli mutants with defects in serine chemotaxis
Authors: Hedblom M L , Adler J
Published: January 2020

In Escherichia coli, taxis to certain chemoeffectors is mediated through an intrinsic membrane protein called methyl-accepting chemotaxis protein I (MCP I), which is the product of the tsr gene. Mutants were selected that are defective in taxis toward all MCP I-mediated attractants (alpha-aminoisobutyrate, L-alanine, glycine, and L-serine) but are normal to MCP I-mediated repellents and to chemoeffectors mediated by other MCPs. The mutants could be divided into two classes based on their ability to respond to various concentrations of L-serine. Two MCP I-mediated L-serine systems appear to function in the wild type: one of high and one of lower affinity. The mutations responsible for the serine taxis defects map at about 99 min on the E. coli chromosome and are not complemented by episomes carrying mutations in the tsr gene; this suggests that they are defective in tsr function. Low concentrations of L-[14C]serine specifically bound to wild-type membranes with a Km of 5 microM; in contrast, there was greatly decreased binding to vesicles prepared from the new mutants or from the tsr mutant AW518. Binding of labeled serine to wild-type vesicles was inhibited by MCP I-mediated attractants, but not by MCP II-mediated attractants. The data suggest that MCP I may function as the L-serine chemoreceptor in E. coli.
Messenger ribonucleic acid and protein metabolism during sporulation of Saccharomyces cerevisiae
Authors: Kraig E , Haber J E
Published: January 2020

To investigate differences between growing yeasts and those undergoing sporulation, we compared several parameters of messenger ribonucleic acid (RNA) transcription and translation. The general properties of messenger RNA metabolism were not significantly altered by the starvation conditions accompanying sporulation. The average messenger RNA half-life, calculated from the kinetics of incorporation of [3H]adenine into polyadenylic acid-containing RNA, was 20 min on both cell populations. Furthermore, 1.3 to 1.4% of the total RNA was adenylated in both growing and sporulating cells. However, the proportion of RNA that could be translated in a wheat germ system slowly decreased during sporulation. Within 8 h after the induction of sporulation, isolated RNA stimulated half as much protein synthesis as the equivalent amount of vegetative RNA. There were significant differences in protein synthesis. The percentage of ribosomes in polysomes decreased threefold as the cells entered sporulation. This decrease began within 5 min of the initiation of sporulation, and the steady-state pattern was attained within 120 min. However, the ribosomes were not irreversibly inactivated; they could be reincorporated into polysomes by returning the sporulating cells to growth medium. Though unable to sporulate, strains homozygous for mating type, MAT alpha/MAT alpha, showed a similar decrease in the number of polysomes when placed in sporulation medium. Furthermore, the same shift toward monosomes was observed during stationary phase of growth. We conclude that the redistribution of ribosomes represents a general metabolic response to starvation. Our data indicate that the loss of polysomes is most likely caused by a decrease in the initiation of translation rather than a severe limitation in the amount of messenger RNA. Furthermore, the loss of polysomes is not due to the decreased synthesis of a major class of abundant proteins. Of the 400 vegetative proteins resolved by two-dimensional gel electrophoresis, only 19 were not synthesized by sporulating cells. Approximately 10 to 20% of the cells in a sporulating culture failed to complete ascus formation. We have shown that [35S]methionine is incorporated equivalently into cells committed to sporulation and cells that fail to form asci. Furthermore, the proteins synthesized by these two populations were indistinguishable, on one-dimensional gels. We compared proteins labeled by various protocols, including long-term and pulse-labeling during sporulation and prelabeling during vegetative growth before transfer to sporulation medium. The resulting two-dimensional gel patterns differed significantly. Many spots labeled by the long-term techniques may have arisen by protein processing. We suggest that pulse-labeling produces the most accurate reflection of instantaneous synthesis of proteins.
Occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes
Authors: Braña A F , Manzanal M B , Hardisson C
Published: January 2020

Evidence of the presence of polysaccharide polymers is shown in Streptomyces sp. for the first time. Cytochemical methods revealed the occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes. Onset of the sporogenesis coincided with the appearance of the granules, which reached a maximum number during the early stages of maturation. The later stages of maturation showed a decrease of these granules, and in mature spores no granules were observed.
The ebg operon consists of at least two genes
Authors: Hall B G , Zuzel T
Published: January 2020

The ebg operon of Escherichia coli includes a second gene designated ebgB. The ebgB gene product is a 79,000-molecular-weight protein and is expressed coordinately with the ebgA gene product, ebg beta-galactosidase. Insertion of the transposable elements Tn5 and Tn9 into ebgA eliminates the expression of ebgB, suggesting that ebgB is distal to ebgA. Ultraviolet light mapping confirms that gene order. The function of the ebgB gene product is unknown.
Genetic and biochemical characterization of D-arabinose dehydrogenase from Neurospora crassa
Authors: Carrasco A , Pincheira G , Ureta T
Published: January 2020

