Detection of fine spiral structures (spirosomes) by weak sonication in some bacterial strains

Fine spiral structures (spirosomes) were observed in cell suspensions of five species of bacteria just after weak sonication. The structure is morphologically indistinguishable from the spirosome reported for Lactobacillus species. The molecular weight of the protein of the spirosomes from nine strains was about 94,000 to 95,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The difference in the molecular weight among these spirosomes was not very great, but there were slight differences among the strains from which the spirosomes were derived.     

Relationship between the production of spirosomes and anaerobic glycolysis activity in Escherichia coli B

The effects of culture conditions (aerobic or anaerobic) and glucose in the medium on the production of spirosomes in Escherichia coli B were studied by SDS-PAGE and electron microscopy. The Mr of the spirosome of E. coli B was estimated to be 97,000. Electron microscopy revealed that the amount of spirosomes derived from anaerobic cultures was about eightfold larger than that from aerobic cultures. In SDS-PAGE, the bands of spirosome protein derived from anaerobic cultures were more intense than those derived from aerobic cultures, either in peptone water or in Davis-Mingioli's minimal medium. With increased glucose concentration under aerobic conditions, the intensity of the band of spirosome protein was similar to that observed under anaerobic conditions in basal media. These results suggest that spirosome production by E. coli B is related to its anaerobic glycolysis activity.     

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.     

Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l -[¹⁴C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg²⁺ for basal incorporation of amino acids into protein was 8 mm ; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg²⁺. The response to nuclear RNA was also enhanced as the K⁺ concentration was increased from 25 to 100 mm . The stimulatory effect of nuclear RNA on incorporation of l -[¹²C]eucine was either unaltered or depressed by addition of a mixture of 19 l -[¹²C]amino acids each at concentrations, of 10^?8, 10^?2, or 10^?1 mm . Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations. Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid-acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid-acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.     

Isolation and chemical characterization of gas-vacuole membranes fromMicrocystis aeruginosa Kuetz. emend. Elenkin

Summary A method involving penicillin treatment was developed to osmotically lyse the cells of the blue-green alga,Microcystis aeruginosa Kuetz. emend. Elenkin, and release the pressure-sensitive gas vacuoles intact. The gas vacuoles were purified by liquid-polymer partitioning or by macromolecular sieving and centrifugation. The degree of purification of the gas vacuoles was followed by observation in the electron microscope and by the use of C¹⁴-labeled vacuolated and nonvacuolated strains ofM. aeruginosa. The gas-vacuole membrane is composed of only protein consisting of 10% basic, 18% acidic and 52% non-polar amino acids.Supported by U.S. Atomic Energy Commission, Contract No. AT(11-1)-1338.     

Rotihibin A,a Novel Plant Growth Regulator,from Streptomyces sp.

A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS–polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85°C, and about 50% of the original activity remained after incubation at 90°C for 10 min in the presence of Ca^{2 +} . The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a poiypeptide of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.     

Isolation and characterization of intracellular protein inclusions produced by the entomopathogenic bacterium Photorhabdus luminescens

Cells of the entomopathogenic bacterium Photorhabdus luminescens contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed to Galleria mellonella larvae.     

Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens

Cells of the entomopathogenic bacterium Photorhabdus luminescens contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed to Galleria mellonella larvae.     

Structure and composition of intracytoplasmic membranes of Ectothiorhodospira mobilis

The lamellar membrane stacks of Ectothiorhodospira mobilis were isolated and purified by a combination of lysozyme and osmotic shock treatment, followed by differential and density gradient centrifugation. Preparations of lamellar membranes were enriched at least 2.4-fold in the ratio of bacteriochlorophyll a to protein.Thin-sectioning, negative staining, platinumcarbon shadowing and freeze-etching were used to study the architecture of the membrane units. Both platinum-carbon shadowing and freeze-etching showed the outer surfaces of the isolated lamellar membrane stacks to be relatively smooth. Particles averaging 7 nm in diameter were seen on several faces following freeze-ctching.Non-polar amino acids amounted to 60% of the total amino acid composition. Lipids constituted 32% of the membrane dry weight. Phosphatidyl ethanolamine and diphosphatidyl glycerol were the major phospholipids. Fatty acids of 10–15 carbons represented a small fraction of both membrane and whole cell fatty acids. Monoenes constituted 36% of the total membrane fatty acids and 38.4% of the total whole cell fatty acids. The major fatty acids of both whole cells and purified membranes were C16:0, C18:1 and cyclopropane C19:0.     

