Strain Dependence of the Cell-expanding Effect of β-1,3-Glucanase in Yeast

The effect of β-1, 3-glucanase on the cell expansion was studied with diploid strains of Saccharomyces ellipsoideus and S. cerevisiae, and their mutants differing in the response to auxin. The following results were obtained. Cell expansion was induced by β-1, 3-glucanase only in auxin-responsive or potentially auxin-responsive strains. β-1, 3-Glucanase induced cell expansion more rapidly than auxin. The cell wall of the auxin-responsive strain was more susceptible to digestion by β-l, 3-glucanase than that of the auxin-unresponsive one. The sensitivity of yeast cells to auxin action is discussed in relation to the nature of cell wall.     

Effect of Auxin on Cell Wall Degrading Enzymes

The effect of auxin on the activities of amylase, cellulase, β-1, 3- and/or β-l, 6-glucanase and hemieellulase were observed using etiolated barley coleoptile and pea epicotyl internode segments. The activities of β-1, 3- and/or β-l, 6-glueanase and hemicellulase of barley were increased by indole-3-acetic acid in a 3 hours' treatment. Amylase activity was not influenced by the auxin. Cellulase activity was not detected under the experimental conditions. 2, 4-Dichlorophenoxyacetic acid increased hemicellulase activity, but not cellulase and amylase activities, in pea epicotyl segments in 3 hours. Fungal β-1, 3-glucanase exogenously applied induced the elongation of barley coleoptile segments. The elongation induced by the enzyme was as high as that induced by indole-3-acetic acid at least for the first 1 to 3 hours.     

Characterization of spinach leaf α-l-arabinofuranosidases and β-galactosidases and their synergistic action on an endogenous arabinogalactan-protein

Two β-galaclosidases (β-Galase-I and -II, EC 3.2.1.23) and two α-l -arabinofuranosidases (α-l -Arafase-I and -II. EC 3.2.1.55). were purified from mesophyll tissues of spinach (Spinacia oleracea L.), using chromatography on DEAE-cellulose, lactose-conjugated Sepharose CL-4B, and Sephadex G-100, or on hydroxylapatite and Sephadex G-150. The apparent molecular mass (M_r) of β-Galase-I and -II, respectively, were estimated to be 38 000 and 58 000 on SDS-PAGE and 64 000 and 60 000 on gel-permeation chromatography, indicating that the former was a dimeric protein. The isoelectric points of β-Galase-I and -II were 6.9 and 5.2, respectively. Both enzymes hydrolyzed maximally p-nitrophenyl (PNP) β-galactoside at pH 4.3, and were activated about 2-fold in the presence of BSA (100 μg ml^?1). The activity of both enzymes was inhibited strongly by heavy metal ions and p-chloromercuriberszoate (p-CMB). d -Galactono-(1→4)-lactone and d -galactal served as potent competitive inhibitors for the enzymes. β-Galase-I and -II could be distinguished from each other in their relative rates and kinetic properties in the hydrolysis of aryl β-galactosides as well as of lactose and galacto-oligosaccharides. In particular. β-Galase-I exhibited a preferential exowise cleavage of β-1,6-galactotriose and β-1.3-galactan. α-l -Arafase-l (M_r 118000) and -II (M, 68 000) were optimally active on PNP α-l -arabinofuranoside at pH 4.8 and gave K_m values of 1.2 and 2.2 mM. respectively. l -Arabino-(1 → 4)-lactone. Ag⁺, and SDS acted as inhibitors for the isozymes. α-l Arafase-I was characterized by its activity to hydrolyze PNP β-d -xylopyranoside besides PNP α-l -arabinofuranoside. inhibition by d -xylose and d -glucono-(1 → 5)-lactone. and less sensitivity to Hg²⁺. Cu²⁺, and p-CMB. Sugar beet arabinan was hydrolyzed rapidly by α-l Arafase-II at one-half the rate for PNP α-l arabinofuranoside, while the polysaccharide was less susceptible to α-l Arafase-I. A spinach leaf arabinogalactan-protein was practically resistant to the action of β-Galases, but its susceptibility to the enzymes increased remarkably after prior hydrolysis with α-l Arafase-Il.     

