Myceloid growth of Arthrobacter globiformis and other Arthrobacter species.

Transitory myceloid growth occurs in certain complex media with Arthrobacter globiformis strain ATCC 8010. This type of growth, however, was not observed in a medium which contained an array of metal ions but did not contain agents able to complex metal ions. Addition of metal-complexing agents to this medium caused an interruption in the life cycle of strain 8010 so that growth occurred only as the myceloid form. It appeared that manganese was the critical metal that was removed by the metal-complexing agents. During growth, the myceloid cells started to fragment, but wall septation was incomplete. A. globiformis strain ATCC 4336 and several other Arthrobacter species and soil isolates, but not Arthrobacter crystallopoietes, responded to metal-complexing agents as did strain 8010. Biotin and vitamin B12 were not involved in this myceloid growth.     

Production of polysaccharides by Arthrobacter globiformis associated with Anabaena azollae in Azolla leaf cavity

Abstract The composition of the capsular polysaccharides (CPS) and exopolysaccharides (EPS) of three strains of Arthrobacter globiformis , isolated from the leaf cavities of Azolla caroliniana (strain B1), A. filiculoides (strains A3 and L1) and A. globiformis ATCC 8010 have been analysed by HPLC and enzymatic assays. Glucose and galactose were detected in the EPS of all the strains, while rhamnose was present only in the EPS of the strain L1 and uronic acids in B1 and ATCC 8010. Traces of fructose were detected by enzymatic assays in all the strains. The CPS contained glucose, galactose and rhamnose, while uronic acids were present only in strain B1. In all the strains the amount of EPS was higher than CPS. The reactivity to different dyes and lectins of the mucilagineous matrix of the algal packets extracted from the fern and of the bacterial mucilage were similar.     

Physical and Biological Properties of Dengue-2 Virus and Associated Antigens

Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients. Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface. Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm(3) and from cell culture at 1.24 g/cm(3). During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm(3). It was shown in electron micrographs to consist of virion fragments. A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm(3) in CsCl, and appeared as "doughnut" forms measuring 13.8 to 14 nm in diameter. A third component, with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.     

黄嘌呤氧化酶产生菌的离子注入诱变及其二步发酵的研究

采用剂量 1.0× 10 1 2 ～ 1.0× 10 1 6ions cm2 的N+注入产黄嘌呤氧化酶的球形节杆菌ATCC80 10 ,研究其诱变效果。结果表明 :细菌的存活曲线呈现特殊的“马鞍形” ,菌落形态和颜色发生了变化 ,且变化与产酶量有关 ,经过筛选获得了遗传性能稳定的高酶活突变株 ,使酶活较出发菌提高了 43.0 %。改进发酵工艺 ,采用二步发酵其酶得率是一步发酵的 4.75倍。     

Structure of Infectious Bursal Disease Virus

Infectious bursal disease virus of chickens was purified, and its structure was examined by the negative-staining technique in the electron microscope. The buoyant density of infectious bursal disease virus in CsCl was found to be 1.34 g/cm(3). The morphological details suggest that the capsid of the virion consists of a single layer of 32 capsomeres arranged in 5:3:2 symmetry. The virion measured about 55 nm in diameter and had no envelope.     

Isolation and characterization of Caulobacter crecentus bacteriophage phi Cd1.

A DNA-containing bacteriophage, phiCd1, was isolated from sewage and shown to infect both stalked and swarmer cells of Caulobacter crescentus strain CB13B1a. phiCd1 is a small, icosohedral bacteriophage, 60 nm in diameter, which possesses a short, noncontractile tail, 10 to 12 nm in length. The bacteriophage particle is composed of at least eight structural proteins. phiCd1 nucleic acid exists as a linear duplex of DNA as judged by: (i) thermal denaturation (Tm), (ii) CsCl density gradient centrifugation, and (iii) chemical analysis of its base composition. The DNA is 61% guanosine plus cytosine, has a buoyant density in CsCl of 1.721 +/- 0.001 g/cm3, and denatures sharply at 78.5 C in 0.1 SSC (standard saline citrate) buffer. The S20, w value for the DNA is 34.3 +/- 0.1S as compared with T7 DNA, indicating a molecular weight of about 29 x 10(6).     

