Reconstitution of Pronase-Susceptible Flocs of Flavobacterium sp.

A component responsible for the aggregation of cells was extracted from Flavobacterium strain B by treatment of cells with 5 m guanidine hydrochloride and partially purified by gel filtration. The guanidine hydrochloride-extracted cells were reaggregated with the component after dialysis against 0.3mm of CaCl₂. Various divalent cations were effective in place of Ca²⁺, but Ca²⁺ was most effective for reconstitution. The reconstituted flocs were deflocculated by the treatment of Pronase or ethylenediaminetetraacetic acid indicating that reconstituted flocs closely resemble natural flocs.     

DNA Binding Activity of Cells of Deoxyribonuclease-susceptible Floc Forming Pseudomonas sp.

Cells of Pseudomonas strain C-120, cultivated under the conditions in which cells do not flocculate naturally, were flocculated with DNA prepared from Escherichia coli, indicating that DNA binding factor was constitutively present on the cell surface. On the other hand, release of DNA into the growth medium was observed accompanying flocculation of cells. The results suggest that release of DNA from cells is an important factor for flocculation. DNA binding activity of cells was abolished by treating cells with proteases, suggesting the DNA binding factor is a proteinaceous component. The effects of salts and 2,4,6-trinitrobenzene sulfonic acid on the cells suggested that amino groups were involved in the DNA binding reaction. The number of DNAs bound per cell was estimated to be about 10 molecules from reconstitution experiments using phage T₄ DNA.     

Amine Oxidases of Microorganisms

An improved purification procedure is described for preparation of crystalline amine oxidase in higher yields from the mycelial extract of Aspergillus niger. The amine oxidase was accumulated in mycelia of the fungus when it was grown in a medium containing n-butylamine as a sole nitrogen source. The n-butylamine was fed from the alkali tank of a pH stat connected with the jar fermenter, to maintain pH of the culture medium at 5.0~5.5 during the incubation.

The amine oxidase was shown to be inhibited by guanidine hydrochloride and dissociation of the enzyme occurred when it was dialyzed against 6 m guanidine hydrochloride containing 0.1 m mercaptoethanol. The native enzyme of its molecular weight of 252,000 dissociated into three subunits of each molecular weight of 85,000 during the dialysis.

Pyridoxal derivatives were detected in digest of the native enzyme by proteolysis followed by acid hydrolysis. The derivatives showed a biological activity to support the growth of Saccharomyces carlsbergensis. Fluorescence property of these derivatives rather resembled to that of the known pyridoxal derivatives. A ¹⁴C-labelled protein was prepared from a solution containing the enzyme and ethylamine-1-¹⁴C, through the reductive action of sodium borohydride. On acid hydrolysis of the protein, a radioactive compound was obtained that has chromatographic and fluorescence properties of pyridoxylethylamine.     

Integrity of polypeptide chains of spectrin from human erythrocytes.

The suggestion that the high molecular weight erythrocyte membrane protein, spectrin, consists of subunits resistant to dissociation by both sodium dodecyl sulfate and 6 m guanidine hydrochloride has been reevaluated. By gel electrophoresis in dodecyl sulfate and thin-layer gel filtration in 6 m guanidine hydrochloride as well as in the much more powerful denaturant guanidine thiocyanate, and by sedimentation velocity in 6 m guanidine hydrochloride, the molecular weight emerges in the range 2–2.5 × 10⁵. Denaturation profiles as a function of guanidine hydrochloride concentration, observed by circular dichroism, reveal that the spectrin conformation is unusually labile, with a mid-point for the unfolding process at a denaturant concentration near 1 m. Complete acylation with succinic anhydride, as well as reaction with citraconic anhydride, leaves the molecular weight unchanged even in 6 m guanidine hydrochloride. The possibility of measuring molecular weights of proteins by viscosity determination in trifluoroacetic acid was explored. A calibration with a series of proteins gave a Mark-Houwink plot with high scatter, which did not result from low precision of viscosity determination or protein degradation. Evidence is adduced from infrared spectra that the scatter is due to a variable degree of protonation of the polypeptide backbone in the acid, leading to altered hydrodynamic characteristics. Within the semiquantitive limits of the method, spectrin is not further disaggregated in trifluoroacetic acid. The presence of refractory noncovalent interactions and of covalent cross-links has been variously invoked to explain an apparent microheterogeneity in spectrin preparations. The results here described appear to render the former explanation untenable.     

