Phospholipids of a bone matrix calcification nucleator.

Phospholipids of a bone matrix calcification nucleator are identified as mono and diphosphoinositides and phosphatidyl serine. The nucleator, a protein-phospholipid complex, was dissociated by acidified-solvent porous-glass column chromatography. Analysis was by gas-liquid chromatography.

Characterization of Bacterionema matruchotii calcification nucleator.

The nucleator of Bacterionema matruchotii calcification was characterized. Parameters examined were: proteolipid purity and singularity, amino acid composition and relative polarity, phospholipid composition, apoprotein homogeneity, essentiality of the complex for nucleation, and ordered structure. The data fulfill a requirement for comparisons among apatite-nucleating proteolipids.

New antibiotics from genetically engineered actinomycetes. I. 2-Norerythromycins, isolation and structural determinations.

Novel macrolide antibiotics have been isolated from a genetically manipulated actinomycete. The major components produced have been isolated and identified as 2-norerythromycins A, B, C and D by mass spectrometry and extensive 1D and 2D NMR experiments.

A new aminoglycoside antibiotic G-367 S1, 2'-N-formylsisomicin fermentation, isolation and characterization.

A new aminoglycoside antibiotic, G-367 S1 (2'-N-formylsisomicin, C20H37N5O8) produced by a rare actinomycetes, Dactylosporangium thailandense G-367 (FERM-P 4840) has been isolated by column chromatography on a cation-exchange resin. G-367 S1 is active against Gram-positive and Gram-negative bacteria.

[Teichoic acid from the cell wall of Actinomadura carminata--a producer of the antibiotic carminomycin]. / Teikhoevaia kislota kletochnoi stenki Actinomadura carminata-- produtsenta antibiotika karminomitsina.

The cell walls of Actinomadura carminata, producing the antibiotic carminomycin, contain a poly(glycerol phosphate) teichoic acid. The polymer belongs to 1,3-type and consists of about 8 glycerol phosphate units, two of them have 2-acetamido-2-deoxy-alpha-D-galactopyranosyl substituent and one--3-O-methyl-beta-D-galactopyranosyl-(1----3)-2- acetamido-2-deoxy-alpha-D-galactopyranosyl residue at C2 of glycerol. The structure of the polymer was established by chemical analysis and 13C-NMR spectroscopy. The teichoic acid accounted for about 10% of the cell wall dry weight. 3-O-methylgalactose in the structure of the teichoic acid was found for the first time.

[Heterogeneity of the genus Actinomadura Lechevalier a. Lechevalier]. / O geterogennosti roda Actinomadura Lechevalier a. Lechevalier

Studies of the morphology of Actinomadura spp. have shown that the genus comprises several morphological types of actinomycetes which differ by the presence and structure of the sporophores on the aerial and substrate mycelium. The actinomycetes differ also by the fatty acid composition of lipids in the mycelium, and can be subdivided into three groups: (1) fatty acids with a straight chain prevail; (2) fatty acids with branched chains of the iso- and anteiso structure predominate; and (3) fatty acids with straight and branched chains are contained in almost equal amounts. Therefore the genus separated on the basis of differences in the composition of the cell wall was heterogeneous according to the properties used in the taxonomy of the actinomycetes at the level of genus. These data prove that morphological properties have to be taken into account in the taxonomy of the actinomycetes.

Bacterial phospholipid analysis by fast atom bombardment mass spectrometry.

The structural analysis of naturally occurring bacterial phospholipids in mixtures by fast atom bombardment (FAB) mass spectrometry are reported. The bacterial strains examined included several genera of actinomycetes, two strains of Escherichia coli, and one strain each of Proteus mirabilis and Pseudomonas aeruginosa. FAB mass spectrometry proved to be a useful tool for the structural identification of phospholipids in mixtures and provided stable pseudo-molecular ions and characteristic fragment ions which permitted the identification of phosphatidylethanolamine and phosphatidyl choline. Information regarding the chain length of the fatty acids, their degree of unsaturation in the chains and the presence of hydroxyl groups was also obtained. The results obtained by FAB mass spectrometry were supported by high-resolution mass spectral data, tandem mass spectrometric studies and FAB mass spectrometry of components which had been separated and partially purified by thin-layer chromatography. Each organism displayed a highly characteristic phospholipid profile suggesting the possible use of FAB mass spectrometry as a method for rapid bacterial detection and identification.

Co-isolation of proteolipids and calcium-phospholipid-phosphate complexes.

This study demonstrates that calcium-phospholipid-phosphate complexes (CPLX) and calcifiable proteolipid are associated in vivo by establishing that they can be co-isolated from calcified bacteria. Both of these membrane constituents, which support apatite formation in vitro, have been isolated independently from Bacterionema matruchotii. However, isolation of proteolipid was preceded by demineralization in 2N formic acid, thereby dissociating bound Ca, whereas isolation of CPLX included sonication of calcified bacteria in 2:1:1.5 chloroform:methanol:Tris buffer, thereby dissociating any protein. Co-isolation is possible by demineralizing the calcified bacteria with 50 mM phthalic acid, pH 5.5, followed by extraction with 2:1 chloroform:methanol, and precipitation of crude phospholipid with acetone. CPLX and proteolipid are present in all Sephadex LH-20 chromatographic fractions of the crude phospholipid and of diethyl ether precipitates of the crude phospholipid. CPLXs contain protein:phospholipid:Ca:Pi but differ in relative composition from each other and from independently isolated CPLX. The Ca:phospholipid:Pi molar ratio of diethyl ether precipitable proteolipid-CPLX is most similar to previously published values for CPLX. The protein content of CPLX accounts for all of the proteolipid apoprotein in each Sephadex LH-20 fraction.

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii.

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-14C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C32:0 mycolic acid by radio-gas-liquid chromatographic (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-14C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C34:0 + C34:1) mycolic acids and the other to (C36:0 + C36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C32:0 mycolic acid synthesized by [1-14C]palmitic acid was pyrolyzed at 300 degrees C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-14C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from 14C-palmitate whereas cerulenin, a specific inhibitor of beta-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-14C]fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.

Fatty and mycolic acid composition of Bacterionema matruchotii and related organisms.

Whole-organism methoanolysates of bacterionemae contained mycolic acids in addition to other long-chain fatty acids. These mycolic acids were similar in general structure and overall size to those found in strains of Corynebacterium diphtheriae and Corynebacterium xerosis. The long-chain fatty acids of bacterionemae, mainly straight-chain saturated and unsaturated acids, were similar to those of certain coryneform bacteria including C. diphtheriae. On the basis of these lipid data, and results of earlier studies, we recommend that the genus Bacterionema be transferred from the family Actinomycetaceae to the Coryneform Group of Bacteria.

Effects of growth conditions on the ion composition of Bifidobacterium bifidum subsp. pennsylvanicum.

The cation content of Bifidobacterium bifidum subsp. pennsylvanicum was markedly influenced by the washing procedure of the cells, by the growth phase and the temperature, and by the composition of the culture medium. Optimal retention of cations was achieved by washing with 0.25 M MgCl2 at 20C. The intracellular Na+ concentration rose during growth in normal medium to a constant value in the stationary phase, the K+ concentration rose in the exponential phase, but fell in the stationary phase. Cells from 29-C cultures contained more Na+ and less K+ in the stationary phase than did cells from 37-C cultures, but the total cation content was the same at 29 and 37C. Intracellular Na+ and K+ concentrations were dependent on the concentrations in the medium and on its osmolarity. The intracellular Na+/K+ ratio varied from 0.04 to 2.3. The concentrations of Na+,K+ and phosphate in the medium hardly affected growth. Mg2+-deficiency of the medium markedly decreased the concentration of Mg2+ within the cell; its concentration in the cell sap was greatly affected, but the amount of sedimentable, bound Mg2+ only slightly. The content of K+ within the cell decreased in Mg2+-deficient medium, but the concentration of Na+ did not. Omission of Tween 80 as well as its substitution by Tween 20 caused a decrease of intracellular K+. Cells from Tween 40 and Tween 60 cultures additionally contained markedly less Na+.

Lipid and wall amino acid composition in the classification of Rothia dentocariosa.

Seven strains of Rothia dentocariosa were degraded by acid methanolysis and the nonhydroxylated fatty acid methyl esters released were examined by thin-layer and gas chromatography. The fatty acid profiles were composed of iso-, anteiso- and straight chain saturated fatty acids with 12-methyltetradecanoic (anteiso-C15), 14-methylpentadecanoic (iso-C16), 14-methylhexadecanoic (anteiso-C17) and hexadecanoic acid (C16) as major components. A small scale integrated procedure was used for the sequential extraction of isoprenoid quinones and polar lipids. The latter were examined by two-dimensional thin-layer chromatography and all of the test strains contained diphosphatidylglycerol, phosphatidylglycerol and two uncharacterised glycolipids. In all cases the major isoprenoid quinones were unsaturated menaquinones with seven isoprene units. Analyses of the cell wall amino acid composition using gas chromatography showed that the strains contained 2.5 to 5 moles of alanine and 1 mole each of glutamic acid and lysine. The chemical data support the integrity of Rothia dentocariosa and can be used to separate it from all other actinomycetes especially those which contain lysine in the wall peptidoglycan.

Relationship between proteolipids and calcium-phospholipid-phosphate complexes in Bacterionema matruchotii calcification.

Calcium-phospholipid-phosphate complexes (Ca-PL-P) were isolated from calcified and uncalcified Bacterionema matruchotii and its calcified lipid extracts. Similar complexes were absent from the noncalcifying bacterium Actinomyces naeslundii. The majority of the Ca-PL-P complexes were associated with the proteolipid acidic phospholipid component. Ca-PL-P complexes isolated from B. matruchotii and from calcified proteolipid contained phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-diphosphate, and phosphatidylserine. They consisted of approximately 52 mole % Ca, 32 mole % organic P, and 15 mole % Pi. During Ca-PL-P extraction from B. matruchotii or its proteolipid-containing calcified lipid extracts, the proteolipid was dissociated and the apoprotein precipitated as fluff at the aqueous-organic solvent interface, thus explaining the failure to detect protein in Ca-PL-P preparations. When the ability of Ca-PL-P complexes and lipid fractions of B. matruchotii to initiate apatite formation from metastable calcium phosphate solution was compared, the yield of hydroxyapatite decreased as follows: Ca-PL-P greater than proteolipid acidic phospholipids greater than proteolipid greater than crude phospholipid greater than total lipids greater than whole cells.

Effects of growth conditions on the lipid composition of Bifidobacterium bifidum subsp. pennsylvanicum.

Lipid-phosphorus and lipid-galactose content and phospholipid and fatty acid composition of Bifidobacterium bifidum subsp. pennsylvanicum were examined under a wide variety of growth conditions. Cells from 29-C cultures contained less lipid-phosphorus than did cells from 37-C cultures, but their lipid-galactose content and phospholipid composition did not differ. At both temperatures, the growth phase influenced the lipid composition similarly. Phosphate, Mg2+ and K+ concentrations in the medium did neither significantly change the cellular lipid-phosphorus content nor the phospholipid composition. Only Mg2+-deficiency markedly reduced growth and lowered the content of cellular lipid-galactose. Omission of Tween 80 from the medium did not affect growth, but lowered the content of lipid-galactose and augmented those of lipid-phosphorus and diphosphatidylglycerol in the cell. Increased osmolarity and substitution of other Tween for Tween 80 caused the same changes in lipid composition, and besides inhibited growth. Omitting Tween 80 and replacing it by other Tweens dramatically reduced the percentage of unsaturated fatty acids. C12- and C14-fatty acids made up about 50% of total fatty acids in cells from Tween 20 cultures and 12-14% in cells from Tween 40 and Tween 60 cultures. The differences in the decline of unsaturated fatty acids and in the degree of replacement of these acids by C12- and C14-fatty acids may be related to the variations in growth in cultures with various Tweens by way of changes in the physical state of the membrane lipids.

On the specificity of the uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: diamino acid ligase of Bifidobacterium globosum.

The peptidoglycan of Bifidobacterium globosum contains ornithine and lysine alternately in the same position of the peptide subunit. The uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: diamino acid ligase of this organism was purified 700-fold. Since the activities for the incorporation of ornithine and lysine into uridine diphospho-N-acetylmuramyl-tripeptide did not separate during purification and since the incorporation of ornithine is competitively inhibited by lysine and vice versa, both ornithine and lysine are assumed to be incorporated by one single enzyme. Studies on the specificity of the ligase toward analogs of ornithine have shown that the enzyme requires a diamino, monocarboxylic acid with 4-6 carbon atoms. Methylation of the epsilon-amino group or hydroxylation of the delta-carbon atom of lysine decreases the competitive properties of the analog, whereas the substitution of the gamma-methylen group by sulfur (S-2-aminoethyl cysteine) results in a highly competitive compound.