Structural studies of the cell wall polysaccharide of Nocardia asteroides R 399

As with other bacteria belonging to the corynebacteria, mycobacteria, and nocardia group, Nocardia possess in their cell walls a neutral polysaccharide. Structural analysis of the cell wall polysaccharide of Nocardia asteroides R 399 was undertaken. The carbohydrate polymer contained D-arabinose and D-galactose as in mycobacteria. Besides these two carbohydrates we pointed out the occurrence of two additional components: D-glucose and a polyol. This polyol, because of its small amount and its uneasy detection, had been for a long time ignored. It has been proven to be the 6-deoxy-D-altritol or 1-deoxy-D-talitol. The polymer consists of a main strand composed of----5 Araf 1----and----4Galp1----or----5Galf1----; oligoarabinosyl side chains were localized on C3 of an arabinosyl residue. Other shorter ramifications also occur on some galactosyl units. A characterization of the linkage between polysaccharide and peptidoglycan inside the cell wall has also been carried out. The two polymers are joined by a phosphodiester bond which involves 6-deoxyaltritol. As some corynebacteria previously analyzed were also shown to contain mannose (and sometimes glucose), we can conclude that the main skeleton of cell wall polysaccharides of the corynebacteria, mycobacteria, and nocardia group of bacteria is an arabinogalactan; however, individual structural features of the polysaccharide are varying according to the bacterial species. These results might be connected with variations that were observed in immunological analysis.     

A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo

Mycobacterium tuberculosis bacilli exhibit cell wall alterations during in vivo growth. Development of ultrasensitive analytical techniques with high specificities is required to analyze the cell wall of M. tuberculosis isolated from experimental animals because of the low amounts of bacteria available and contamination by host tissue. Here we present a novel methodology to analyze all three major components (mycolic acids, arabinogalactan, and peptidoglycan) of the mycobacterial cell wall from mycobacteria isolated from animal tissue. In this procedure, the cell wall carbohydrates are analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) of alditol acetates, the peptidoglycan by GC/MS (mass spectrometry) analysis of the unique amino acid diaminopimelic acid (after derivatization with isopropyl chloroformate), and the mycolic acids by liquid chromatography (LC)/MS (negative ion) without derivatization. The procedure was designed so that all three analyses could be performed starting with a single sample given the difficulty of preparing multiple aliquots in known ratios. Linkage analysis, including an enantiomeric specific procedure, of the arabinogalactan polymer is also presented. These procedures will enable the determination of the cell wall alterations known to occur in the important nongrowing "dormant" M. tuberculosis present during disease. With some adaptations, the methodology is also applicable to the analysis of small amounts of in vivo grown bacteria of other species.     

Biosynthetic origin of mycobacterial cell wall arabinosyl residues.

Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases. However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known. To obtain information on this issue, mycobacteria growing in culture were fed glucose labeled with 14C or 3H in specific positions. The resulting radiolabeled cell walls were isolated and hydrolyzed, the arabinose and galactose were separated by high-pressure liquid chromatography, and the radioactivity in each sugar was determined. [U-14C]glucose, [6-3H]glucose, [6-14C]glucose, and [1-14C]glucose were all converted to cell wall arabinosyl residues with equal retention of radioactivity. The positions of the labeled atoms in the arabinose made from [1-14C]glucose and [6-3H]glucose were shown to be C-1 and H-5, respectively. These results demonstrated that the arabinose carbon skeleton is formed via the nonoxidative pentose shunt and not via hexose decarboxylation or via triose condensations. Since the pentose shunt product, ribulose-5-phosphate, is converted to arabinose-5-phosphate as the first step in 3-keto-D-manno-octulosonic acid biosynthesis by gram-negative bacteria, such a conversion was then searched for in mycobacteria. However, cell-free enzymatic analysis using both phosphorous nuclear magnetic resonance spectrometry and colorimetric methods failed to detect the conversion. Thus, the conversion of the pentose shunt intermediates to the D-arabino stereochemistry is not via the expected isomerase but rather must occur via novel metabolic transformations.     

Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls

The long-posed question of the nature of the link between the mycolylarabinogalactan and the underlying peptidoglycan of the cell walls of Mycobacterium sp. has been addressed. The insoluble cell wall matrix of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis was partially hydrolyzed with acid either before or after per-O-methylation and the resulting oligosaccharides further derivatized and analyzed by gas chromatography/mass spectrometry. The structures of fragments arising from the reducing end of arabinogalactan demonstrated the existence of the terminal sequence----5)-D-Galf-(1----4)-L-Rhap-(1---3)-D-GlcNAc. Other analyses confirmed the presence of muramyl-6-P within the peptidoglycan of these mycobacteria. Based on the acid lability of the 3-linked GlcNAc unit, the presence of about equimolar amounts of Rhap-(1----3)-D-GlcNAc and muramyl-6-P in an isolated cell wall fragment, and 31P NMR analysis, it was concluded that the GlcNAc residue of the terminal triglycosyl unit of arabinogalactan is joined by 1-O-phosphoryl linkage to the 6-position of some muramyl residues within the peptidoglycan. Thus, it is reasoned that the massive mycolylarabinogalactan of mycobacteria, responsible for aspects of disease pathogenesis and much of the antibody response in infections, is attached to the peptidoglycan framework by the actinomycete-specific diglycosylphosphoryl bridge, L-Rhap-(1----3)-D-GlcNAc-(1----P, perhaps thereby providing a unique target for site-directed chemotherapy of mycobacterial infections.     

An arabinogalactan protein isolated from medium of cell suspension cultures of Silybum marianum (L.)Gaernt

Cell suspension cultures of Silybum marianum secreted polymers extracellularly containing 97% carbohydrates and 3% proteins. Fractionation of polysaccharides by anion-exchange chromatography yielded an unbound neutral fraction composed of glucose, xylose, galactose, arabinose and rhamnose and a bound fraction in which galactose and arabinose were predominantly found. The bound fraction specifically bind to Yariv phenylglycoside suggesting the presence of an arabinogalactan protein (AGP). Further purification of the AGP was done by precipitation of the culture medium with the Yariv reagent. The precipitated AGP eluated as single peak by gel permeation with an average molecular weight of 100. Eighteen aminoacids were detected, Ser, Gly, Glu, Asp, Thr and Hyp being the major ones. Linkage analysis showed terminal and 1,3-linked arabinose and almost all galactose was present in the 1,3-galactopyranoside form. The NMR spectral data revealed residues of galactopyranose and arabinofuranose as constituents of AGP. This study is the first examination of an AGP secreted by S. marianum cells in suspension culture.     

Biochemical characterization of the Mycobacterium tuberculosis phosphoribosyl-1-pyrophosphate synthetase

Mycobacterium tuberculosis arabinogalactan (AG) is an essential cell wall component. It provides a molecular framework serving to connect peptidoglycan to the outer mycolic acid layer. The biosynthesis of the arabinan domains of AG and lipoarabinomannan (LAM) occurs via a combination of membrane bound arabinofuranosyltransferases, all of which utilize decaprenol-1-monophosphorabinose as a substrate. The source of arabinose ultimately destined for deposition into cell wall AG or LAM originates exclusively from phosphoribosyl-1-pyrophosphate (pRpp), a central metabolite which is also required for other essential metabolic processes, such as de novo purine and pyrimidine biosyntheses. In M. tuberculosis, a single pRpp synthetase enzyme (Mt-PrsA) is solely responsible for the generation of pRpp, by catalyzing the transfer of pyrophosphate from ATP to the C1 hydroxyl position of ribose-5-phosphate. Here, we report a detailed biochemical and biophysical study of Mt-PrsA, which exhibits the most rapid enzyme kinetics reported for a pRpp synthetase.     

Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses

The peptidoglycan-bound arabinogalactan of a virulent strain of Mycobacterium tuberculosis was per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides reduced and O-pentadeute-rioethylated. The per-O-alkylated oligoglycosyl alditol fragments were separated by high pressure liquid chromatography and the structures of 43 of these constituents determined by 1H NMR and gas chromatography/mass spectrometry. The arabinogalactan was shown to consist of a galactan containing alternating 5-linked beta-D-galactofuranosyl (Galf) and 6-linked beta-D-Galf residues. The arabinan chains are attached to C-5 of some of the 6-linked Galf residues. The arabinan is comprised of at least three major structural domains. One is composed of linear 5-linked alpha-D-arabinofuranosyl (Araf) residues; a second consists of branched 3,5-linked alpha-D-Araf units substituted with 5-linked alpha-D-Araf residues at both branched positions. The non-reducing terminal region of the arabinan was characterized by a 3,5-linked alpha-D-Araf residue substituted at both branched positions with the disaccharide beta-D-Araf-(1----2)-alpha-D-Araf. 13C NMR of intact soluble arabinogalactan established the presence of both alpha- and beta-Araf residues in this domain. This non-reducing terminal motif apparently provides the structural basis of the dominant immunogenicity of arabinogalactan within mycobacteria. A rhamnosyl residue occupies the reducing terminus of the galactan core and may link the arabinogalactan to the peptidoglycan. Evidence is also presented for the presence of minor structural features involving terminal mannopyranosyl units. Models for most of the heteropolysaccharide are proposed which should increase our understanding of a molecule responsible for much of the immunogenicity, pathogenicity, and peculiar physical properties of the mycobacterial cell.     

Isolation of polysaccharides from callus culture of Lemna minor L

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minor L.) with water and ammonium oxalate. Residues of galactose and arabinose in the 2.0-2.5:1 ratio were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glucuronic acids, galactose, and arabinose. The percentage of arabinogalactan and pectin was similar. The yield of polysaccharide fractions did not depend on the method for their isolation. Extraction with water, treatment of the biomass with an aqueous solution of formalin and diluted hydrochloric acid, and extraction with an aqueous solution of ammonium oxalate allowed us to obtain the highest-purity pectin polysaccharide.     

The effect of ultraviolet radiation on the growth and polysaccharide composition of a callus culture of Silene vulgaris

Ultraviolet radiation (wavelength, 280-315 nm; power, 0.2-13.0 W/m2; exposure, 1 or 3 h) was shown to change the growth of campion callus and the polysaccharide (pectin and arabinogalactan) composition of cell walls. An increase in the concentration of polysaccharides and a decrease in the content of arabinose and galactose residues in pectin and arabinogalactan were noted. For the majority of calluses, growth indices, specific growth rate, and biomass productivity (per 11 medium) were almost the same as in nonirradiated control cells. Maximum values of the growth index and specific growth rate, determined for dry biomass, were observed at a low dose of irradiation (0.2 W/m2) and an exposure of 3 h. A considerable decrease in the content of arabinose and galactose in pectin was noted at high doses of irradiation (exposure, 3 h). Samples of arabinogalactan were characterized by variable arabinose to galactose ratios, which were in the range 1 : (3.4-8.3).     

Arabinogalactan utilization in continuous cultures of Bifidobacterium longum: effect of co-culture with Bacteroides thetaiotaomicron

Studies showed that the plant cell wall polysaccharide arabinogalactan supported growth of Bifidobacterium longum in batch culture. Galactose was also utilized, but not arabinose, the other major constituent sugar of the polymer. Enzymes required for hydrolysis of arabinogalactan ('arabinogalactanase', alpha-arabinopyranosidase, beta-galactosidase) were inducible and cell-associated in B. longum, and their expression was repressed by glucose. Considerable amounts of alpha-arabinopyranosidase and beta-galactosidase were synthesized during growth on arabinogalactan, but only low levels of arabinogalactanase were detected. B. longum only grew on arabinogalactan in continuous culture under putative carbon-excess conditions. In C-limited chemostats, the bifidobacterium could not establish unless Bacteroides thetaiotaomicron was present in co-culture. The relationship between the two organisms was not simply commensal; at low specific growth rates, bacteroides cell population densities were approximately 30% lower than those recorded in axenic culture, indicating the existence of competitive interactions with the bifidobacterium. In contrast, at high specific growth rates, a mutualistic association was observed, in that Bact. thetaiotaomicron was maintained in the chemostats at high dilution rates if bifidobacteria were also present. Measurements of residual carbohydrate in spent culture fluid from C-limited chemostats indicated that a large part of the arabinogalactan molecule could not be broken down by either B. longum or Bact. thetaiotaomicron alone, or in co-culture. Formate and acetate were the major fermentation products of B. longum cultured in the presence of high concentrations of arabinogalactan, confirming that these bacteria were growing under energy-limited conditions.     

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.     

