Search PubMed⌕ Search

Biomedical subjects

E W Nester

Publications and source records attributed to E W Nester.

At least 145 records · Page 8Linked to original sources
PubMed1977

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.

Arginine↗
PubMed1976

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.

Bacillus subtilis↗
PubMed1976

RP4 promotion of transfer of a large Agrobacterium plasmid which confers virulence.

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.

Arginine↗
PubMed1976

Evidence for diverse types of large plasmids in tumor-inducing strains of Agrobacterium.

Homology between the large plasmids of 15 pathogenic Agrobacterium strains isolated from various parts of the world has been measured and was found to vary over a wide range, from 3 to 100%. Two genetically distinct groups of plasmids can be identified: one closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain, and the other closely related to the plasmid of A. tumefaciens C-58, a nopaline-utilizing strain. The plasmids of four Agrobacterium strains do not belong to either group. One of these four strains utilizes octopine, one utilizes nopaline, and two utilize neither. Three strains contained two large plasmids. In one of these strains, the two plasmids were not homologous to one another. Chromosomal homologies for the Agrobacterium strains surveyed also vary over a wide range, but do not correlate with plasmid homologies. Neither do plasmid homologies correlate with any numerical classification scheme. The significance of these plasmid homology studies for crown gall tumorigenesis is considered.

Chromosomes, Bacterial↗
PubMed1976

An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis.

Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis. One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine. The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids. The other gene has not yet been mapped. Mutants have been isolated that lack one or the other enzyme activity. These mutants are prototrophic for tyrosine and phenylalanine. However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain. The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation. The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways.

Bacillus subtilis↗
PubMed1976

Conjugation in Agrobacterium tumefaciens in the absence of plant tissue.

A general, reliable conjugation system for Agrobacterium tumefaciens in the absence of plant tissue is described in which A. tumefaciens can serve either as the donor or recipient of plasmid deoxyribonucleic acid with reasonable efficiency. Plasmid RP4 was transferred from Escherichia coli to A. tumefaciens and from strain of A. tumefaciens. Both RP4 and the A. tumefaciens virulence-associated plasmids were detected by alkaline sucrose gradients in A. tumefaciens strains A6 and C58 after mating with E. coli J53(RP4). The pathogenicity (tumor foramtion) of strains A6 and C58 and the sensitivity of strain C58 to bacteriocin 84 were unaffected by the acquistion of RP4 by the Agrobacterium strains. Plasmid R1drd-19 was not transferred to A. tumefaciens. Transformation experiments with plasmid deoxyribonucleic acid were unsuccessful, even though, in the case of RP4, conjugation studies showed taht the deoxyribonucleic acid was compatible with that of the recipient strains.

Anti-Bacterial Agents↗
PubMed1976

Regulation of histidinol phosphate aminotransferase synthesis by tryptophan in Bacillus subtilis.

The effect of tryptophan on the synthesis of histidinol phosphate aminotransferase and prephenate dehydrogenase has been examined. The genes specifying two enzymes for tryptophan biosynthesis (anthranilate synthase and tryptophan synthase-B) were found to be derepressed in a temporal sequence according to their chromosomal location. The genes for histidinol phosphate aminotransferase and prephenate dehydrogenase were derepressed simultaneously approximately 8 min after tryptophan synthase-B. When excess tryptophan was added to a derepressed culture, the pattern of repression of trpE (anthranilate synthase), trpB (tryptophan synthase-B), hisH (histidinol phosphate aminotransferase), and tyrA (prephenate dehydrogenase) was found to be simultaneous. Methyl tryptophan-resistant mutants, which synthesize elevated levels of the tryptophan enzymes, also synthesized elevated levels of histidinol phosphate aminotransferase. Qualitatively similar data were obtained in a temperature-sensitive tryptophanyl-transferase ribonucleic acid synthetase mutant grown at elevated temperatures. The time at which messenger ribonucleic acid was synthesized for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase in the presence of actinomycin D indicated that ordered enzyme synthesis was a result of ordered transcription of the corresponding portion of the genome. The effect of the drug rifampin on enzyme synthesis was also examined. The addition of this drug halted the transcription of anthranilate synthase very rapidly, but later regions of the tryptophan region continued to be transcribed. The transcription of the hisH and tyrA genes was also shut off rapidly after rifampin was added. The significance of these observations to the control of transcription of the hisH gene by tryptophan is discussed.