Purification and Some Properties of Acidocin 8912, a Novel Bacteriocin Produced by Lactobacillus acidophilus TK8912

Acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912, was purified by ammonium sulfate fractionation and successive chromatographies on CM-cellulose, Sephadex G-50, Sephadex G-25, and reversed-phase HPLC on Aquapore RP-300. The purified acidocin 8912 migrated as a single band on SDS–PAGE. The molecular weight was estimated to be 5200 by SDS–PAGE, and 5400 by HPLC gel filtration on TSKgel G3000PW_XL. Both the amino acid composition and the N-terminal amino acid sequence analysis indicated that acidocin 8912 was a peptide composed of presumably 50 amino acids containing a Lys residue at the N-terminus. The purified acidocin 8912 showed a bactericidal effect on sensitive cells but not a bacteriolytic effect.     

Demonstration and characterization of the cell wall carbohydrate and protein antigens from Clostridium botulinum type E Saroma

Two different cell wall antigens, carbohydrate (CHO) and protein (P), from Clostridium botulinum type E Saroma were extracted with sodium dodecyl sulfate (SDS) and purified by chromatography on DEAE-Sepharose CL-6B and Sephadex G-75 or G-100. The CHO antigen was composed of glucose, galactose, glucosamine, galactosamine, alanine and phosphorus with a molar ratio of 1.5:1.5:0.25:0.25:1:1. The P antigen was an acidic protein with a molecular weight of 60 kDa, in which the major amino acids were aspartate, glutamate and serine, while the minor ones were cysteine and methionine. Thin sections of the intact or SDS-extracted cells of the organism demonstrated that the cell wall was composed of a two-layered structure, an inner layer about 20 nm thick and an outer layer about 10 nm, and by the extraction with SDS, the outer layer disappeared from the cell surface, leaving the inner layer. Immunogel diffusion tests demonstrated that either CHO antigen or P antigen was common among the nonproteolytic strains of C. botulinum.     

Isolation and Partial Properties of a Porin-Like Protein from Vibrio parahaemolyticus Cell Envelope

The cell envelope of Vibrio parahaemolyticus pilot strain K-11 contains a major protein with an apparent molecular weight of 35,000 which was not solubilized with 2% sodium dodecyl sulfate (SDS) at 50 C for 30 min and was resistant to trypsin. The protein was extracted from the SDS-insoluble envelope with SDS containing 0.4 m NaCl and purified by acetone precipitation and gel filtration. The purified protein was completely dissociated into a monomer with a molecular weight of 35,000 in SDS at 60 C. The amino acid composition of the protein was nearly the same as that of porins from Escherichia coli and Salmonella typhimurium. Thus the protein seems to be porin-like.     

Extracellular Lytic Enzymes of Myxococcus virescens

An easy and rapid method for the purification of a bacteriolytic endopeptidase produced by Myxococcus virescens is described. The bacteria were grown in casitone media and the cells were sedimented by centrifugation. About 1.2 g of montmorillonite were added per liter of cell-free culture solution. The clay was sedimented by centrifugation and the enzyme was then eluted by 0.05 M Na-phosphate buffer pH 6.0, containing 0.4 M NaCl. The enzyme was diluted with water and chromatographed on carboxymethyl-cellulose columns. The purified enzyme liberated free amino groups but no reducing sugars or N-acetylhexosamines when acting on purified N-acetylated cell walls of Micrococcus lysodeikticus. Analysis of N- and C-terminal amino acids in the digestion products showed that the enzyme had liberated about 110 nmoles of lysine ε-amino groups and 60 nmoles of alanine carboxyl groups per mg of cell wall. When it acted on a bisdisaccharide pentapeptide dimer isolated from M. lysodeikticus cell walls, it cleaved about 30% of the alanyl-lysine linkages. Consequently the enzyme was an alanyl-lysine endopeptidase. It had no muramyl-alanine amidase activity.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Methyl 2,3-Di-O-(l-alanyl)-α-d-glucopyranoside,a New Sweet Substance