Detailed structural and quantitative analysis reveals the spatial organization of the cell walls of in vivo grown Mycobacterium leprae and in vitro grown Mycobacterium tuberculosis

The cell wall of mycobacteria consists of an outer membrane, analogous to that of gram-negative bacteria, attached to the peptidoglycan (PG) via a connecting polysaccharide arabinogalactan (AG). Although the primary structure of these components is fairly well deciphered, issues such as the coverage of the PG layer by covalently attached mycolates in the outer membrane and the spatial details of the mycolic acid attachment to the arabinan have remained unknown. It is also not understood how these components work together to lead to the classical acid-fast staining of mycobacteria. Because the majority of Mycobacterium tuberculosis bacteria in established experimental animal infections are acid-fast negative, clearly cell wall changes are occurring. To address both the spatial properties of mycobacterial cell walls and to begin to study the differences between bacteria grown in animals and cultures, the cell walls of Mycobacterium leprae grown in armadillos was characterized and compared with that of M. tuberculosis grown in culture. Most fundamentally, it was determined that the cell wall of M. leprae contained significantly more mycolic acids attached to PG than that of in vitro grown M. tuberculosis (mycolate:PG ratios of 21:10 versus 16:10, respectively). In keeping with this difference, more arabinogalactan (AG) molecules, linking the mycolic acids to PG, were found. Differences in the structures of the AG were also found; the AG of M. leprae is smaller than that of M. tuberculosis, although the same basic structural motifs are retained.     

An alternate carbon source for enhancing production of polysaccharides by Silene vulgaris callus

Pectin termed silenan and acidic arabinogalactan were isolated as cell-wall polysaccharides of Silene vulgaris callus in the presence of various carbon sources as components of the media. The maximum yields, productivity per litre of medium and production per day of acidic arabinogalactan, were achieved using glucose or galactose as the carbon source. Sucrose was found to increase the production of the polysaccharides. Yields, productivity and rate of production of arabinogalactan per day were decreased in the presence of arabinose. Yields of silenan, productivity and rate of production per day were closely related irrespective of the sugar used as the carbon source in the media (sucrose, glucose or galactose) and yields of silenan from the callus growing on arabinose were comparable. A concentration of sucrose in the 20-50 g/L range enhanced the biosynthesis of silenan and at 50 g/L the silenan contained the linear backbone and the ramified regions of the macromolecule.     

The effect of ultraviolet radiation on the growth and polysaccharide composition of a callus culture of Silene vulgaris

Ultraviolet radiation (wavelength, 280–315 nm; power, 0.2–13.0 W/m²; exposure, 1 or 3 h) was shown to change the growth of campion callus and the polysaccharide (pectin and arabinogalactan) composition of cell walls. An increase in the concentration of polysaccharides and a decrease in the content of arabinose and galactose residues in pectin and arabinogalactan were noted. For the majority of calluses, growth indices, specific growth rate, and biomass productivity (per 11 medium) were almost the same as in nonirradiated control cells. Maximum values of the growth index and specific growth rate, determined for dry biomass, were observed at a low dose of irradiation (0.2 W/m²) and an exposure of 3 h. A considerable decrease in the content of arabinose and galactose in pectin was noted at high doses of irradiation (exposure, 3 h). Samples of arabinogalactan were characterized by variable arabinose to galactose ratios, which were in the range 1: (3.4–8.3).     

Enzymatic degradation of cell wall polysaccharides from soybean meal

Soybean meal, soybean water unextractable solids (WUS) and extracts thereof, which contain particular cell wall polysaccharides, were incubated with a number of cell wall degrading enzymes. The intact cell wall polysaccharides in the meal and WUS were hardly degradable, while the extracts from WUS were well degraded. The arabinogalactan side chains in the pectin-rich ChSS fraction (Chelating agent Soluble Solids) could to a large extent be removed from the pectins by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase and arabinofuranosidase B. The remaining polymer was isolated and represented 30% of the polysaccharides in the ChSS fraction. Determination of the sugar composition showed these polymers to be very highly substituted pectic structures. It still contained 5 mol% of arabinose and 12 mol% of galactose, representing 7% and 12%, respectively, of the arabinose and galactose present in the ChSS fraction before degradation. Further, the presence of uronic acid (50 mol%) and of xylose (18 mol%) indicated the presence of a xylogalacturonan.     

