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.

Interconversion of radical and nitrone forms of lysodektose--a new trisaccharide from Micrococcus lysodeikticus.

Isolated from Micrococcus lysodeikticus, 6-O-(2-deoxy-2-(N-methyl)hydroxilamino-beta-D-glucopyranosyl)-alph a-alpha- trehalose (lysodektose) is oxidized by K3Fe(CN)6 in a stepwise manner to become a nitroxyl radical and a nitrone with a double bond in the fragment O-N = CH2 which could be reduced to the original hydroxylamine form with sodium borohydride. Thus derivatives of lysodektose specifically labelled with 2H and (or) 3H in the methyl group are easily obtained. When oxidized in cells poisoned with vitamin K analogues, lysodektose is transformed into nitrone concomitant with modification of its methyl group. Participation in the antioxidant defence of the bacteria is suggested for this new trisaccharide.

Polymer length of teichuronic acid released from cell walls of Micrococcus luteus.

Teichuronic acid released from its phosphodiester linkage to peptidoglycan in the cell walls of Micrococcus luteus by mild acid treatment is resolved into a ladderlike series of bands by electrophoresis on polyacrylamide gels in the presence of borate. Each band of the ladder differs from its nearest neighbor by one disaccharide repeat unit, ----4)-2-acetamido-2-deoxy-beta-D-mannopyranuronosyl-(1----6)- alpha-D-glucopyranosyl-(1-. Acid-fragmented teichuronic acid, after conversion to the phenylamine derivative, was fractionated by preparative-scale molecular sieve column chromatography, which produced a series of elution peaks. Fast-atom-bombardment mass spectrometry of the smallest member of the series determined its molecular weight and established its identity as the phenylamine derivative of one disaccharide repeat unit of teichuronic acid. Homologous fractions of the same series were used to index the ladder of bands obtained by polyacrylamide gel electrophoresis from samples containing a more extensive distribution of polymer lengths. Nearly native teichuronic acid consists of polymers with a broad range of molecular sizes ranging from 20 to 55 disaccharide units. The most abundant species are those which have 25 to 40 repeat units. Prolonged treatment of teichuronic acid with the acid conditions used to release it from peptidoglycan causes gradual fragmentation of the teichuronic acid.

Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus.

Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB3H4 followed by complete acid hydrolysis yielded [3H] glucosaminitol from the true reducing end residue of teichuronic acid and [3H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.

[Polyamines in representative taxa of coryneform and other bacteria]. / Poliaminy predstavitelei nekotorykh taksonov korinepodobnykh i drugikh bakterii.

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.

Chemical treatments of the cell packets induced from a Micrococcus luteus mutant and teichuronic acids on the packet surface.

Cell packets (MT packets) induced from a tetrads-forming mutant (strain MT) of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of MT packets with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the MT packets, mediated with the antiserum, was also observed with SEM. Gold particles were observed uniformly on the whole packet surface and also on the bridging structure formed by the outermost layer of the cell wall. Mild acid treatment, NaIO4-NaBH4 treatment and mild Smith degradation of the MT packets extremely decreased the agglutinability and binding of protein A-gold particles. The treatments gave a little influence on the surface feature and appreciably destroyed the regular packet structure. It was supposed that teichuronic acids distributed uniformly on the whole packet surface, naturally on the surface of the bridging structure too, and appreciably participated in the maintenance of the regular packet structure.

Determination of G + C content of DNA using high-performance liquid chromatography for the identification of staphylococci and micrococci.

The guanine-plus-cytosine (G + C) content of different species of Staphylococcus and Micrococcus was determined by high-performance liquid chromatography. Purified bacterial DNA was hydrolysed by nuclease P1. The nucleotides were separated by chromatography and quantified by measurement of the optical density at 260 nm. The G + C content of staphylococci ranged from 31.5 to 37.9 moles %, and that of micrococci from 68.7 to 75.2. Most of our results were comparable to those obtained with the thermal method.

Quantitative determination of phospholipids using the dyes Victoria blue R and B.

Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm. Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes. This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube. Because of its high sensitivity (about 24.00 OD units/mumol of phosphatidic acid and about 10.25 OD units/mumol of other phospholipids), specificity, and simplicity, the proposed phospholipid assay appears to be very useful for rapid analyses of lipid extracts as well as TLC spots or suspensions of biological materials, as demonstrated for membranes and cells of Micrococcus lysodeikticus. The applicability of the dye Victoria blue B to the quantitative determination of phospholipids, except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, at 605 nm using chloroform/ethylene glycol/glycerol/water and pentane (hexane)/ethyl acetate/isopropanol/water biphasic solvent systems with similar sensitivities and of sodium dodecyl sulfate in the pentane-containing system with high sensitivity (22.96 OD units/mumol) is also shown. The adaptation of this phospholipid assay to the determination of phospholipases C and D and to the differential quantitation of choline-containing phospholipids using additional phospholipid estimation techniques is discussed.

Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus.

The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.

Determination of the A-T content of DNA by second-derivative ultraviolet spectroscopy.

A method for the determination of the A-T content of DNA based on second-derivative ultraviolet spectra is presented. It allows measurement in a wide range of pH values, ionic strengths, and buffer media. It is nondestructive for the sample and requires not more than 10 micrograms of DNA.

Cell surface of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface.

Micrococcus luteus IFO 3333 cells, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. The surface of a M. luteus cell consisted of two or three areas with borders--the rough and the smooth areas, or the rough, the slightly rough, and the smooth areas; fluffy materials were clearly seen in the rough area. Gold particles were observed uniformly and densely on the whole cell surface. However, either mild acid treatment or mild Smith degradation of the cells altered the fluffy rough area to a rough one, and extremely decreased the agglutinability and the binding of protein A-gold particles. Teichuronic acids appeared to be distributed uniformly on the whole cell surface of M. luteus IFO 3333.

An economical large-scale procedure to purify Micrococcus plasmid DNA.

A reproducible and economical procedure for obtaining a large yield of highly purified covalently closed circular (ccc) plasmid DNA from an industrially important strain of Micrococcus is described. The procedure adopted here departs in several ways from commonly used protocols for isolation of plasmid DNA from Gram (positive) and Gram (negative) bacteria. The plasmid DNA prepared by this procedure is free of contaminants and is pure enough to be used for electron microscopy, DNA transformation, sequencing, in vitro transcription and mutagenesis.

Pitted keratolysis. The role of Micrococcus sedentarius.

Though pitted keratolysis of the foot is generally viewed to be caused by bacteria, there is confusion regarding the identity of the causative organism. Species of Corynebacterium, Actinomyces, Dermatophilus, and Micrococcus have been proposed by various investigators. We have studied eight cases of pitted keratolysis and have cultured an organism identified as Micrococcus sedentarius on the basis of colonial morphology, micromorphology, biochemical reactions, and chemical analysis of whole-cell components. Furthermore, pitted keratolysis was produced experimentally in a human volunteer by applying M sedentarius under an occlusive dressing onto the surface of the heel.

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.

Fatty acids and carbohydrate-containing lipids in four Micrococcaceae strains.

Fatty acid composition and lipidic carbohydrate to lipidic phosphorus molar ratio of yellow pigmented micrococci are compared to red pigmented ones and may be summarized by three indexes. These bacteria show wide differences in their fatty acid composition: three strains possess saturated branched chain fatty acids and one has unsaturated straight chain ones. A significant increase in 'anteiso/iso indexes' is observed between pink (M. roseus) and yellow colored bacteria (M. lysodeikticus, S. lutea). There is no significant difference (P greater than 0.05) between the 'unsaturation indexes' of the red pigmented parental D. radiodurans strain and its colorless mutant. Radioresistant strains exhibit a higher 'carbohydrate/phosphorus index' than other strains. There seems to be a relationship between a high carbohydrate-containing lipid content and a high resistance to physical and chemical agents, in particular to radiations. These differences observed in the lipid composition have implications in taxonomy and in establishing an evolutionary scheme.

Mannose incorporation and lectin recognition of pronase-sensitive components in Micrococcus lysodeikticus (M. luteus) membranes.

Isolated cytoplasmic membranes from Micrococcus lysodeikticus were able to incorporate [14C]mannose from GDP-[14C]mannose. Labelled mannose remained in the membrane fraction after its repeated washing and lipid extraction. Sodium dodecyl sulfate gel electrophoresis in 12% acrylamide showed a set of bands with molecular weights ranging from 230 000 to 19 000 which stained for protein and carbohydrate, and incorporated [14C]mannose. Some of these bands reacted with different lectins (concanavalin A, wheat germ agglutinin and ricin). Furthermore, the mannose was incorporated via a glycosylation pathway similar to that followed in eukaryotic system as shown by the preliminary identification of a lipid intermediate transferring the sugar to proteins and by the differential sensitivity to bacitracin and tunicamycin. These complex membrane components were sensitive to digestion with pronase. All the results presented suggest their glycoprotein nature.

High levels of glycolipid and low levels of phospholipid in a marine caulobacter.

Studies of the lipid composition of the marine bacterium Caulobacter halobacteroides revealed the presence of glycolipid as the predominant lipid constituent. The presence of minor amounts of phospholipid was confirmed with the incorporation of 14C- and 32P-labeled compounds. Other marine caulobacters had similar lipid compositions. Five chromatographically separable glycolipids were detected, two of which were identified as mono- and diglycosyldiglycerides. Glycolipid constituted 90 to 99% of the total extractable lipid based on 14C-acetate incorporation into six marine caulobacter strains. In addition, comparisons were made with the lipid extracts of the nonmarine Caulobacter crescentus and Micrococcus lysodeikticus, which contain substantial amounts of phospholipid. Studies of lipid composition during growth showed the maximum amount of phospholipid during early logarithmic growth (2.9%) with a decrease to 0.3% in the early stationary phase. The finding of a group of organisms in which phospholipid is not a major constituent of the lipid fraction is unique and generates many questions about the lipid requirements for membrane structure and function.

The phosphate diester linkage of the peptidoglycan polysaccharide moieties of Micrococcus lysodeikticus cell wall.

The external polysaccharide is a major component of Micrococcus lysodeikticus cell wall and displays distinct composition. The complete structure of the external polysaccharide had been elucidated as a basis for investigation of the cell wall structure-function relation. However, the mode of attachment of the polysaccharide to the peptidoglycan through a phosphodiester was not clear due to limitations in structural and biosynthetic studies. The present study describes purification of a lysozyme-resistant nondialyzable high-molecular-weight fragment of cell wall and identifies the sugar, D-glucose, as the point of external polysaccharide attachment to the peptidoglycan through a phosphate diester. Kinetic studies for the acid-catalyzed release of external polysaccharide from the peptidoglycan were performed in parallel with synthetic [methyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-alpha-D- glucopyranoside-6-yl]-alpha-D-glucopyranosyl phosphate and alpha-D-glucopyranosyl phosphate and showed the presence of a phosphodiester linkage between external polysaccharide and peptidoglycan. In addition, type of phosphate residue and cross-linking between muramic acid and protein part have been determined.