Characteristics of a new bacteriophage, Psp231a, infecting Pseudomonas phaseolicola HB10Y.

Bacteriophage Psp231a infects Pseudomonas phaseolicola, strain HB10Y, which is the host cell for the enveloped bacteriophage phi 6. This paper describes the biophysical characteristics of Psp231a and the physical properties of its nucleic acid. In electron micrographs the virion appears as an icosahedral structure, approximately 55 nm in diameter, with a short tail. The virion density is 1.48 g/cm3 in CsCl, and the sedimentation coefficient is approximately 407S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 12 polypeptides ranging in molecular weight from 5,000 to 117,000. The nucleic acid of Psp231a is linear, double-stranded DNA of molecular weight 28 X 10(6). Its density in CsCl is 1.716 g/cm3, and its sedimentation coefficient in 3 M CsCl is 20.0S, corresponding to an S020,W of 34S.     

Bacteriophages of Rhodopseudomonas spheroides: Isolation and Characterization of a Rhodopseudomonas spheroides Bacteriophage

A DNA-containing bacteriophage, designated RS1, infecting Rhodopseudomonas spheroides 2.4.1, has been isolated from sewage. The buoyant density of RS1 in CsCl equilibrium centrifugation is 1.50 g/cm(3), and the buoyant density of RS1 DNA is 1.706. The phage possesses a polyhedral head, approximately 65 nm in diameter, and a tail 60 nm long. When grown on aerobic cells, RS1 has a latent period of 120 min and an average burst size of 20. When grown on anaerobic cells, RS1 has a latent period of 150 min, and a burst size similar to that observed during aerobic infection. The adsorption rate constant of RS1 to aerobic cells is 1.2 x 10(-9) ml/min, and 0.58 x 10(-9) ml/min to anaerobic cells. Adsorption of RS1 to R. spheroides requires the presence of divalent cations.     

Characterization of Polyoma DNA-Protein Complexes I. Electrophoretic Identification of the Proteins in a Nucleoprotein Complex Isolated from Polyoma-Infected Cells

A study was undertaken to examine polyoma DNA-protein complexes. A biophysical characterization of the complexes was made, and the proteins found in such complexes were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison was made between a 52S nucleoprotein complex isolated from nuclei of 26-h polyoma-infected cells and a 28S virion core complex ejected out of mature virus particles. It was found that both complexes were reduced to a 20S viral DNA component plus free protein after incubation in 1 M NaCl or Sarkosyl. Treatment of the complexes with either Pronase or 0.5 M NaCl resulted in only partial removal of proteins from the viral DNA. After fixation in formaldehyde, the 52S nucleoprotein complex had a buoyant density of 1.45 g/cm³, and the virion core complex had a buoyant density of 1.59 g/cm³. Sodium dodecyl sulfate-polyacrylamide gel profiles of purified polyoma virion proteins, used as a reference marker, demonstrated three capsid proteins, V1 to V3, as well as four histones, V4 to V7, which constituted about 7% of the total virion protein. Electrophoretic analysis of the proteins comprising the 52S nucleoprotein complex revealed that the same seven proteins present in the mature virion were also found in this complex. However, the ratios of the proteins in the complex were quite different from that of the mature virion, with the four histones comprising 48% of the total complex protein. A pulse-chase experiment of the nucleoprotein complex demonstrated that the 26-h complex was chased into mature virions.     

Isolation and characterization of a Pseudomonas aeruginosa bacteriophage with a very limited host range

A Pseudomonas aeruginosa bacteriophage, phi PLS743, with extremely limited host range has been isolated. It belongs to the virus family Podoviridae, morphological type C1, and possesses a head diameter of 45 nm. The phage has a buoyant density in CsCl of 1.516 g/cm3, and its mass is 45 x 10(6) daltons. The phage particles are composed of double-stranded DNA (49.9 mol% G + C; 42.4 kilobase pairs) and 11 structural proteins (66% by weight). The major head protein, P5, has a Mr of 34,500. The DNA is not cut by SalI or XhoI restriction endonucleases, but is cut by PvuII (1 site), KpnI and BglII (2 sites), PvuI (4 sites), BamHI (7 sites), EcoRI (9 sites), and HindIII (12 sites). A restriction endonuclease map is presented.     

