Production in vitro, on different solid culture media, of two distinct exopolysaccharides by a mucoid clinical strain of Burkholderia cepacia.

The production of exopolysaccharides (EPSs) by a mucoid clinical isolate of Burkholderia cepacia involved in infections in cystic fibrosis patients, was studied. Depending on the growth conditions, this strain was able to produce two different EPS, namely PS-I and PS-II, either alone or together. PS-I is composed of equimolar amounts of glucose and galactose with pyruvate as substituent, and was produced on all media tested. PS-II is constituted of rhamnose, mannose, galactose, glucose and glucuronic acid in the ratio 1:1:3:1:1, with acetate as substituent, and was produced on either complex or minimal media with high-salt concentrations (0.3 or 0.5 M NaCl). Although this behavior is strain-specific, and not cepacia-specific, the stimulation of production of PS-II in conditions that mimic those encountered by B. cepacia in the respiratory track of cystic fibrosis patients, suggests a putative role of this EPS in a pathologic context.

Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG hsp60 proteins, and to the Escherichia coli homologue GroEL.

Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.

Exopolysaccharide production by mucoid and non-mucoid strains of Burkholderia cepacia.

Thirteen strains of Burkholderia cepacia from various origins with mucoid and non-mucoid phenotypes were assayed for exopolysaccharide (EPS) production. The EPS were characterized by glycosyl composition analysis and examination of the products resulting from lithium-ethylenediamine and Smith degradations. The results showed that all strains, including the non-mucoid strains, were able to produce EPS exhibiting the same structural features, i.e. presence of one rhamnosyl, three galactosyl, one mannosyl, one glucosyl and one glucuronosyl residues, suggesting that this EPS is representative of the B. cepacia species.

Structural elucidation of a novel exopolysaccharide produced by a mucoid clinical isolate of Burkholderia cepacia. Characterization of a trisubstituted glucuronic acid residue in a heptasaccharide repeating unit.

The structure of the exopolysaccharide (EPS) produced by a clinical isolate of Burkholderia cepacia isolated from a patient with fibrocystic lung disease has been investigated. By means of methylation analyses, carboxyl reduction, partial depolymerization by fuming HCl and chemical degradations such as Smith degradation, lithiumethylenediamine degradation and beta-elimination, supported by GC/MS and NMR spectroscopic analyses, the repeat unit of the EPS has been identified and was shown to correspond to the acidic branched heptasaccharide with the following structure: [formula: see text]. This partially acetylated acidic polymer, distinguished by the presence of the less usual D-isomer of rhamnose and of a trisubstituted glucuronic acid residue, could represent the main EPS produced by this bacterial species.

Effects of characterised Pseudomonas aeruginosa exopolysaccharides on adherence to human tracheal cells.

This study evaluated, in vitro, the role of different Pseudomonas aeruginosa exopolysaccharides (EPS) in mediating adherence to human respiratory epithelial cells. Two mucoid and non-mucoid isogenic pairs of P aeruginosa strains isolated from patients with cystic fibrosis (CF) and bronchiectasis were used. Adherence was tested with human tracheal epithelial cell lines from CF and normal fetuses. The CF cells bound significantly more bacteria than the normal cells. The strain from the bronchiectasis patient was significantly more adherent than that from the CF patient and this difference was consistently most marked with the non-mucoid variant and with normal epithelial cells. The differing behaviour of mucoid CF and non-mucoid bronchiectasis strains reflected the chemical composition of their EPS: mainly alginate in the former and neutral polysaccharides in the latter. Additive inhibition experiments with chemically characterised EPS indicated that neutral polysaccharides associated with alginate may act as ligands for the adherence of P. aeruginosa to CF epithelial cells.

Structural elucidation of two polysaccharides present in the lipopolysaccharide of a clinical isolate of Burkholderia cepacia.