An inhibitor of plant virus infection from leaves of Yucca recurvifolia was purified by a method using gel filtration on Sephadex G-75, and column chromatography on CM-Toyopearl 650M. The purified inhibitor thus obtained was homogeneous on disc and SDS disc electrophoreses, and the molecular weight of the inhibitor was 23,000. The inhibitor consisted of 17.7% nitrogen, which was found to be a basic simple protein, and contained no neutral sugar, hexosamine nor sialic acid. The inhibitor was estimated to be composed of about 208 amino acid residues. This inhibitor was named “yucca leaf protein (YLP).”     

Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP⁺ to NAD⁺ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.     

2-O-methyl-d-xylose containing sheath in the cyanobacterium Gloeothece sp. PCC 6501

The sheath of the unicellular cyanobacterium Gloeothece sp. PCC 6501 was isolated from cell homogenates by differential and sucrose gradient centrifugation, followed by lysozyme treatment and hot sodium dodecyl sulfate extraction. The sheath contains a major fraction of carbohydrate consisting of galactose, glucose, mannose, rhamnose, 2-O-methyl-d-xylose, xylose, glucuronic and galacturonic acids, but only traces of fatty acids and phosphate. A protein content of about 2% (of fraction dry weight) could not be removed by the detergent treatment.Abbreviation SDS sodium dodecyl sulfate     

A Protease Inhibitor from Penicillium cyclopium

A protease inhibitor produced by Penicillium cyclopium on solid cultures of wheat bran was purified by means of column chromatography on Duolite A-2 and DEAE-cellulose, acetone precipitation and lyophilization. The purified inhibitor obtained as a white, floccose and hygroscopic substance was monodisperse by ultracentrifugal analysis. It was found to be an acidic macro-molecule of a molecular weight of about 5000. The chemical analyses rejected the possibility of the presence of amino acids, peptides, sugars, amino sugars, or uronic acids in the inhibitor molecule.

Properties of a protease inhibitor from Penicillium cyclopium were studied. The pH range of the inhibitor action is restricted to acid pH, optimally at pH 3. Increasing temperature accelerates its action upon enzyme. The inhibitor causes enzyme inactivation in proportion to its concentration. It is fairly stable in an acid solution but unstable in an alkaline solution. It undergoes destruction by heat, hydrogen peroxide and ascorbic acid. The inhibitor reversibly combines with Al³⁺, Fe³⁺, Ag⁺ and Cu²⁺ to produce a precipitate. Salts interfer with the inhibitor activity. Generally, acid proteases from various penicillia are susceptible to the inhibitor while those from other genera are resistant.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

Pig liver monoamine oxidase: Studies on the subunit structure

The molecular weight of pig liver MAO has previously been shown to be about 115,000 with 1 mole of covalently bound FAD per mole of enzyme. Gel filtration of purified enzyme on Sepharose 4B in 6 m guanidine and 0.1 m mercaptoethanol (MCE) and analytical ultracentrifugation in 0.1% sodium dodecyl sulfate (SDS) and 0.1% MCE yielded molecular weights of 55,000 and 63,000, respectively. By polyacrylamide electrophoresis in 0.1% SDS + MCE one band of 60,000 MW appeared. These results seem to imply that the enzyme is composed of two subunits of which one carries the active site. If MCE was omitted during the gel electrophoresis two equally large bands of about 60,000 MW were formed. By using enzyme inhibited by [¹⁴C]pargyline, a MAO-inhibitor blocking the active site of the enzyme in a 1:1 molar ratio, it was found, however, that both bands contained pargyline. Furthermore, amino acid analyses yielded the same amino acid composition of the two bands. The results are interpreted that the enzyme is composed of two subunits of identical molecular size (about 60,000) of which only one contains the active site and that the enzyme preparation contained two forms of the enzyme presumably differing in the number of disulfide bonds.