Buoyant Density of the Hepatitis A Virus-Like Particle in Cesium Chloride

We recently visualized by immune electron microscopy a virus-like particle in the stools of patients with hepatitis A. The particle measured approximately 27 nm in diameter and morphologically resembled a picornavirus or parvovirus. To further characterize this particle, we have determined its buoyant density in cesium chloride (CsCl) by ultracentrifugation. Hepatitis A particles from three positive stool specimens were isopycnically banded in separate experiments, and the gradient fractions were examined for particles by immune electron microscopy by using hepatitis A convalescent sera. In each experiment, the particles were observed in a normal distribution about a peak fraction with a mean density of approximately 1.4 g/cm(3). The buoyant density of 1.4 g/cm(3) in CsCl together with its morphology and the reported resistance of hepatitis virus to acid, ether, and heat suggest that this particle is parvovirus-like.     

Isolation and Characterization of a Bacteriophage for Thiobacillus novellus

A DNA-containing bacteriophage for Thiobacillus novellus has been isolated from sewage and purified by ammonium sulfate precipitation, differential centrifugation, and cesium chloride equilibrium centrifugation. The buoyant density of this phage in CsCl is 1.51 g/cm(3). Electron microscopy studies have revealed a polyhedral head about 60 nm in diameter and a tail surrounded by a number of fine filaments. It has an adsorption rate constant of 1.1 x 10(-9) ml/min, a latent period of 45 min, and an average burst size of 150. The mole guanine and cytosine content in its DNA has been estimated to be 57 to 58%. Five structural proteins with molecular weights of 62,000, 42,500, 30,500, 17,750, and 13,500 have been detected by polyacrylamide gel electrophoresis techniques.     

Biophysical Properties of Australia Antigen

Biophysical studies with Australia complement-fixing (CF) antigen showed it to be a particle with a buoyant density of 1.20 g/cm(3) in CsCl, a sedimentation coefficient of 110, and an average diameter of 25 nm. The CF antigen was not inactivated by ether, 1% deoxycholate, 1% Tween 80 or overnight heating at 56 C. The antigen was unstable when treated with 1% sodium dodecyl sulfate. A procedure is described for the isolation and partial purification of Australia antigen from serum by using isopycnic banding and rate separation techniques. Treatment of the 1.20 g/cm(3) Australia antigen with 1% Tween 80 yielded a minor peak of CF activity with a buoyant density of 1.39 g/cm(3) in CsCl.     

Interaction of a DNA-binding protein, the gene product of D5 of bacteriophage T5, with double-stranded DNA. Analysis by metrizamide gradient centrifugation

Interactions of DNA and the gene product D5 (gpD5) of bacteriophage T5, a DNA-binding protein that binds preferentially and cooperatively to double-stranded DNA, were analyzed by metrizamide gradient centrifugation. Conditions were set so that DNA and DNA protein complex sedimented to apparent equilibrium positions. DNA has a buoyant density of 1.12 g/cm3, and DNA saturated with gpD5 has a buoyant density of 1.17 g/cm3. These values are independent of DNA size and base composition in the range studied. At gpD5 concentration below the saturation value in a low ionic strength buffer, DNA distribution is bimodal, indicating cooperative binding of gpD5 to DNA. However, in the presence of 10 mM MgCl2, the binding process becomes distributive, with the buoyant density increasing linearly with the amount of gpD5 added until the saturation. From these data, one molecule of gpD5 is calculated to cover 40 base pairs at saturation. The technique as described has general applicability to the study of any interaction between DNA and dNA-binding proteins that bind in sufficient amount to cause detectable changes in buoyant density.     