Based on the sugar composition, methylation analyses and Smith degradation, supported by NMR spectroscopic analyses and fast-atom-bombardment MS experiments, the lipopolysaccharide produced by a clinical isolate of Burkholderia cepacia was shown to contain two distinct polymers, both with linear trisaccharide repeating units; a major, containing D-rhamnose and D-galactose residues (2:1) with the structure -->3)-alpha-D-Rhap(1-->3)-alpha-D-Rhap(1-->4)-alpha-D-Galp(1 --> (major), and a minor repeating unit, constituted by D-rhamnosyl residues, with the structure -->3)-alpha-D-Rhap(1-->3)-alpha-D-Rhap(1-->2)-alpha-D-Rha p(1--> (minor).

Structural studies of the acidic exopolysaccharide produced by a mucoid strain of Burkholderia cepacia, isolated from cystic fibrosis.

The acidic exopolysaccharide produced by a mucoid strain of Burkholderia cepacia isolated from a cystic fibrosis patient, was purified by cetyltrimethylammonium bromide precipitation and/or anion-exchange chromatography. Based on the sugar composition and permethylation analyses, supported by GLC-MS and NMR spectroscopy analyses, the repeating-unit of the polysaccharide was established as -->3)-beta-D-Glcp-(1-->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Gal p-(1-->.

Influence of nutrient media on the chemical composition of the exopolysaccharide from mucoid and non-mucoid Pseudomonas aeruginosa.

Two mucoid Pseudomonas aeruginosa strains and their non-mucoid revertants isolated from two different clinical origins (cystic fibrosis and bronchiectasis) were grown in various chemically defined media. The extracted exopolysaccharide was characterized by gas-liquid chromatography and 1H-NMR spectroscopy. The exopolysaccharide was always heterogeneous, with an alginate fraction and a neutral fraction essentially composed of glucose, galactose, rhamnose and hexosamines. The alginate composition (mannuronate/guluronate ratio and O-acetylation degree) changed according to the carbon source in nutrient media and whether the strains tested were responding differently to these environmental stimuli. In all cases, the best carbon source for the alginate production was glycerol: the two cystic fibrosis strains produced a predominantly O-acetylated alginate whereas only the mucoid bronchiectasis strain produced a polymannuronate exopolysaccharide.

High heterogeneity of the exopolysaccharides of Pseudomonas solanacearum strain GMI 1000 and the complete structure of the major polysaccharide.

The exopolysaccharide of Pseudomonas solanacearum, which is believed to play an important role in bacterial virulence, was considered by most authors as a homogeneous entity essentially composed of N-acetylgalactosamine. The present work demonstrates the high degree of heterogeneity of this exopolysaccharidic material, which consists of a high molecular weight acidic polysaccharide and a mainly noncarbohydrate structure as major subfractions. Rhamnose-rich polyoside and glucan fractions are also present as minor components. We report the complete structure of the acidic heteropolymer involving, in addition to N-acetylgalactosamine, equimolar ratios of two rare amino sugars, 2-N-acetyl-2-deoxy-L-galacturonic acid and 2-N-acetyl-4-N-(3-hydroxybutanoyl)-2,4,6-trideoxy-D-glucose. The structure of this acidic exopolysaccharide provides the first precise basis for the analysis of the correlation exopolysaccharide structure with pathogenicity in P. solanacearum.

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei.

Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long-chain 3-hydroxyacids, branched at position 2), confirming previous proposals. Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids: di-unsatured mycolates, oxomycolates and wax-ester mycolates (ester of dicarboxymycolic acid and 2-icosanol or 2-octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer-Villiger oxidation step, as has been proposed on the basis of structural considerations. By observing the labelling of the different mycolate pools in the cell, i.e. the organic-solvent-extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cell-wall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall-linked mycolates while wax-ester mycolates appeared first as wall-linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall-linked arabino-galactan.

[Structural and metabolic study of the mycolic acids of Mycobacterium fortuitum]. / Etude structurale et métabolique des acides mycoliques de Mycobacterium fortuitum.

