Microbial lipids and stable foam formation in the activated sludge process.

The presence of fats and oils in sewage has been related to the formation of stable foams in activated sludge treatment systems. Foam forming microbes can utilise and, in some cases, store lipid substrates. Since surface lipids would confer the hydrophobicity necessary for flotation on the sludge biomass, the extractable lipids in foaming and non-foaming biomass samples were examined. Both pure mono-cultures and sludge samples were used. The results showed that, whilst there were some differences in the lipid profiles of the mono-cultures, the different sludge types did not show any significant pattern or variation which could be used as a lipid-based explanation for foam formation.

High-performance liquid chromatography of sialic acid-containing oligosaccharides and acidic monosaccharides.

Many sialic acid-containing oligosaccharides and five acidic monosaccharides have been separated by high-performance anion-exchange chromatography using a Dionex AS6 ion-exchange column eluted with aqueous 50 mM NaOH plus 50-175 mM sodium acetate. Using a pulsed amperometric detector, as little as 50 pmol oligosaccharide can be detected. Many factors, such as the presence of fucosyl groups or sialyl groups, glycosidic linkage positions, and branching structure, can have a tremendous influence on overall acidity of the oligosaccharide, which can lead to excellent separations and make this method an important addition to existing alternatives for the separation of sialic acid-containing oligosaccharides.

Structural analysis of the specific capsular polysaccharide of Rhodococcus equi serotype 2.

The specific capsular polysaccharide produced by Rhodococcus equi serotype 2 is a high-molecular-weight acidic polymer composed of D-glucose, D-mannose, D-glucuronic acid and 3-O-[(S)-1-carboxyethyl]-L-rhamnose in equimolar proportions. Structural analysis, employing a combination of chemical and n.m.r. techniques, established that the polysaccharide is composed of linear repeating tetrasaccharide units. (formula; see text) in which the beta-D-mannose residues carry O-acetyl groups at O-2 and O-3 to the extent of 1.7 mol equivalents. Unequivocal determination of the absolute chirality of the 3-O-[(S)-1-carboxyethyl]-alpha-L-rhamnose residues was achieved by chemical correlation with an authentic synthetic sample. The 1H and 13C-n.m.r. resonances of the native and O-deacetylated serotype 2 polysaccharides were fully assigned by homo- and heteronuclear chemical-shift correlation methods.

Mass spectrometric analysis of O-peracetylated derivatives of 1-monomycoloylglycerol isolated from Corynebacterium pseudotuberculosis, Rhodococcus rhodochrous and R. lentifragmentus.

1-Monomycoloylglycerols from Corynebacterium pseudotuberculosis, Rhodococcus rhodochrous and R. lentifragmentus were reacted with acetic acid ahydride in the presence of pyridine. On infrared spectra the reaction products showed a sharp characteristic absorption of the acetyl ester group at 1235 cm-1; the hydroxyl group absorption (3400 cm-1) was absent. O-Peracetylated monomycoloylglycerols were analyzed by mass spectrometry under electron impact mode. The most common and representative peaks were associated to the following remarkable fragments: (a) peak at m/z 159 represented the backbone of the glycerol unit of peracetylated monomycoloylglycerols; it constituted the diagnostic and base peak of that group of compounds; (b) peak representing the glycerol moiety together with the alpha-subunit of the mycolic acid moiety; it gave the size of the chain length of the hydrocarbon side chain of that mycolic acid; and (c) a series of peaks of acylium ions minus 60 mass units (acetic acid) indicated the size of the chain length of the esterified mycolic acid. Therefore, acetylation of monomycoloylglycerol becomes the derivatized compound suitable for mass spectrometry. Moreover, the derivatives are more thermostable and resistant to pyrolysis. By associating these properties, separation and identification of homologs of monomycoloylglycerols are further expected.

Structural analysis of the specific capsular polysaccharide of Rhodococcus equi serotype 1.

The specific capsular polysaccharide produced by Rhodococcus equi serotype 1 was found to be a high molecular weight acidic polymer composed of D-glucose, D-mannose, and D-glucuronic acid. Structural analysis of the polysaccharide employed a combination of chemical and nuclear magnetic resonance techniques, from which it was determined that the polysaccharide possessed a linear repeating tetrasaccharide unit containing a single O-acetyl substituent and and acetal-linked pyruvic acid moiety: [formula: see text] The 1H and 13C nuclear magnetic resonances of O-deacetylated and pyruvic-free serotype 1 polysaccharides were fully assigned by homo- and hetero-nuclear chemical shift correlation methods.

