Involvement of an N-Acetylglucosaminidase in Autolysis of Propionibacterium freudenreichii CNRZ 725

Propionibacterium freudenreichii plays an important role in Swiss cheese ripening (it produces propionic acid, acetic acid, and CO₂). Moreover, autolysis of this organism certainly contributes to proteolysis and lipolysis of the curd because intracellular enzymes are released. By varying external factors, we determined the following conditions which promoted autolysis of both whole cells and isolated cell walls of P. freudenreichii CNRZ 725: (i) 0.1 M potassium phosphate buffer (pH 5.8) at 40°C and (ii) 0.05 to 0.1 M KCl at 40°C. We found that early-exponential-phase cells possessed the highest autolytic activity. It should be emphasized that the pH of Swiss cheese curd (pH 5.5 to 5.7) is near the optimal pH which we determined. Ultrastructural observations by electron microscopy revealed a 16-nm-thick homogeneous cell wall, as well as degradation of the cell wall that occurred concomitantly with cell autolysis. In the presence of 0.05 M potassium chloride, there was a great deal of isolated cell wall autolysis (the optical density at 650 nm decreased 77.5% ± 7.3% in 3 h), and one-half of the peptidoglycan material was released. Finally, the main autolytic activity was due to an N-acetylglucosaminidase activity.     

Auxin-enhanced glucan autohydrolysis in maize coleoptile cell walls

Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1→3), (1→4)-β-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the β-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles.     

Use of secondary ion mass spectrometry to image44calcium uptake in the cell walls of apple fruit

Summary Calcium, an important agent in regulating cell wall autolysis during fruit ripening, interacts with pectic acid polymers to form cross-bridges that influence cell separation. In the present study, secondary ion mass spectrometry (SIMS) was used to determine whether the cell walls of apple fruit were able to take up exogenously applied⁴⁴Ca, which was infiltrated into mature fruit. SIMS, which has the ability to discriminate between isotopes, allowed localization of the exogenously applied⁴⁴Ca and the native⁴⁰Ca. The results indicated that the total amount of calcium present in the cell walls was enriched with⁴⁴Ca and that heterogeneity of⁴⁴Ca distribution occurred in the pericarp. Isotope ratio images showed microdomains in the cell wall, particularly in the middle lamella intersects that oppose the intercellular spaces. These domains may be the key areas that control cell separation. These data suggest that exogenously applied calcium may influence cell wall autolysis.Abbreviations SIMS secondary ion mass spectrometry     

Cell Wall Autolysis During Kiwifruit Development

Cell walls prepared from developing kiwifruits showed autolyticactivity. The proteins extracted from active walls were alsoable to release carbohydrates from inactive cell walls. Analysisof the sugars released, using both procedures, showed that uronicacids were the major component, especially during the firsthours of incubation, although neutral sugars such as glucoseand galactose were also present. Most of the carbohydrates autolyticallyreleased from the cell wall eluted in the void volume on a BioGel P2 column. However, carbohydrates released from inactivecell walls by the protein extract mostly eluted in the monosaccharideuronic acid and glucose peaks. The autolytic activity of isolatedcell walls, as well as the glycosylhydrolase activity of theproteins extracted from the cell walls, showed important changesduring fruit development. The differences between autolyticactivity and the glycosylhydrolase activity against the cellwall suggest that the glycosylhydrolases ‘in muro’are subjected to some regulatory mechanism which disappearswith their extraction. Finally, the role of glycosylhydrolases,such as polygalacturonases and galactosidases, in relation tocell wall changes in fruits, is discussed.Copyright 1998 Annalsof Botany Company Actinidia deliciosa; autolysis; cell wall enzymes; fruit growth; glycosylhydrolases; kiwifruit.     

Phenolic components and degradability of cell walls of grass and legume species

Cell walls separated from the aerial parts of Lolium multiflorum, Lolium perenne and Phleum pratense contained bound cis and trans ferulic and p-coumaric acids and diferulic acid which were released from the walls by treatment with sodium hydroxide. The total content of these acids in L. multiflorum ranged from 5 to 16.8 mg/g of wall, the trans-ferulic acid content varying between 2.8 and 8.9 mg/g of wall. In addition, small amounts of p-hydroxybenzoic acid were released from senescent leaf blade plus sheath parts. Cell walls from legume species gave much smaller amounts of the acids, the total content of aerial parts of Trifolium pratense being <0.8 mg/g of wall. The degra dability of the cell walls with a commercial cellulase preparation was determined and the water-soluble phenolic compounds released were estimated by UV absorption spectroscopy.     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