The Effect of Gravity Compensation on Growth and Cell Wall-loosening Enzymes in Helianthus annuus Hypocotyls

The effect of gravity compensation by the clinostat on the elongation, weight, and activity of two cell wall-loosening enzymes (cellulase and β-1,3-glucanase) in Helianthus annuus hypocotyls was examined. Gravity compensation increases elongation (28.1 %) and weight (18.3 %). The activity of cellulase extracted from the apical sections is raised, but there is no significant effect on β-1,3-glucanase. The relationship between gravity compensation, changes in auxin level, and function of these two enzymes in respect to elongation is discussed.     

Auxin-Induced Expansion Growth of Cells and Protoplasts of Yeast

Using an auxin-responsive mutant of Sacchairomyces ellipsodeus, expansion growth of cells caused by auxin was studied especially in comparison with that of protoplasts.

• 1 Indole-3-acetic acid induced detectable cell expansion growth in 3 hours in a buffered simple solution where no cell division occurred.
• 2 The auxin-induced expansion growth was inhibited by an antiauxin, trans-cinnamic acid.
• 3 Actinomycin D, chloramphenicol and cycloheximide inhibited the auxin-induced cell expansion growth.
• 4 Protoplasts did not expand in response to auxin under the condition where intact cells did.
• 5 The stability of protoplasts was not changed by the low auxin concentration (20 mg/1) which induced cell expansion.
• 6 High concentrations (100–1000 mg/1) of auxin caused protoplasts to burst even under an osmotically stable condition.

     

Lysis of Yeast Cell Wall by Enzymes from Streptomycetes

This paper deals with yeast cell-wall lytic enzymes formed by Streptomyces with regard to the connection with the cell-wall structure.

In the first place, 29 organisms of β-glucanase-producing Streptomycetes were selected among 777 strains belonging to genus Streptomyces by means of a cylinder-plate method employing the yeast glucan as a substrate. As for these organisms, the depolymerizing activity against the yeast glucan was considered to be mainly due to β-1,3-glucanase activity. Against the heat-treated cell of bakers’ yeast, the crude enzymes merely showed poor lytic activities, however, in the combined employment with some protease preparations, especially with an alkaline protease from St. satsumaensis nov. sp., a remarkable increase of the lytic activities was demonstrated. On the other hand, the intact cell wall of bakers’ yeast, or both the heat-treated and the intact cells of Sacch. cerevisiae 18.29 strain were dissolved very easily by a sole action of β-glucanase or of protease, respectively. In consequence, it seemed that the lysis occurred with different mechanisms in response to differences of substrates. On this subject, the results of investigations and discussions were described in special measure. In addition, the possibility, that some other enzymes than β-glucanase or protease might concern to the lysis of the cell wall, was also investigated and discussed.     

Effect of 2,4-Dichlorophenoxyacetic Acid on Growth and on β-Glucan Synthetases of Peroxyacetyl Nitrate Pretreated Avena Coleoptile Sections

Coleoptile sections from Avena sativa L. were exposed to non-lethal concentrations of peroxyacetyl nitrate (PAN). The sections were then incubated in solutions of 50 mM glucose plus 2.5 mM potassium phosphate with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). Growth after 4 hours was measured. A corresponding series of experiments was carried out and the effect of the 2,4-D treatments on enzymes utilizing uridine diphosphate glucose (¹⁴C-glucose) to form glucolipid and β-glucans including cellulose was determined. Growth in the PAN-treated sections was inhibited less at optimal and superoptimal auxin levels than at low auxin levels. Glucolipid synthetase activity was only slightly inhibited by PAN pretreatment and was reduced by increasing levels of auxin. Responses of alkali-soluble glucan and cellulose synthetases were similar to growth in both control and PAN treated tissues. It was concluded that the earlier reported response of cell wall metabolism in vivo probably is due to effects on these enzyme levels.     

Induction of β-Glucosidase in Botrytis cinerea by Cell Wall Fractions of the Host Plant

The pathogenicity of Botrytis cinerea has been found to correlate positively with the β-glucosidase activity. In this report, the relationship between the induction of β-glucosidase and the components of host plant tissues was studied by the use of tissue fractions and cellulose-related compounds.

The most active enzyme induced by the crude fiber fraction and Avicel was β-glucosidase, among the cell wall degrading enzymes tested. The β-glucosidase was very inducible in the strains with strong pathogenicity, and intensively degraded the fiber fraction made from apple fruit tissues. The same degradation of the cell wall fraction was demonstrated with the purified enzyme.     