Induction of interferons (IFNs) and tumor necrosis factor (TNF) in mice by a novel glycolipid trehalose 2,3,6'-trimycolate from Rhodococcus aurantiacus (Gordona aurantiaca).

The immunomodifying activity of a novel mycoloyl glycolipid, trehalose 2,3,6'-trimycolate (GaGM), from a unique psychrophilic acid-fast bacterium, Rhodococcus aurantiacus, was examined. ICR mice were primed intravenously (i.v.) or intraperitoneally (i.p.) with liposomes containing GaGM (300 micrograms/mouse), and were administered LPS dissolved in saline (25 micrograms/mouse, i.v.) 2 weeks later. Two hours after injection of LPS, interferons (IFNs) and tumor necrosis factor (TNF) were induced significantly in mice sera. The increase in activities of IFNs and TNF was approximately paralleled with granuloma formation in spleen of mice primed with GaGM. However, IFNs and TNF were not induced either in mice primed with GaGM but not elicited with LPS, or in those primed with GaGM and elicited by GaGM. Both activities induced were lower in mice primed with trehalose mono- or dimycolate from R. aurantiacus (GaTMM, GaTDM) or TDM from Nocardia rubra than in GaGM-primed mice. Time course study showed that the maximum activity of each interferon (alpha, beta, or gamma) was observed at different stages after LPS administration; IFN-alpha, IFN-beta, and IFN-gamma appeared 3, 2, and 6 hours most abundantly after LPS administration, respectively.

Lipid composition in the classification of Rhodococcus equi.

The fatty acid, menaquinone and polar lipid composition of representatives of Rhodococcus equi and related taxa were determined. All of the R. equi strains had major proportions of straight chain saturated, monounsaturated and 10-methyl branched fatty acids, dihydrogenated menaquinones with eight isoprene units as the predominant isoprenologue, and characteristic polar lipid patterns that contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and glycolipids including a "cord factor"-like compound that was most pronounced in fresh isolates. The mycolic acids of these strains fell within the range C24 to C48, had 0 to 4 double bonds and released major amounts of C14:0 esters on pyrolysis. These lipid data provide further evidence that R. equi strains form a distinct taxospecies within the genus Rhodococcus. The remaining strains also gave lipid profiles consistent with their assignment to the genus Rhodococcus. These organisms included strains identified as R. sputi.

Carotenoid pigments of genus Rhodococcus.

A study of carotenoid pigments of the genus Rhodococcus was carried out. According to carotenes contained, Rhodococcus species were divided into three groups: the first group of Rhodococcus luteus, R. coprophilus, R. lentifragmentus, and R. maris, which formed beta-carotene; the second group of R. equi, R. rubropertinctus, R. aichiensis, R. sputi, R. chubuensis, R. obuensis, R. bronchialis, R. roseus, R. rhodochrous, R. rhodnii, and R. terrae, which formed gamma-carotene-like substance; and the third group of R. aurantiacus, which formed neither carotene. Other carotenoid pigments were different according to the species.

Menaquinone composition in the classification and identification of aerobic actinomycetes.

Menaquinones were the only isoprenoid quinones found in 36 strains representing different species of the genera Nocardia, Mycobacterium, Rhodococcus, Amycolatopsis, Saccharothrix, Streptomyces, Nocardiopsis and Actinomadura. Dihydrogenated menaquinones with nine isoprene units [MK-9(H2)] were the main components isolated from Mycobacterium. Dihydrogenated and tetrahydrogenated menaquinones with eight isoprene units were the predominant compounds identified in typical Rhodococcus and Nocardia strains, respectively. "Nocardia phenotolerans" differed from all of the other Nocardia species included in the study, in that it contained the MK-9(H2) [MK-8(H2)] menaquinone system. Nocardioform bacteria lacking mycolic acids contained tetrahydrogenated menaquinones with nine isoprene units as the main component. The Streptomyces strains studied exhibited complex mixtures of partially saturated menaquinones with nine isoprene units with the hexa- and/or octahydrogenated components predominating. Actinomadurae contained major amounts of hexahydrogenated menaquinones with nine isoprene units. In contrast, the single Nocardiopsis strain examined possessed complex mixtures of menaquinones with ten isoprene units, the dihydrogenated components being main constituents.

High-performance liquid chromatography analysis of mycolic acids as an aid in laboratory identification of Rhodococcus and Nocardia species.