Production and release of peptidoglycan and wall teichoic acid polymers in pneumococci treated with beta-lactam antibiotics

Autolysin-defective pneumococci treated with inhibitory concentrations of penicillin and other beta-lactam antibiotics continued to produce non-cross-linked peptidoglycan and cell wall teichoic acid polymers, the majority of which were released into the surrounding medium. The released cell wall polymers were those synthesized by the pneumococci after the addition of the antibiotics. The peptidoglycan and wall teichoic acid chains released were not linked to one another; they could be separated by affinity chromatography on an agarose-linked phosphorylcholine-specific myeloma protein column. Omission of choline, a nutritional requirement and component of the pneumococcal teichoic acid, from the medium inhibited both teichoic acid and peptidoglycan synthesis and release. These observations are discussed in terms of plausible mechanisms for the coordination between the biosynthesis of peptidoglycan and cell wall teichoic acids.     

Response of the leaf cell wall to desiccation in the resurrection plant Myrothamnus flabellifolius

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.     

Modification of polysaccharides from callus culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G. using carbohydrases <Emphasis Type="Italic">in vitro</Emphasis>

Polysaccharides (pectin and intracellular and extracellular arabinogalactans) were isolated from campion callus culture cultivated on medium with varied concentrations of pectinase and beta-galactosidase. A decrease in contents of arabinose residues in pectin and arabinogalactans and of galactose residues in arabinogalactans was associated with an increase in the activities of alpha-L-arabinofuranosidase and beta-galactosidase upon addition of pectinase into the medium. Pectinase destroyed the high-molecular-weight (more than 300 kD) fraction of pectin and decreased the content of galacturonic acid residues. alpha-L-Arabinofuranosidase transformed arabinogalactan into galactan, and galactan was destroyed under the influence of galactosidase. The contents of arabinogalactan and/or galactan in the cells were decreased, and it was released into the culture medium. Pectin samples with low contents of arabinose and galactose in the side chains and galactan samples were obtained from the callus grown on the medium with beta-galactosidase. Cultivation of the plant cells on medium containing carbohydrases resulted in modification of pectin and arabinogalactan of the cell walls.     

Influence of ultraviolet-C on the compositions of cell-wall polysaccharides and carbohydrase activities of Silene vulgaris callus

UV-C irradiation (254 nm) was found to enhance the secretion of some cell-wall-degrading enzymes, especially the following carbohydrases: beta-galactosidase, alpha-L-arabinofuranosidase, polygalacturonase, pectinesterase, cellulase, xylanase, and beta-xylosidase, in the campion callus, contributing thereby to an alteration in the polysaccharide structure. The relative amounts of the galactose and arabinose residues in pectin (silenan) and of arabinose in arabinogalactan of calli irradiated during the exponential phase were shown to decrease during the stationary phase. A decrease in the degree of SV methylesterification was found for the irradiated callus. These alterations were found to persist over a long period of culturing time. Decreasing the relative amounts of the arabinose residues in arabinogalactan and pectin and the galactose residues in silenan corresponded to increasing activity of alpha-L-arabinofuranosidase and beta-galactosidase, respectively, due to treatment with UV-C. UV-C irradiation may be used as a tool for modifying the structural features of the cell-wall polysaccharides, such as the relative amounts of galactose and arabinose residues in the side chains of polysaccharides, with the purpose of obtaining physiologically active polysaccharides with the desired properties and structural features.     

Purification and Properties of Several Galactanases of Bacillus subtilis var. amylosacchariticus

Two crystalline and one highly purified galactanases were obtained from the culture broth of Bacillus subtilis var. amylosacchariticus (1043) and their chemical and enzymatic properties, especially, their specificities were comparatively studied. Their molecular weights were almost the same, but the isoelectric points were considerably different from each other. The galactanases were sensitive to metal chelators and stabilized by Ca²⁺. The pH optimum of the enzymes were between 6.0 and 7.0. All the galactanases investigated here attacked soybean arabinogalactan without liberation of arabinose, though they were inactive against coffee bean arabinogalactan. In digestion of soybean arabinogalactan, all the galactanases purified here formed galactose, galactobiose and galactotriose whereas the galactanase previously isolated from Bacillus subtilis K–50 produced galactobiose as the main final product.