Sporangila autolysis in Clostridium perfringens type A

The autolytic system functioning in the release of mature spores and enterotoxin from sporangia of Clostridium prefringens was partially characterized. After sporangial autolysis in buffer, the supernatant fluid of the suspension contained autolysin active against purified sporangial walls. The autolysin was most active at pH 8 and 37°C, in the presence of Co²⁺ (0.3 · 10⁻³ M CoCl₂) and trypsin (48 μg/ml). Sodium dodecyl sulfate-treated sporangial walls further extracted with trichloroacetic acid to remove teichoic acid were a better enzyme substrate than walls treated only with sodium dodecyl sulfate. N-Acetylmuramyl-l-alanine amidase activity which released N-terminal alanine, and endopeptidase activity which hydrolysed the d-alanyl-glycine linkage liberating N-terminal glycine and C-terminal alanine, were both functional at pH 8. It is not known if one or two enzyme are involved. Autolysin appeared in cells as early as 2 h after inoculation into sporulation medium. Two asporogenic Stage 0 mutants grown in sporulation medium also produced autolysin identical in mode of action to that of the sporogenic wild type. Although the active cellular autolysin concentration subsequently decreased as cells sporilated, the walls of 8-h-old sporangia containing refractile heat-resistant spores were more susceptible to digestion by autolysin, than those of 2-, 4-, or 6-h-old cells grown in sporulation medium or of 4- or 14-h vegetative cells from growth medium. The results suggest that a progressive change may occur in the structure of the sporangial wall during spore morphogenesis, thus increasing its susceptibility to autolysis.     

A gas chromatographic analysis of the release of arabinose from coleoptile cell walls under the influence of auxin and dextranase preparations

An analysis by gas chromatography of the products of digestion of cell wall pellets of Avena sativa L., coleoptiles shows that after short term autolysis of pellets from auxin-rich plants, arabinose is released. Addition of arabinan as a substrate increases the yield of arabinose considerably, suggesting that a similar substance is broken down in the walls.In pellets from auxin-poor coleoptiles much less arabinose is released and addition of arabinan has no effect. Dextranase preparations stimulate this release and can also break down arabinan. This suggests that auxin and a dextranase-like enzyme are involved in the process.     

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25°C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l⁻¹, respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties. Journal of Industrial Microbiology & Biotechnology (2001) 26, 235–240. Received 02 August 2000/ Accepted in revised form 15 December 2000     

Tomato fruit cell wall : I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins

Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat-inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces β-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exogenous polygalacturonase (4-7%) remained constant throughout fruit development.     

Autolytic Enzyme System of Streptococcus faecalis III. Localization of the Autolysin at the Sites of Cell Wall Synthesis

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 autolyzed in dilute buffers. Walls were isolated from cultures grown in the presence of (14)C-lysine for about 10 generations and then on (12)C-lysine for 0.1 to 0.8 of a generation (prelabeled). These walls released (14)C to the soluble fraction more slowly than they lost turbidity during the initial stages of autolysis. Walls isolated from cultures grown in the presence of (14)C-lysine for only the last 0.1 to 0.4 of a generation (postlabeled) released (14)C to the supernatant fluid more rapidly than they lost turbidity. Autolysin in both pre- and postlabeled walls was inactivated, and such walls were then incubated in the presence of unlabeled walls containing active autolysin. The inactivated walls lost their (14)C label only very slowly until autolysis of the unlabeled walls was virtually complete and release of soluble autolysin was expected. When this experiment was done in the presence of trypsin, a fourfold increase in the autolysis rate resulted, but the same pattern of (14)C release was observed. A parallel release of (14)C and loss of turbidity from pre- or postlabeled walls was observed upon trypsin "activation" and by addition of isolated soluble autolysin to inactivated walls. We conclude that the wall-bound autolysin acts first on the more recently synthesized portion of the wall. Trypsin appears to speed wall autolysis by activating additional latent autolysin in situ at sites in the older portion of the wall.     

Differential effects of pectolytic enzymes on tomato polyuronides in vivo and in vitro

There are marked differences between polyuronide degradation in vivo during tomato ripening and in vitro during the autolysis of cell wall preparations. Experiments using purified enzymes and enzymically inactive wall preparations show that the combined action of polygalacturonase (E.C. 3.2.1.15) and pectinesterase(E.C. 3.1.1.11)can mimic this in vitro autolysis of cell walls. Assuming these two enzymes are also responsible for polyuronide degradation in vivo their combined action must be restricted in some way.     

Autolysis in Staphylococcus aureus: Preferential Release of Old Cell Walls

The kinetics of release of old versus new cell wall in two strains of Staphylococcus aureus were studied during autolysis. In both strains the autolytic enzyme is an amidase. Cells were double labeled with (3)H and (14)C, and the distribution of radioactivity in the cell walls was monitored during autolysis. In all cases the rate of release of steady-state lable from peptidoglycan was significantly higher than that of pulse label. Identical results were obtained with whole cells or isolated cell walls. The results suggest that in S. aureus the old cell wall is preferentially released during autolysis.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Studies on the Pectic Substances of Plant Cell Walls: III. DEGRADATION OF CARROT ROOT CELL WALLS BY ENDOPECTATE LYASE PURIFIED FROM ERWINIA AROIDEAE

Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented.     