Studies on the structure of the acetylcholine receptor from Torpedo marmorata

The purified acetylcholine receptor of Torpedo marmorata has been characterized by sedimentation velocity measurements on dilute solutions using an ultracentrifuge and scanner. Several preparations were studied and all exhibited sedimentation coefficients in the vicinity of 24S. In a number of experiments the receptor could be resolved into two sedimenting boundaries of 18S and 26S, corresponding to minimum molecular weights of about 5 × 10⁵ and 10⁶, respectively. Additions of sodium dodecyl sulfate or Triton X-100 resulted in marked decreases in sedimentation coefficient, while treatment with Lubrol-WX had only a slight effect on the S values. Small changes in S_20,w were produced by guanidine hydrochloride alone, although addition of dithiothreitol with 6 M guanidine hydrochloride resulted in an 8.8S component. Electrophoresis in sodium dodecyl sulfate gave one principal band with a molecular weight of 46,000.     

Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe

We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe.     

Behavior of Glutenin in Different Concentrations of Guanidine Hydrochloride

In order to determine the limiting dispersion glutenin in concentrated guanidine hydrochloride, the behavior of glutenin was observed in different concentrations of guanidine hydrochloride. Increase of intrinsic viscosity and decrease of turbidity were observed with increasing concentrations of guanidine hydrochloride up to 6 m and a limiting state was attained above 6 m. The molecular weight determination also gave a limiting value above 6 m. The values obtained were 850,000 for one preparation and 640,000 for another. While the turbidity and viscosity in 6.5 m guanidine hydrochloride were constant in the pH region from 4 to 5.5, they increased slightly around pH 6 and decreased toward pH 7. The viscosity of glutenin around pH 7 was lower than that around pH 4, but the same molecular weight was obtained at pH 4.0 and 6.9.     

Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line,BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL₁ cells to sequentially deproteinized BCL₁ chromatin-cellulose was investigated. [³H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAF-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [³H]TA-receptor complexes to chromatin-cellulose extracted with 0–8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocortical receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [³H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL₁ cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL₁ cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6–8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Structure of Deoxyribonucleic Acid on the Cell Surface During Uptake by Pneumococcus

We exposed competent cells of Diplococcus pneumoniae to high-molecular-weight donor deoxyribonucleate (DNA) and examined the state of the DNA bound to them in forms sensitive to deoxyribonuclease I. The portion elutable with 5 M guanidine hydrochloride was shown to be native, of much lower molecular weight (4 x 10(6) to 5 x 10(6)) than the donor, and as active in further transformation as sheared DNA of the same size. The portion resistant to release by guanidine hydrochloride was also shown to be native and active in transformation. These results, along with previous ones, imply that the breaks produced outside the cell are not at genetically specific sites. Furthermore, it was found that entry past the cell barrier to deoxyribonuclease could occur at 0 C by a process sensitive to ethylenediaminetetraacetate.     

Studies on the posterior silk gland of the silkworm Bombix mori. IV. Ultracentrifugal analyses of native silk proteins, especially fibroin extracted from the middle silk gland of the mature silkworm

Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S°_20,w values of 10S, 9–10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.71₆, the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 – 4.2 x 10⁵, with a tendency to lie fairly close to 3.7 x 10⁵.     

Concentration of viruses in beef extract by flocculation with ammonium sulfate

Bacteriophages and enteroviruses in water were adsorbed to positively charged filters (Virosorb 1MDS [AMF Cuno, Inc., Meriden, Conn.] or Seitz S [Republic Filters, Milldaler, Conn.]). Adsorbed viruses were eluted by treating the filters with 10% beef extract, pH 9. Organic flocculation of the beef extract at pH 3.5 permitted recovery of more than 40% of the enteroviruses tested but less than 15% of the bacteriophages present. A method was developed that uses salts at pH 7 to flocculate beef extract. Two volumes of saturated ammonium sulfate were added to beef extract, and both enteroviruses and bacteriophages were adsorbed to the flocs that formed. Greater than 70% of the enteroviruses and bacteriophages were recovered by centrifuging the sample and suspending the flocs in a small volume of distilled water.     

