One-step purification of bacterial lipid macroamphiphiles by hydrophobic interaction chromatography.

Bacterial lipid macroamphiphiles extracted with phenol/water can be purified in one step by hydrophobic interaction chromatography. Lipids and the major part of protein are separated from macroamphiphiles during phenol/water extraction. Coextracted nucleic acids, polysaccharides, and residual protein are effectively removed by column chromatography on octyl-Sepharose whereby macroamphiphiles are primarily adsorbed and later eluted with a buffered propanol gradient. The procedure is applicable to macroamphiphiles with various lipid structures as was demonstrated using the diacylglycerol-containing lipoglycan of Micrococcus luteus, the lipid A-containing lipopolysaccharide of Salmonella typhimurium, and the diglyceryl tetraether lipoglycans of Thermoplasma acidophilum and Thermoplasma volcanicum. On elution from octyl-Sepharose, separation into molecular species of different compositions was observed with the lipopolysaccharide of S. typhimurium and the lipoglycan of T. volcanicum. It was also shown that, after phenol/water extraction, membrane lipids are completely recoverable from the phenol layer, which makes it possible to isolate lipids along with macroamphiphiles from the same sample of bacteria.

Structural and immunochemical characterization of a ribosomal protein from gram-positive Micrococcus luteus which is functionally homologous to Escherichia coli ribosomal protein S1.

Ribosomes from gram-positive Micrococcus luteus contain an acidic protein (ML-S1). ML-S1 has been purified by chromatography of ribosomes on a poly(U)-Sepharose column and the purified protein has a mobility in sodium dodecyl sulphate/polyacrylamide gels similar to that of ribosomal protein S1 of Escherichia coli (apparent Mr 72,000). Protein ML-S1 reacted with E. coli anti-S1 serum with an immunological partial-identity reaction. ML-S1 also reacted with antibodies raised against two structural domains of E. coli S1 (the N-terminal ribosome-binding domain and central and C-terminal nucleic-acid-binding domain). Weak reaction with antiserum to the nucleic-acid-binding domain of E. coli S1 was observed. ML-S1 was digested with trypsin under mild and exhaustive conditions. Mild digestion resulted in the production of a trypsin-resistant core (ML-S1F1) like E. coli S1. The fragment pattern obtained after exhaustive digestion differed appreciably from that obtained with E. coli S1. ML-S1 bound to poly(U) as strongly as E. coli S1 and also showed appreciable binding to denatured DNA. Addition of ML-S1 to S1-depleted ribosomes from E. coli and M. luteus markedly stimulated the poly(U)-directed polyphenylalanine synthesis. Phage MS2-RNA-dependent translation was also found to be stimulated by ML-S1 although to a much lesser extent than the stimulation by E. coli S1. At a molar excess of ML-S1 to ribosomes the protein showed a similar inhibitory effect to E. coli S1 on polypeptide synthesis. Our data indicate that ML-S1 retained the structural domains important for its function despite certain structural differences from E. coli S1.

Characterization of mesosomes of Micrococcus luteus: isolation and properties of mesosomal ribosomes, and localization of penicillin-binding proteins in mesosomal membranes.

Mesosomes were isolated and purified from Micrococcus luteus under hypertonic conditions throughout preparation processes. The purified mesosomal preparation was composed of closed tubules and vesicles. Electron-dense ribosome-like particles were observed within the isolated mesosomal vesicles by electron microscopy. The ribosome-like particles were isolated from the purified mesosomes by a procedure involving solubilization of the membranes with detergents followed by centrifugation on a linear density gradient of sucrose. The isolated particles have a sedimentation coefficient of 70S in the presence of 10 mM Mg2+, when Mg2+ concentration was lowered to 0.1 mM, the particles were dissociated into two sub-particles of 30S and 50S. The 70S particles had the same appearance as cytoplasmic 70S ribosome particles upon observations of negatively stained preparations. These findings indicate that mesosomal tubules contain ribosomes. The isolated mesosomal ribosomes had the ability for protein synthesis when polyuridylic acid-directed polyphenylalanine synthesis was assayed. The sensitivity of mesosomal ribosomes to inhibitors, chloramphenicol and streptomycin, for protein synthesis was significantly lower than that of both cytoplasmic and cytoplasmic membrane-bound ribosomes. In addition, three penicillin-binding proteins were detected in the mesosomal membranes. One of these was localized predominantly in the mesosomal membranes and the other two were distributed almost equally in both mesosomal and cytoplasmic membranes.

Isolation and characterization of a succinylated polysaccharide from the cell wall of Micrococcus agilis.

A polysaccharide consisting of rhamnose, galactose, glucosamine and ester-linked succinic acid was extracted from the isolated cell walls of Micrococcus agilis by the hot water-phenol and 5% trichloroacetic acid (TCA) extraction methods. The hot water-phenol extractable polysaccharide accounted for 30% of the weight of the wall, with 23% by the TCA method. Phosphorus contents were less than 0.01% of the polysaccharide. Succinyl residues released by alkali treatment (0.1 N NaOH, 30 min, 37 degrees C) were identified by gas-liquid chromatography, and accounted for 6.3% and 5.1% of the polysaccharide purified from the hot water-phenol and TCA extracts, respectively. The polysaccharide was not bound when chromatography on Concanavalin A-Sepharose 4B (Con A/Sepharose 4B) columns was performed and it could thus be separated from any residual membrane lipomannan. The purified polysaccharide behaved as a negatively-charged polymer on electrophoresis in 1% agarose (at pH 8.6). A strong cross-reaction, unaffected by removal of the succinyl groups, was observed with type XXIII pneumococcal polysaccharide antiserum indicating the presence of L-rhamnose, linked through non-reducing, lateral end groups.

Mechanism of rat liver DNA methyltransferase interaction with anti-benzo[a]pyrenediol epoxide modified DNA templates.

We investigated the methylation reaction catalyzed by 1500-fold purified rat liver DNA methyltransferase (DMase) on native Micrococcal luteus DNA (ML-DNA) and poly(dC-dG) templates containing covalently bound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the strongly carcinogenic, principal metabolite of benzo[a]pyrene. Since eukaryotic DNA methyltransferases recognize the dinucleotide 5'd[CG] in DNA as a substrate for methylation, the model polynucleotide poly(dC-dG) was used to study in more detail the mode of interaction and effect on incorporation. With either of these BPDE-modified templates, a progressive inhibition of methylation was correlated with increasing amount of BPDE substitution. The effect of BPDE-dG adducts did not alter the apparent km with respect to the concentration of d[CG] in either unmodified or BPDE-modified poly(dC-dG) (km = 10 microM) but lowered the relative apparent Vmax. In assays in which perturbation by salt of preformed enzyme-DNA complex is measured, no change in the relative stability to either unsubstituted or the carcinogen-modified template was noted, thus, excluding any change in the ionic component of this interaction. However, in competition-type experiments, BPDE-DNA is an inhibitor of the methylation reaction on native DNA. When BPDE-DNA is allowed to interact with the enzyme before the addition of native competitor DNA, the methylation rate is not stimulated, suggesting very tight hydrophobic binding of the enzyme to BPDE-DNA and an inhibition in the dissociation of DMase from the template following a methylation event.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immunochemical determination of DNA damage induced by high doses of ionizing radiation]. / Opredelenie immunokhimicheskim metodom povrezhdenii, voznikaiushchikh v DNK v rezul'tate deistviia vysokikh doz ioniziruiushchei radiatsii.

It was shown by the immunochemical method that DNA of X-irradiated E. coli cells of a radiosensitive mutant ABA88uvr A6 can react with the antiserum to thymine dimers which, in all appearance, are induced by ionizing radiation in bacterial DNA. The number of thymine dimers in DNA of E. coli AB1886uvr A6 increased with the dose increase. No dimers were detected in radioresistant cells of M. radioproteolyticus probably due to the effective excision thereof.

[Localization of polyphosphates in cells of microorganisms using 31-P-NMR-145, 75 MHz of high resolution]. / Issledovanie lokalizatsii polifosfatob v kletkakh mikroorganizmov metodom 31-P-iaMP-145, 78 MGts vysokogo razresheniia.

Using the method of 31P-NMR of high resolution at 145,78 MHz the presence of mobile inorganic polyphosphates in the cells of actinomycetes (Mycobacterium smegmatis), yeasts (Candida albicans and Endomyces magnusii) and bacteria (E. coli) was established. A considerable increase in the intensity and a low field shift of the polyphosphate signal after addition of EDTA to the M. Smegmatis cells can be indicative of possible localization of inorganic polyphosphates in the periplasmic space. The lack of effect of exogenous EDTA and Mn2+ on the polyphosphate signal in the spectrum of E. magnusii cells is probably due to localization of polyphosphates inside the cells. A comparison of 31P-NMR spectra of living cells and bacterial extracts of Staphylococcus aureus, Micrococcus lysodeikticus, Bacillus antracoides, B. megaterium and Salmonella typhimurium is given.

Solubility characteristics of Micrococcus lysodeikticus membrane components in detergents and chaotropic salts analyzed by immunoelectrophoresis.

In order to evalute the effectiveness and selectivity of various reagents in the solubilization of bacterial membranes, membranes of Micrococcus lysodeikticus were treated with detergents and chaotropic agents. The composition of the extracts so obtained was analyzed by rocket and two-dimensional immunoelectrophoretic techniques. Recoveery of succinate-, malate-, and reduced nicotinamide adenine dinucleotide- (NADH) dehydrogenases, ATPase, succinylated lipomannan and cytochromes in the extracts was measured. Treatment with a variety of non-denaturing detergents produced extracts that were generally qualitatively uniform although quantitative differences were observed. The degree of extraction of various components was correlated with the hydrophile-lipophile balance. Several chaotropic agents were also evaluated as reagents for membrane solubilization. These agents were less effective in extraction of bulk protein, but produced extracts enriched in some membrane components.

Interaction of phenosafranine with nucleic acids and model polyphosphates. III. Heterogeneity in phenosafranine interactions with DNA base pairs.

Fluorescence and circular dichroism spectral measurements, thermal denaturation studies and binding competition experiments with netropsin and actinomycin D were carried out in systems containing phenosafranine bound to DNA's differing in base composition. The investigated properties exhibit a heterogeneity related to the content of A.T and G.C pairs in DNA and to the nature of phenosafranine binding modes. At low level of saturation of binding sites (r less than 0.1) phenosafranine does not show strong preference for any of the DNA base pairs in the overall binding. However, the strong monomer non-cooperative binding outside the helix (mode I1) occurs predominantly, even though not exclusively in G.C rich regions. The strong binding modes involving intercalated dye molecules (mode I2 and eventually mode II1) prevail in A.T rich regions. These binding modes become the principal types of strong phenosafranine interaction with DNA when the level of saturation of binding sites increases, i.e. at r greater than 0.1.20

Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans.

The complexity of the genome of Micrococcus radiodurans was determined to be (2.0 +/- 0.3) X 10(9) daltons by DNA renaturation kinetics. The number of genome equivalents of DNA per cell was calculated from the complexity and the content of DNA. A lower limit of four genome equivalents per cell was approached with decreasing growth rate. Thus, no haploid stage appeared to be realized in this organism. The replication time was estimated from the kinetics and amount of residual DNA synthesis after inhibiting initiation of new rounds of replication. From this, the redundancy of terminal genetic markers was calculated to vary with growth rate from four to approximately eight copies per cell. All genetic material, including the least abundant, is thus multiply represented in each cell. The potential significance of the maintenance in each cell of multiple gene copies is discussed in relation to the extreme radiation resistance of M. radiodurans.

Primary structure of a high potential iron sulfur protein from a moderately halophilic denitrifying coccus.

The occurrence of a specific high potential iron-sulfur protein in a halophilic, denitrifying coccus provisionally assigned to the genus Paracoccus has been confirmed through primary structure determination. This protein, known as "HiPIP", has been found previously only in photosynthetic bacteria. The sequence of this 71-residue protein is as similar to the HiPEPs from photosynthetic bacteria as they, in turn, are to one another (average number of identically matching residues is 38%). Paracoccus HiPIP is a highly acidic protein in accord with general observations on the proteins of halophilic bacteria. A possible explanation for its occurrence in Paracoccus is that gene transfer is involved.

A membrane-associated lipomannan in micrococci.

Membranes of Micrococcus lysodeikticus, Micrococcus flavus and Micrococcus sodonensis contain acidic lipomannans. Lipoteichoic acids could not be detected in these organisms, and the suggestion that they are substituted for by the lipomannans is strengthened by the chemical and physical resemblances between the two polymers. The mannans contain glycerol, ester-linked fatty acids and mono-esterified succinic acid residues, giving them both hydrophobic and charged properties. The M. lysodeikticus mannan has a chain of about 60 hexose units with two branch points, and is joined at its reducing end to the 1-position of a glycerol moiety bearing two fatty acid residues. Succinic acid on the mannan enables it to bind Mg2+ efficiently, and the polymer is firmly associated with the cytoplasmic membrane, probably by intercalation of its fatty acids with those of the membrane lipids.