Anthranilate Synthase↗
PubMed1975

Purification and characterization of shikimate kinase enzyme activity in Bacillus subtilis.

In Bacillus subtilis shikimate kinase enzyme activity can be demonstrated when a small polypeptide forms a trifunctional complex with the bifunctional enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase-chorismate mutase. The shikimate kinase polypeptide whoch carries the catalytic site has been purified to homogeneity by a five-step procedure. The skikimate kinase was determined to have a molecular weight of 10,000 by superfine Sephadex G-75 thin layer chromatography and by calculation of the minimum chemical molecular weight from its amino acid composition. This number corresponds closely to the molecular weight determined by the mobility of the protein following electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The enzyme aggregates with itself forming larger molecular weight proteins. Thes aggregational pattersn depend on protein concentration and sulfhydryl bridges. The enzyme activity is completely inhibited by EDTA and the requirement for Mg2+ can be partially replaced by Mn2+, Ca2+, and Co2+. The inhibition of shikimate kinase activity by p-hydroxymercuribenzoate is reversed completely when the enzyme complex is treated with dithiothreitol, suggesting the sulfhydryl groups may be involved with the active site. The trifunctional complex is relatively unstable, and the nonidentical subunits dissociate readily. This dissociation results in a 99% loss in shikimate kinase activity and a 30% decrease in the chorismate mutase-DAHP synthetase activities. Shikimate kinase activity is subject to a variety of controls. It is inhibited by the allosteric effectors chorismate and prephenate, the products of the reaction, ADP, and shikimate 5-phosphate. The activity responds to changes in the energy charge of the cell. Because of the variety of controls exerted on this enzyme, this member of the regulatory complex may represent the key enzyme in the allosteric control of the synthesis of the common precursors of aromatic acid synthesis.

Adenosine Diphosphate↗
PubMed1975

Plasmid required for virulence of Agrobacterium tumefaciens.

The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

Amino Acids↗
PubMed1974

Detection and quantitation of octopine in normal plant tissue and in crown gall tumors.

Octopine has been detected in normal tobacco leaf and stem tissue, normal sunflower stem tissue, pinto bean leaves, and normal tobacco callus tissue in culture. Octopine was identified in extracts by means of electrophoresis and chromatography in several solvent systems. Tobacco and sunflower tumor lines induced by various strains of Agrobacterium tumefaciens were found to contain from 1 to 240 times as much octopine as the normal plant tissues examined. Several strains of A. tumefaciens produce undifferentiated tobacco tumors containing high levels of octopine, but produce undifferentiated sunflower tumors containing normal levels of octopine and high levels of arginine. Further, strain CGIC of A. tumefaciens produces in tobacco an undifferentiated tumor which contains high levels of octopine and a teratoma which contains normal levels of octopine. This evidence shows that there is no consistent relationship between the causative strain of A. tumefaciens and the octopine content of the resulting crown gall tumor.

Journal Article↗
PubMed1974

Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors.

Renaturation kinetics of labeled Agrobacterium tumefaciens DNA are not influenced by addition of 10(4)-fold excess of crown gall tumor DNA. Reconstruction experiments demonstrated that 0.01% added bacterial DNA produces a detectable increase in rate of renaturation of labeled DNA. Crown gall tumor DNA therefore cannot contain as much as 0.01% A. tumefaciens DNA (one entire bacterial genome per three diploid tumor cells). By the same technique, PS8 bacteriophage DNA is not detected in crown gall tumor DNA under conditions that allow detection of 0.0007-0.001% added phage DNA. Crown gall tumor DNA cannot contain as much as one entire phage genome per diploid tumor cell. The presence of a small fraction of either genome (less than 5%), even if present as multiple copies, would escape detection by this method.