High-performance liquid chromatography analysis of the p-bromophenacyl esters of mycolic acids from whole organisms gave chromatographic patterns that were useful in differentiation of Rhodococcus and Nocardia species. Rhodococcus equi, R. erythropolis, and R. rhodochrous contained more-polar mycolic acids and were easily separated from the less-polar mycolic acid-containing species of R. sputi, R. bronchialis, R. corallinus, R. rubropertinctus, and R. terrae. The less-polar mycolic acid-containing Rhodococcus species showed chromatographic patterns that partially overlapped (in elution times) the patterns of Nocardia asteroides, N. otitidiscaviarum, and N. brasiliensis, but the larger number of peaks in the last species made separation between the genera possible. Distinct chromatographic patterns were found for most species, except for R. equi strains that showed two different patterns. Strains of R. rubropertinctus and R. terrae appeared identical. N. asteroides and N. otitidiscaviarum showed similar mycolic acid patterns.

Polyacrylamide gel electrophoresis of whole-cell preparations of Rhodococcus equi.

The whole-cell proteins of ten strains of Rhodococcus equi isolated from horses, pigs, or humans, including the type strain ATCC 6939, were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein profiles of seven different capsular serotypes and the type strain were very similar when bacteria were cultured under the same conditions. Protein profiles were largely unaffected by incubation at two temperatures (30 degrees C, 37 degrees C) or times (12 h, 48 h). There were generally minor differences in protein profiles between strains grown in different media (brain heart infusion, nutrient, minca broths, tryptic soy-blood agar) with the marked exception of a prominent diffuse 17.5 kd protein which was expressed in nutrient broth. This protein was not produced by the type strain and was lost on repeated passage in vitro (50th, 100th passage) in two of three other strains examined.

Antitumour activity of purified arabinogalactan-peptidoglycan complex of the cell wall skeleton of Rhodococcus lentifragmentus.

Antitumour activity of arabinogalactan peptidoglycan (AP) complex (peptidoglycan and arabinogalactan liberated by an acid or alkaline treatment from Rhodococcus lentifragmentus AN-115 cell wall skeleton) was examined in mice and compared with that of the cell wall skeleton. The growth of syngeneic fibrosarcoma Meth A cells after implantation in BALB/c mice was significantly suppressed by AP complex, and also regressed after intratumoral injection of AP complex on days 1, 4 and 7 after tumour implantation. Although the activity of peptidoglycan was less than that of AP complex, peptidoglycan also showed both tumour-suppressive and regressive activities. Arabinogalactan did not show antitumour activity. It is interesting that peptidoglycan has an important role in the effect against tumours.

Possible existence of a novel amphipathic immunostimulator in the phenol-water extracts of Mycobacteriaceae.

The extracts having diverse immunostimulating activities were obtained as a water-phase fraction from four bacterial species representing the 4 genera (Mycobacterium, Nocardia, Gordona, and Rhodococcus) of Mycobacteriaceae by the phenol-water method, which is commonly used for extraction of endotoxic lipopolysaccharides (LPS) from gram-negative bacteria and amphipathic substances from gram-positives. These fractions, especially those of G. aurantiaca and R. terrae, showed strong stimulatory effects on murine splenocytes, macrophages of mice and guinea pigs, the immunoadjuvant activities in guinea pigs and mice, and the distinct activities inducing a tumor necrosis factor and interferons alpha/beta and gamma in primed mice. The fractions from G. aurantiaca and R. terrae exhibited potent pyrogenicity and the ability to activate the clotting enzyme cascade of the horseshoe crab (Tachypleus tridentatus). Some of these biological activities were not very different from the potency of the reference endotoxic LPS derived from Escherichia coli or Fusobacterium nucleatum. But the test fractions neither showed the activity to prepare rabbit skin to the local Shwartzman reaction, nor reacted with anti-lipid A conventional and monoclonal antibodies. Furthermore, unlike LPS, these fractions stimulated the splenocytes of C3H/HeJ mice (LPS-Nonresponder). Although the fractions showing the above biological activities have not yet been adequately purified, they contained polysaccharides, whose main constituent sugar is mannose with a smaller amount of arabinose, fatty acids consisting primarily of palmitic, stearic, and tuberculostearic acids, and small amounts of peptides and amino sugars. Since components characteristic of known immunomodulators of bacterial origin, namely endotoxins (lipid A's), cell wall peptidoglycans, lipoteichoic acids, cord factors (trehalose dimycolates), or deoxyribonucleic acids, were practically not detected in these fractions, the agent responsible for the above bioactivities is considered to be a novel substance different from the known, bacterial immunomodulators.