Dynamics of the accumulation of virus particles with different physico-chemical properties in FRhK-4 cells infected with hepatitis A virus

The propagation time-course of hepatitis A virus (HAV, strain HAS-15) in continuous culture of the foetal rhesus monkey kidney cells (FRhK-4) was investigated. The HAV infectivity and viral RNA content in the infected cells reached the maximal level 5-8 days after infection, while accumulation of hepatitis A antigen (HAAg) continued for 2-3 weeks more. Viral particles with the densities 1.27-1.28 g/cm3 and 1.18-1.22 g/cm3 were isolated from the infected cells as well as the mature virions with the buoyant density 1.33-1.34 g/cm3 in CsCl. The concurrent accumulation of mature virus and "light" particles (1.18-1.22 g/cm3) was registered during infection. Viral particles with the density 1.27-1.28 g/cm3 accumulated predominantly from the 14th to the 21st-24th days after infection. The mature virions (1.34 g/cm3) as well as the particles with the density 1.24-1.25 g/cm3 were isolated from supernatant precipitated by ammonium sulphate. The HAAg activity of both fractions increased progressively in equal proportion in course of infection.     

Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl.

Although hepatitis A virus (HAV) released by infected BS-C-1 cells banded predominantly at 1.325 g/cm3 (major component) in CsCl, smaller proportions of infectious virions banded at 1.42 g/cm3 (dense HAV particles) and at 1.27 g/cm3 (previously unrecognized light HAV particles). cDNA-RNA hybridization confirmed the banding of viral RNA at each density, and immune electron microscopy demonstrated apparently complete viral particles in each peak fraction. The ratio of the infectivity (radioimmunofocus assay) titer to the antigen (radioimmunoassay) titer of the major component was approximately 15-fold greater than that of dense HAV particles and 4-fold that of light HAV particles. After extraction with chloroform, the buoyant density of light and major component HAV particles remained unchanged, indicating that the lower density of the light particles was not due to association with lipids. Light particles also banded at a lower density (1.21 g/cm3) in metrizamide than did the major component (1.31 g/cm3). Dense HAV particles, detected by subsequent centrifugation in CsCl, were indistinguishable from the major component when first banded in metrizamide (1.31 g/cm3). However, dense HAV particles recovered from CsCl subsequently banded at 1.37 g/cm3 in metrizamide. Electrophoresis of virion RNA under denaturing conditions demonstrated that dense, major-component, and light HAV particles all contained RNA of similar length. Thus, infectious HAV particles released by BS-C-1 cells in vitro consist of three distinct types which band at substantially different densities in CsC1, suggesting different capsid structures with varied permeability to cesium or different degrees of hydration.     

Cloning, sequence analysis, and purification of choline oxidase from Arthrobacter globiformis: a bacterial enzyme involved in osmotic stress tolerance

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, one of a limited number of compounds that accumulate to high levels in the cytoplasm of cells to prevent dehydration and plasmolysis in adverse hyperosmotic environments. In the present study, the highly GC rich codA gene encoding for choline oxidase was cloned from genomic DNA of Arthrobacter globiformis strain ATCC 8010 and expressed to high yields in Escherichia coli strain Rosetta(DE3)pLysS. The resulting enzyme was purified to high levels in a single chromatographic step using DEAE-Sepharose, as shown by SDS-PAGE analysis. Denaturation and mass spectroscopic analyses showed that the covalent linkage between the FAD cofactor and the protein is preserved in recombinant choline oxidase, consistent with protein flavinylation being a self-catalytic process. The enzyme was shown to be a homodimer of 120,000 Da by size-exclusion chromatography and to be active with both choline and betaine aldehyde as substrate. Sequencing analysis indicated that the nucleotide sequence of codA originally reported in GenBank contains seven flaws, resulting in a translated protein with a significantly altered amino acid sequence between position 298 and 410.     

Properties of hepatitis A virus particles produced during infection in vitro

Four types of virus-specific particles with different sedimentation coefficients and buoyant densities in CsCl were shown to be accumulated in hepatitis A virus (strain HAS-15) infected fetal rhesus monkey kidney cells (FRhK-4 line). Unlike the mature virions (155S, 1.34 g/cm3), cell-associated isosedimenting 92 S-particles (buoyant densities of 1.30 and 1.20 g/cm3) proved to be sensitive to lipase action. Particles of all four types were shown to contain similar sets of polypeptides, and, with the exception of "empty" 1.30 g/cm3-particles, appeared to be "full" under the immune electron microscopic examination. The viral RNA was unequivocally identified by the molecular hydridization test only in the mature virions.     

Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100-250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.