D-Arabinose dehydrogenase has been purified to homogeneity from wild-type Neurospora crassa 74-A (FGSC 262) and from two colonial mutants, col-15a (FGSC 1391) and col-16a (FGSC 1349), found to contain more of the enzyme. The enzymes were characterized by measurement of several kinetic and physicochemical parameters. The enzymes were the same in all characteristics studied thus far. Immunological studied performed with enzyme preparations from the three strains showed antigenic identity and indicated that those colonial strains contain more normal enzyme, rather than the usual amount of an altered "improved" enzyme. Quantitation of the enzyme in crude extracts, performed by single radial immunodiffusion, showed that the colonial strains have twice the level of enzyme as the wild-type strain. Genetic characterization, performed by analysis of meiotic products, heterokaryosis, and reversions, indicated that the difference in D-arabinose dehydrogenase activity detected among the three strains is probably determined by one gene. The genetic control, structural or regulatory of this enzyme activity is different from that determining the morphological alterations exhibited by mutant strains carrying the col-15 or col-16 gene.
Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus
Authors: Kaboev O K , Luchkina L A , Akhmedov A T , Bekker M L
Published: January 2020

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.
Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase
Authors: Meganathan R , Bentley R , Taber H
Published: January 2020

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects. Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis. The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei. Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M. phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE. The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with [2,3-14C2]OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent.
Methyl-accepting chemotaxis protein III and transducer gene trg
Authors: Hazelbauer G L , Engström P , Harayama S
Published: January 2020

A comparison of the two-dimensional gel patterns of methyl-3H- and 35S-labeled membrane proteins from trg+ and trg null mutant strains of Escherichia coli indicated that the product of trg is probably methyl-accepting chemotaxis protein III. Like the other known methyl-accepting chemotaxis proteins, the trg product is a membrane protein that migrates as more than one species in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that it too is multiple methylated. It appears likely that all chemoreceptors are linked to the tumble regulator through a single class of membrane protein transducers which are methyl-accepting proteins. Three transducers are coded for by genes tsr, tar, and, probably, trg. Another methyl-accepting protein, which is not related to any of these genes, was observed.
Incomplete flagellar structures in Escherichia coli mutants
Authors: Suzuki T , Komeda Y
Published: January 2020

Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative precursors of the basal body were detected in mutants with defects in flaM, flaU, flaV, and flaY. No structures homologous to the flagellar basal body or its parts were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI, flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbA, flbB, and flbD. One flaZ mutant had an incomplete flagellar basal body structure and another formed no significant structure, suggesting that flaZ is responsible for both basal body assembly and the transcription of the hag gene.
Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains
Authors: Tyrell D J , Bulla L A , Andrews R E , Kramer K J , Davidson L I , Nordin P
Published: January 2020

Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.
Genetic dissection of catalytic activities of the Salmonella typhimurium mannitol enzyme II
Authors: Leonard J E , Saier M H
Published: January 2020

Approximately 60 mutants of Salmonella typhimurium were isolated which exhibited altered levels of the activities of the mannitol enzyme II. The mutants were grouped into six distinct categories based on their mannitol fermentation, transport, chemotaxis, and phosphorylation activities.
Catabolite repression of Escherichia coli heat-stable enterotoxin activity
Authors: Martínez-Cadena M G , Guzman-Verduzco L M , Stieglitz H , Kupersztoch-Portnoy Y M
Published: January 2020

The Escherichia coli heat-stable enterotoxin (ST) coded for by plasmid pYK007 (Apr ST+) showed a dependence for cyclic adenosine 3',5'-monophosphate (cAMP) to express ST activity in an adenyl cyclase (cya) deletion mutant; no ST activity was detected in the presence of cAMP in a cAMP receptor protein (crp) deletion mutant or in a double deletion mutant (delta cya delta crp). The cya-crp effect on ST activity could not be accounted for by a modification of the copy number of plasmid deoxyribonucleic acid per chromosome equivalent or by an alteration in the secretion of an active intracellular enterotoxin.
Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase
Authors: Fujita Y , Freese E
Published: January 2020

A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes.
Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions
Authors: Weston A , Brown M G , Perkins H R , Saunders J R , Humphreys G O
Published: January 2020

Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations. During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells. Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant. Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid. The results suggest that transport of DNA across the inner membrane is a limiting step in transformation. After transformation the bulk of labeled plasmid DNA remained associated with outer membranes. However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present. Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+. This parallels their relative efficiencies in promoting transformation. Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation.