Biomedical subjects
Publications and source records attributed to J M Shively.
Carboxysomes were isolated from Nitrobacter winogradskyi and Nitrobacter agilis. The icosahedral particles contained double-stranded deoxyribonucleic acid (DNA). In the presence of ethidium bromide and cesium chloride, the particle-bound DNA had a buoyant density of rho 25 = 1.701 g/cm3. Electron microscopy revealed the DNA to be a 14-micron circular molecule.
The electron impact mass spectrum of a previously identified ornithine-containing lipid from Thiobacillus thiooxidans has been interpreted using exact mass measurements, low and high energy ionization, and defocused metastable studies. The spectrum, which did not contain a molecular ion for the intact lipid, was consistent with cyclization of the ornithine zwitterionic moiety with elimination of water to give 3[3'-(11,12-methylene-2-hydroxyoctadecanoxy)hexadecanylamine]-2-piperidone. Production of this sufficiently volatile species for mass spectral analysis was accomplished by gentle pyrolysis in the mass spectrometer source. The spectrum can be understood to arise by three primary decompositions which serve to separate the two fatty acid constituents. The remainder of the spectrum is consistent with the expected fragmentations of these constituents.
Paracoccus denitrificans grows on methanol as the sole source of energy and carbon, which it assimilates aerobically via the reductive pentose phosphate cycle. This gram-negative bacterium grew rapidly on 50 mM methanol (generation time, 7 h, 30 degrees C) in excellent yield (3 g of wet-packed cells per liter of culture). Electron microscopic studies indicated that the late-log-phase cells were coccoid, having a thick envelope surrounding a layer of more diffuse electron-dense material and a relatively electron-transparent core. Ribulose bisphosphate carboxylase in the 15,000 X g supernatant of fresh cells had specific activities (micromoles of CO2 fixed per minute per milligram of protein) of 0.026, 0.049, 0.085, 0.128, and 0.034 during the lag, early, mild-, and late log, and late stationary phases, respectively. The enzyme was purified 40-fold by pelleting at 159,000 X g, salting out, sedimentation into a 0.2 to 0.8 M linear sucrose gradient, and elution from a diethylaminoethyl-Sephadex column. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels polymerized from several acrylamide concentrations and sedimentation behavior. The molecular weight of the native enzyme, as measured by gel electrophoresis and gel filtration, averaged 525,000. Sodium dodecyl sulfate dissociated the enzyme into two types of subunits with molecular weights of 55,000 and 13,600. The S20,w of the enzyme was 14.0 Km values for ribulose bisphosphate and CO2 were 0.166 and 0.051 mM, respectively, and the enzyme was inhibited to the extent of 94% by 1 mM 6-phosphogluconate.
The icosahedral bodies of Nitrobacter agilis are about 120 nm in diameter and, as viewed by electron microscopy, consist of an outer shell enclosing 10-nm particles. The inner 10-nm particle is the enzyme D-ribulose 1,5-bisphosphate carboxylase. The bodies isolated from cells incubated 1 month without nitrite had a specific activity for the enzyme of 0.54 mu mol of CO2 fixed per min per mg of protein.
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The polyhedral inclusions of Thiobacillus neapolitanus have been isolated; they contain ribulose diphosphate carboxylase.
Escherichia coli O111a is a thermosensitive strain which, when grown at 40 C, accumulates large quantities of intracellular membranes. The ultrastructure of these membranes in cells which have been chemically fixed, embedded, and examined as thin sections has been compared with that of membranes in cells negatively stained or freeze-fractured. Results indicate that the extra membranes are present in the three types of preparations examined and, therefore, clearly are not artifacts of chemical fixation. Negative staining has proved also to be a valuable tool as a rapid means of monitoring cells for the accumulation of large amounts of extra membranes. Also, examination of thin sections has shown that distinct continuities between the plasma membrane and the extra membranes exist. In general, membrane surfaces in freeze-fractured cells containing extra membranes appear smooth and lack the particles associated with the plasma membranes of many cells.
The carboxysomes of Thiobacillus neapolitanus are shown, by electron microscopy, to consist of a paracrystalline array of 10-nm particles surrounded by a "membrane." The 10-nm particles have a center hole or depression and have been previously identified as ribulose diphosphate carboxylase. The membrane is a monolayer approximately 3.5-nm thick.
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The ultrastructure of seven Thiobacillus species was studied. The structure of their cell envelopes is similar, if not identical, to that found in other gram-negative bacteria. Obvious differences were noted in the middle layer of the cell envelope of the seven cultures. Polyhedral inclusion bodies were apparent in four of the organisms: T. thioparus, T. neapolitanus, T. intermedius, and T. thiooxidans. Lamellar bodies, similar to those present in certain photosynthetic bacteria were found in a few cells of T. thioparus. Structures resembling mesosomes were discovered in T. dinitrificans. A few cells of T. intermedius possessed paracrystalline bodies. Other inclusions, probably volutin and polysaccharide, were present in some of the cultures.
A temperature-sensitive strain of Escherichia coli (strain 0111a(1)) was shown to accumulate membranous structures at 40 C. These "extra membranes" appeared as vesicles or whorls (or both), depending on the time of growth at 40 C. After 2 hr of growth at 40 C, only vesicles were observed in E. coli 0111a(1) cells; both vesicles and whorls were apparent after 6 hr. The number of cells which contained both types of extra membrane reached a maximum value (75%) after 10 hr of growth at 40 C. Extra membrane production was also studied by using temperature shifts. In shift-up experiments, cells grown at 30 C into early stationary phase accumulated extra membrane after a shift to 40 C. The percentage of E. coli 0111a(1) cells containing extra membrane decreased significantly after a shift from 40 to 30 C. Phase- and electron-microscopic observations indicated that E. coli 0111a(1) cells grown at 40 C were larger than E. coli 0111: B(4) cells grown at either temperature. The ratio of optical density per cell and cell measurements obtained from quantitative electron microscopy confirmed that E. coli 0111a(1) cells grown at 40 C were about twice as large. Microdensitometer traces indicated that the dimension of a single membrane of either whorls or vesicles was 5.4 nm in peak-to-peak distance (8.8 nm total thickness).
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The ornithine-containing lipid was separated from the other lipids of Thiobacillus thiooxidans by thin-layer chromatography. The aminolipid possesses both amide and ester linkages.
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Phosphatidyl glycerol, disphosphatidyl glycerol, and phosphatidyl ethanolamine were found in all of the Thiobacillus species studied. T. thioparus possessed only these phospholipids. T. intermedius, T. neapolitanus, and T. thiooxidans contained phosphatidyl-N-monomethylethanolamine, and T. novellus lipids contained phosphatidyl-N-monomethylethanolamine, phosphatidyl-N-N-dimethylethanolamine, and phosphatidyl choline, in addition to the three phospholipids common to all of the thiobacilli. Methionine was found to act as a methyl donor in the biosynthesis of the methylated forms of phosphatidyl ethanolamine. Phosphatidyl inositol was not detected in any of the organisms. Changing the nutrient medium did not result in a qualitative change in the phospholipid spectrum of the cultures.
Cells and spent growth media from sulfur- and thiosulfate-grown cultures of Thiobacillus thiooxidans were analyzed. The phosphatides were examined by thinlayer chromatography, and the products of their hydrolysis by hydrochloric acid and methanolic potassium hydroxide were separated by paper chromatography. The phospholipids in both cells and spent growth media were identified as phosphatidyl ethanolamine, phosphatidyl-N-monomethylethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. These comprised about 97% of the total lipid phosphorus. Lyso-phosphatidyl-N-monomethylethanolamine and lysophosphatidyl glycerol accounted for the remaining 3%. The percentage of the total lipid phosphorus accounted for by each phospholipid depended on the age of the culture.
Thompson, T. L. (University of Nebraska, Lincoln), and J. M. Shively. Lethal action of ribonuclease for thermophilic bacilli. J. Bacteriol 91:673-676. 1966.-Exposure of a thermophilic bacillus, NU 47, to 0.2 mug/ml of pancreatic ribonuclease resulted in a drastic reduction of viable cells. The observed lethality is thought to be associated specifically with the ribonuclease, since heat treatment did not diminish activity and the addition of ribonucleic acid to the cells completely eliminated the action of the enzyme. Cells from 2-hr cultures, buffered between pH 6.0 and 7.0, were most sensitive. The lethal action was most pronounced when cells were exposed to the enzyme at temperatures between 25 and 45 C. A greater proportion of cells survived at higher temperatures. The release of ultraviolet-absorbing materials into the medium after exposure to enzyme could not be detected.