The biosynthesis of mycolic acids was studied in whole cells of Mycobacterium fortuitum. At first the structures of the main mycolates produced by the used strain were established as diunsaturated and epoxymycolates. By using [1-14C]acetate as a radiotracer of the lipid synthesis, it was observed that the turnover of the mycolates during the exponential phase of growth of M. fortuitum is fast enough to make very difficult the identification of their precursors. If the growth of the bacterial cells is stopped or highly diminished, by the removal of a large part of their nutritional medium, mycolate synthesis, in contrast to the synthesis of other fatty acids, is stopped as shown by incubation of the concentrated bacterial culture with [1-14C]acetate. After removal of aliquots of the sedimented bacteria at intervals, during several hours, mycolate synthesis resumes when the cell concentration becomes lighter. In these conditions the sequence of radiolabeling of mycolates and of their potential precursors (tetracosanoate and meromycolates) can be observed. In spite of their low accumulation, tetracosanoate and meromycolates were isolated and purified and their specific radioactivity, after different incubation times, could be measured. The results are in agreement with the hypothesis that meromycolates are condensed with tetracosanoate to produce mycolates. However, because of the large differences of isotopic dilution of these two precursors inside the mycolate molecule, this hypothesis, generally taken as evidence, has to be modified. A hypothetical pathway of the mycolate synthesis is proposed, taking into account all these observations.

Glutamate excretion triggering mechanism: a reinvestigation of the surfactant-induced modification of cell lipids.

Lipid alterations induced by surfactants to trigger glutamate excretion were investigated with an industrial strain of Corynebacterium glutamicum. The lipid composition of this strain was determined for cultures in a synthetic medium and in a complex medium. A distribution of complex lipids between the cell wall and the cell membrane is proposed. Depending on growth conditions, 70-85% of the cell fatty acids had saturated chains in cells grown with surfactants. In the synthetic medium up to 80% of the straight-chain fatty acids may have come from the acylated surfactant added to the induce excretion. In industrial fermentation, the maximal excretion rate corresponded to the highest saturated fatty acid content of cells. It was shown by radioactive labelling in the synthetic medium that the addition of the acylated surfactant induced the degradation of more than 50% of the phospholipids. Some phospholipid synthesis occurred at that time using the surfactant (saturated) fatty acids, but the membrane did nor recover its previous phospholipid content. A model is proposed to explain the mechanism of glutamate excretion triggering by surfactants.

[Study of the biosynthesis of phleic acids, polyunsaturated fatty acids synthesised by Mycobacterium phlei (author's transl)]. / Etude du processus de biosynthèse des acides phléiques, acides polyinsaturés synthétisés par Mycobacterium phlei

Because of their structures, phleic acids (general formula: CH3-(CH2)m-(CH=CH-CH2-CH2)n-CO2H; main component: m = 14, n = 5) cannot be synthesized by the same kinds of enzymatic systems as other natural polyunsaturated fatty acids. By using specifically labelled 14C compounds, we have tested the ability of different molecules to be incorporated in the phleate skeletons by Mycobacterium phlei. The localisation of radioactive carbon atoms has been studied by chemical degradation of labelled phleates, isolation and purification of the degradation products, and determination of their specific radioactivity. When M. phlei cells are incubated with labelled acetate, the unsaturated and saturated parts of the molecules of phleic acids are unequally labelled. The radioactivity of succinate monoester on the one hand and fatty acids (mixture of myristic and palmitic acids) on the other hand, measured after oxidative degradation of phleate esters, shows a constant ratio under definite conditions. Whether [1-14C]acetate or [2-14C]acetate is used for incubation, the same ratio is observed. Therefore acetate is the precursor of the unsaturated part as well as of the saturated part of the phleate molecules. By using labelled fatty acid esters, it has been found that palmitic acid is the precursor of phleates with m = 14, while myristic acid is the precursor of phleates with m = 12. Stearic and eicosanoic acids are not incorporated without degradation. The hypothesis of a condensation of a saturated fatty acid with a preformed polyunsaturated molecule was examined. Search for such a molecule in the lipids of M. phlei gives negative results. Pentaunsaturated phleate arising from palmitate is more abundant than pentaunsaturated phleate arising from myristate, while the reverse is true for hexaunsaturated phleates. These observations make very unlikely such an hypothesis. An elongation process fits well with the observed facts provided that this process involves elongation by two acetate units simultaneously, making elongation by four carbon atoms at a time. Such a requirement would be easily satisfied if two molecules of acetate are condensed together before their utilization in the elongation process. In such a hypothetical process, crotonate would be the most probable substrate of the elongation reaction.