Bacteriophages↗
PubMed1974

Attempts to detect deoxyribonucleic acid from Agrobacterium tumefaciens and bacteriophage PS8 in crown gall tumors by complementary ribonucleic acid-deoxyribonucleic acid-filter hybridization.

Labeled ribonucleic acid (RNA) complementary to Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA (cRNA) were used in a systematic study of the sensitivity of cRNA/deoxyribonucleic acid (DNA)-filter hybridization for detection of small amounts of phage or bacterial DNA immobilized on filters. A. tumefaciens cRNA of specific activity 10(6) to 2 x 10(6) counts per min per mug reacted to a significant extent when the DNA-filter contained 1% A. tumefaciens DNA in a salmon DNA background, but 0.1% A. tumefaciens DNA was not detectable. PS8 phage cRNA of the same specific activity reacted to a significant extent when the DNA-filter contained as little as 0.01% PS8 DNA in a salmon DNA background. Both kinds of cRNA were found to bind to tobacco crown gall tumor DNA-filters. Similar reaction was found with control normal callus DNA-filters but not with tobacco seedling DNA-filters. The "hybrids" formed by cRNA with normal callus and tumor DNA-filters had low thermal stability. Attempts to purify the tumor and normal callus DNA prior to immobilization on the filter resulted in elimination of this spurious binding. No evidence was found for bacterial or phage DNA in crown gall tumor DNA.

Bacteriophages↗
PubMed1973

Deoxyribonucleic acid-membrane complexes in the Bacillus subtilis transformation system.

The fate of transforming deoxyribonucleic acid (DNA) in Bacillus subtilis was studied by isolating the DNA-membrane complex on Renografin gradients. Soon after uptake, transforming DNA binds to the cell membrane and displays a greater resistance to shear than the recipient genome-membrane complex. Ten minutes after uptake, a portion of the donor DNA is released from the membrane. Most of the released donor radioactivity represents unintegrated, biologically inactive DNA. Recombinant or integrated DNA is enriched 1.5- to 1.7-fold in the membrane. This enrichment last at least 30 min after termination of DNA uptake, and probably much longer. The data suggest that transforming DNA may be integrated into the recipient genome on, or close to, the cell membrane.

Bacillus subtilis↗
PubMed1973

Ribonucleic acid polymerase in Allomyces arbuscula.

Three distinct species of ribonucleic acid (RNA) polymerase were resolved from Allomyces arbuscula by diethylaminoethyl-cellulose chromatography and characterized as to ionic strength and divalent cation preference. alpha-Amanitin specifically inhibited enzyme II; neither rifampin nor cycloheximide had any effect on the three enzymes. RNA polymerase was isolated from three stages of the diploid life cycle: the hyphal growth stage, mycelia in the process of forming sporangia, and the mitospores. The same three enzyme species could be resolved from each stage. Thus, there is no evidence from this work that RNA polymerase plays a major role in the control of development.

Cell Nucleus↗
PubMed1973

Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis.

Mutants have been isolated which correspond to every step concerned with the biosynthesis of the aromatic amino acids in Bacillus subtilis. Each mutant has been characterized, and the lesion it bore was analyzed by deoxyribonucleic acid transformation and PBS-1 mediated transduction. The biochemical analysis revealed that each of the mutations appears to have affected a single enzyme, except for two groups of pleiotropic mutations. All aroF mutants (chorismic acid synthetase) lack dehydroquinic acid synthetase (aroB) activity. The gene that specifies aroB is closely linked to the gene coding for the aroF enzyme. Both genes are a part of the aro cluster. Mutants lacking chorismate mutase activity also lack d-arabino-heptulosonic acid-7-phosphate synthetase and shikimate kinase activity, presumably as a result of these three activities forming a multi-enzyme complex. Another mutant, previously undescribed, had been isolated. The affected gene codes for the tyrosine and phenylalanine aminotransferase activity. All of the mutations have been located on the B. subtilis genome except those in the genes specifying shikimate kinase activity and tyrosine-phenylalanine aminotransferase activity.

Alcohol Oxidoreductases↗