Rapid linkage of indole carboxylic acid to the plant cell wall identified as a component of basal defence in Arabidopsis against hrp mutant bacteria

Changes occurring to plant cell walls were examined following inoculation of Arabidopsis leaves with pathogenic and non-pathogenic (hrpA mutant) strains of Pseudomonas syringae pv. tomato. We have targeted low molecular weight, cross-linked phenolic and indolic compounds that were released from wall preparations by alkaline hydrolysis at 70 °C and in a microwave bomb. Significantly higher concentrations of syringaldehyde, p hydroxybenzaldehyde and indole carboxylic acid were recovered from cell walls isolated from leaves 24 h after challenge with the hrpA mutant compared with wild-type DC3000. Time course experiments showed that the accumulation of indole carboxylic acid and the other group of differentiating metabolites had occurred within 12 h of inoculation. The callose synthase deficient mutant pmr4-1 was more resistant than wild-type Columbia plants to P. syringae pv. tomato. Restricted bacterial multiplication was associated with increased accumulation of indole carboxylic acid on the plant cell wall. In the absence of callose deposition in the pmr 4-1 mutant, indolic derivatives may serve as a structural scaffold for wall modifications following bacterial challenge.     

Activity of cell-wall degradation associated with differentiation of isolated mesophyll cells of Zinnia elegans into tracheary elements

Cell walls were prepared from cultured mesophyll cells of Zinnia elegans L. that were transdifferentiating into tracheary elements and incubated in a buffer to undergo autolysis. The rate of autolysis of cell walls was determined by measuring the amount of carbohydrate released from the cell walls into the buffer during incubation. During the course of culture of mesophyll cells, the autolysis rate increased markedly at the time when thickenings of secondary cell walls characteristic of tracheary elements became visible (after 48-72 h of culture), and thereafter the rate remained at a high level. Comparative studies on the autolysis rate of cell walls using various control cultures, in which tracheary element differentiation did not take place, revealed a close relationship between the autolysis rate around the 60th hour of culture and differentiation. Sugar analysis by colorimetric assays and gas chromatography of carbohydrates released from the cell walls detected uronic acid, arabinose, galactose, glucose, xylose, rhamnose, fucose, and mannose. Among these sugars, uronic acid was the most abundant, and accounted for approximately half of the total released sugars. The decrease of acidic polysaccharides in the primary cell walls during tracheary element differentiation was visualized by staining cultured cells with alcian blue at pH 2.5. These results suggest that active degradation of components of primary cell walls, including pectin, is integrated into the program of tracheary element differentiation.     

Cell wall degradation in the autolysis of filamentous fungi

A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes.Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present.In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.     

Characterization and localization of the cell wall autolysis substrate in Pisum sativum epicotyls

The products released in cell wall autolysis from 4-day-old epicotyls of Pisum sativum elute in gel filtration chromatography (Bio Gel P.2) as two components, mono and polysaccharides, in a practically constant ratio over the time of incubation. The polysaccharides are mainly composed of arabinose and galactose, with smaller amounts of xylose and glucose, whereas the monosaccharide are almost exclusively composed of galactose. The same results were obtained when inactive cell walls were hydrolyzed by the enzymes extracted from the cell wall with LiCl. The hydrolysis of the different cell wall fractions by these enzymes shows that the autolytic substrates are preferentially located on the pectic fractions.     

Release of carboxymethyl-cellulase and β-glucosidase from cell walls of Trichoderma reesei

Summary Carboxymethyl-cellulase and -glucosidase activities were determined in the cytosole, cell walls and extracellular culture fluid of Trichoderma reesei QM 9414 cultivated on cellulose and cellobiose. By means of carboxymethylcellulose as a specific desorbens for cellulose bound CM-cellulase and -glucosidase it was found that these enzymes are cell wall bound during consumption of the carbon source, but are excreted during the subsequent cultivation stage. Treatment of intact cell walls with various chemical agents could not cause a release of the enzyme. Treatment of intact cell walls with -mannanase or trypsin released CM-cellulase, whereas, treatment with laminarinase or chitinase released -glucosidase. Both enzymes were also released during autolysis in phosphate buffer. This autolysis was accompanined by the appearance of extracellular mannanase, laminarinase and proteinase. The results suggest that cleavage of chemical bonds of certain cell wall polymers of T. reesei could be responsible for the appearance of CM-cellulase and -glucosidase in the culture fluid during later stages of growth.