Ejectisins: tough and tiny polypeptides are a major component of cryptophycean ejectisomes

Fragments of discharged ejectisomes were isolated from two Cryptomonas and a Chroomonas species by detergent treatment followed by Percoll density gradient centrifugation. The fragments withstand high concentrated detergent solutions, reducing agents and freeze-thawing. Disintegration was achieved in 6 M guanidine hydrochloride. Reassembly into long, filamentous, ejectisome-like structures occurred after dialysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the polypeptide patterns of isolated ejectisome fragments and of reconstituted ejectisome-like structures were dominated by polypeptides with relative molecular weights of approximately 6 kDa. The polypeptides were not glycosylated and did not cross-react with antisera directed against recombinant Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis. A polyclonal antiserum directed against reconstituted, ejectisome-like filaments cross-reacted with the 6-kDa polypeptides and immunolabeled extruded ejectisome filaments. Twenty amino acid residues, obtained by N-terminal amino acid sequence analysis, matched to polypeptide sequences deduced from cDNA sequences of the cryptophyte Guillardia theta. The term “ejectisins” is introduced for the 6-kDa polypeptides which represent a major component of cryptophycean ejectisomes.     

Over‐expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons

The ability of a yeast cell to propagate [PSI⁺], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI⁺] propagation in dividing cells and they get diluted out as cells multiply. Over‐expression of Hsp104 also leads to [PSI⁺] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over‐expression in [PSI⁺] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion‐free [psi^‐] cells present, no change in the molecular weight of Sup35‐containing SDS‐resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over‐expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over‐expression of Hsp104 causes malpartition of [PSI⁺] propagons between mother and daughter cells in a sub‐population of cells during cell division thereby generating prion‐free [psi^?] cells.     

Subunit structure of oyster paramyosin

Paramyosin from the oyster Crassostrea commercialis was studied by equilibrium sedimentation. In non-denaturing solvents the minimum molecular weight is 208000. Dissociation into subunits requires complete disruption of the alpha-helix. This occurs at pH7 in guanidine hydrochloride solutions of concentration greater than 7m in the presence of a disulphide-bond-reducing agent. Solutions of the protein in concentrated guanidine hydrochloride are polydisperse and contain species of low molecular weight (approx. 25000) comprising approx. 5% to 10% of the protein. The molecular weight of the main component is estimated to be 97000 and the paramyosin molecule contains two of these subunits. From the present observations no decision can be made as to whether or not the small component (or components) represents part of the paramyosin molecule. Preferential binding of guanidine hydrochloride to the extent of 0.13g./g. of protein was shown in solutions of paramyosin in 7.85m-guanidine hydrochloride.     

Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA

Summary Donor deoxyribonucleic acid (molecular weight 5-8×10⁷) introduced into competent cells of Bacillus subtilis is converted to molecules with a weight average molecular weight of 9×10⁶. These molecules, having little transforming activity, constitute in all probability eclipse phase DNA. At least part of the DNA is transiently complexed with a cellular component, changing its buoyant behaviour in CsCl gradients. When shortly after uptake of donor DNA the total DNA extracted from recipient cells is sheared to a molecular weight of 8×10⁶ or less, no eclipse phase is discernable. Donor marker frequencies in sheared, reisolated DNA mixtures decrease by a factor of 4 as a function of time of incubation of the transforming cells. This indicates that only 25% of the irreversibly absorbed DNA is finally integrated into the recipient genome.     

Flocculation of Chlorella vulgaris by Lactobacillus casei

Summary The flocculation of Chlorella vulgaris by Lactobacillus casei was studied to determine whether the latter could act as a suitable flocculant for the removal of Chlorella from algal ponds. The flocculating activity of the Lactobacilli was caused by the bacterial cells themselves, and not by diffusible products of bacterial metabolism. Diffusible products of algal metabolism inhibited flocculation. For algae resuspended in water, the best flocculation occurred at pH values less than 3.5 where the charges on the bacterial and algal cells were opposite. For flocculation at least one bacterium was required for every algal cell; in terms of cell concentrations,10 mg/l of bacteria were required to flocculate an algal suspension of 1,000 mg/l. The mechanism of flocculation implied by the results is that positively charged cells of L. casei adsorb to the surface of negatively charged cells of C. vulgaris neutralizing the charge and thus destabilizing the algal suspension. Because of the low pH required and because diffusible products of algal metabolism inhibit the flocculation, it is unlikely that L. casei could be usefully employed as a flocculant of Chlorella from algal ponds.     

Flocculant Production by Paecilomyces sp. Taxonomic Studies and Culture Conditions for Production

Screening of microorganisms producing a new kind of flocculating substance was carried out. A fungus that produced a large amount of a strong microbial cell flocculant was isolated from soil. The fungus was classified as a Paecilomyces species. The substance produced showed very interesting and unique flocculation characteristics. It could efficiently flocculate all tested suspended solids in aqueous solution, and flocculation was practically not affected by ionic strength, pH or temperature.

The influence of components of the culture medium upon the flocculant production by Paecilomyces sp. 1-1 was studied. Addition of hydrolysates of casein such as casamino acid and polypeptone stimulated the production of the flocculant. A high concentration of calcium ions also significantly increased the mycelial growth and the production of the flocculant. Under the optimum cultural conditions, a 500-fold dilution of the culture filtrate could efficiently flocculate and sediment E. coli cells.     

Dilute solution properties of proteoglycan fractions from bovine nasal cartilage

Fresh proteogycans (adult bovine nasal cartilage) isolated from the densest portion of a dissociative density gradient had a weight-average molecular weight of ca. 10⁶ in 4M guanidine hydrochloride (GdnHCI) by light scattering. Fractions of such material obtained by elution with 4M GdnHCI from 2% agarose gel, both normal and cross-linkd, has proteoglycan subunit molecular weights ranging from 0.8 to 2.6 × 10⁶ and root-mean-square radii ranging from 35 to 52 nm in the same solvent. The protein molecular weight per proteoglycan subunit was about 1.2 × 10⁵ and that of keratan sulfate about 1.8 × 10⁵, both independent of total molecular weight. A random-flight “graft copolymer” model having uniform side chains of chondroitin sulfate (40 disaccharides) and keratan sulfate (15 disaccharides) and a random-coil polypeptide back bone was used to estimate the unperturbed radius, whihc was about 19 nm for a mol wt of 1.5 × 10⁶. Experimental light-scattering data for fractions were fitted very well by theoretical curves for the particale scattering factor for both linear and appropriate branched polymers. Examination of coil expansion on the basis of perturbation calculations for branched polymer models suggested that expansion did not account for the experimentally observed radii in terms of unperturbed radii calculated from the model. A possible explanation is that substantial local stiffening of the polypeptide chain due to substitution of side-chain clusters increases the unperturbed radii. The intrinsic viscosity [η] is 4M GdnHCI ranged from 120 to 180 ml/g, and could be interpreted in terms of th eequivalent sphere model; the Flory number has approximately its normal value for flexible linear polymers. The treatment of the sedimentation coefficient by this is less successful, since the Man delkern-Flory parameter β apparently increases with increasing molecular weight; average value are similar to those for flexible polymers, but the variation in β makes this method useful only for rough estimation of molecular weight of proteoglycans. Molecular weights of purified proteoglycans are the same in 0.2M NaCI as in 4M GdnHCI, while crude preparations gave higher molecular weights in 0.2M NaCI, probably because of association due to incomplete removel of “linking” proteins.     

The proteoglycans of bovine nasal cartilage and human articular cartilage: a sedimentation equilibrium analysis

Cartilage proteoglycan was isolated from bovine nasal septum and fractionated according to buoyant density after dissociative CsCl density gradient centrifugation. Gel-exclusion chromatography showed that hyaluronic acid was present in fractions of density lower than 1.69 g/mL. The molecular weight, assessed by sedimentation equilibrium analysis, of the proteoglycan present in the fractions with density > 1.69 g/mL, which appeared chromatographically homogeneous and constituted 54% of the preparation, ranged from 1.0 to 2.6 × 10⁶ for v = 0.55 cm³ g^?1. Carbodiimide-induced modification of the carboxyl groups by methylamine resulted in a reduction of the molecular weight to 0.74 – 1.25 × 10⁶. An analogous reduction in molecular weight was obtained after equilibration of this proteoglycan fraction with hyaluronic acid oligomers containing five disaccharide units. Since both procedures are known to cause inhibition of the interaction between proteoglycans and hyaluronic acid, it is suggested that this lower molecular-weight range represents the true degree of polydispersity of the sub-units of hyaline cartilage proteoglycan constituting this fraction, while the higher values obtained for the intact proteoglycan are the result of the presence of hyaluronic acid in the sample. The molecular-weight range of the whole proteoglycan subunit preparation, assessed after carboxyl group modification, was 0.5–1.2 × 10⁶. Apparently normal and abnormal cartilage was excised from single human osteoarthrosic femoral heads. Proteoglycans extracted by 4M guanidine hydrochloride were isolated after dissociative density gradient centrifugation and subjected to carboxyl group modification. Preparations from normal tissue exhibited molecular-weight averages ranging from 5 to 9 × 10⁵. A molecular-weight reduction was observed with proteoglycans isolated from abnormal areas.