Purification,Crystallization, and Properties of an Endo β-1,3-Glucanase from Rhizopus chinensis R-69

An endo β-l,3-glucanase was purified in crystalline form from a culture filtrate of Rhizopus chinensis R-69. Molecular weight of the enzyme was determined to be 23,000 by molecular sieve chromatography and the mode of action of the enzyme was suggested to be a less random type of β-1,3-glucanase. Km and V_max of the enzyme for laminarin are 3.4 g/liter and 1541. U., respectively. The enzyme does not decompose the cell walls of living yeast; it decomposes, however, the preparation of yeast glucan.     

EFFECT OF YEAST SEXUAL HORMONES ON ELONGATION OF AVENA COLEOPTILE

Effect of yeast (Saccharomyces cerevisiae) sexual hormones on the elongation of etiolated Avena coleoptile segments was studied. The elongation was promoted by a hormone excreted by cells of mating type a, but not by α hormone excreted by cells of α type. The effect of the former was as great as that of 5 mg/1 indole-3-acetic acid in the first hour of application. The optimal concentration of a hormone was 10 units/ml. Its growth promoting effect was greatly inhibited by an antiauxin, 2,4,6-trichlorophenoxyacetic acid. a Hormone increased cell wall extensibility just as auxin does. Testosterone, β-estradiol, progesterone and ergosterol showed very little effect on the elongation of coleoptile segments.     

Stress-relaxation Properties of the Avena Coleoptile Cell Wall

Changes in the cell wall properties of Avena coleoptile segments were studied under various conditions by stress-relaxation analysis. Rheological models consisting of four or an infinite number of Maxwell viscoelastic components were used. The stress-relaxation parameters of these models, t₁, t_o, T, Gi and stress/strain ratio, were determined. The following results were obtained. 1. The 1/T₁ increased and stress/strain ratio decreased with the age of the coleoptiles. Decapitation caused a decrease in l/t₁. 2. Auxin increased I/T₁ but decreased t_o and stress/strain ratio within 5 minutes after application. 3. Treatment with a fungal β-l,3-glucanase increased 1/T₁ both in living and methanol-killed, pronase-treated coleoptiles. Cellulase did not cause the changes observed in the parameters of the isolated cell wall of the coleoptile segments. This held true for all treatments (with and without auxin, killed and pronase-treated). The results obtained suggest that auxin primarily causes a partial degradation of the non-cellulosic physaccharide components of the cell wall.     

Effect of Cell Lysis (CLs) Products on Acidophilic Chemolithotrophic Microorganisms and the Role of Acidocella Species

Acidophilic chemolithotrophic microorganisms (CMs) are widely used for bioleaching of mineral resources. However, the growth of bacteria and their leaching activity are often inhibited (restricted) by organic components, e.g. lysates and exudates. The aims of this study were to examine the extent of cell lysis (CLs) inhibition on acidophilic microorganisms and to identify microorganisms that can utilize CLs products and eliminate their inhibition effect on acidophilic microorganisms. Specifically, it was revealed that Acidithiobacillus caldus was severely inhibited at 5% CLs products, whereas A. ferrooxidans and Leptospirillum ferriphilum are severely inhibited at 20%. It has been found that strains RBA and RBB of heterotrophic bacteria, isolated from anaerobic sludge, can biodegrade CLs products and when co-cultured with A. ferrooxidans, they can alleviate the toxic effect of CLs products under low pH (2–3). It has been shown that besides CLs, isolated strains can grow on glucose, glycerol, yeast extract, citric acid, and tryptone soya broth with an optimum temperature of 35°C and a pH of 3. The strains showed the ability to reduce ferric ions to ferrous ions when glycerol was used as a substrate after 2 days under both aerobic and anaerobic conditions. On the basis of morphophysiological and molecular biological studies, the isolated strains RBA and RBB were identified as Acidocella spp.     

Physiological Responses of Tomato Plants Grown in Fusarium Suppressive Soil

Tomato plants grown in a Fusarium wilt suppressive soil and in the same soil steamed and amended with non-pathogenic Fusarium strains were protected from subsequent infection with Fusarium oxysporum f. sp. lycopersici. Protected plants had higher laminarinase, chitinase, N-acetyl-glucosaminidase and β-l, 4-glucosidase activity than unprotected plants grown in steamed unamended soil. Higher peroxidase and polyphenoloxidase activity and higher content of phenols were also associated with suppressive soil conditions, although in a less consistent way. Among 5 non-pathogenic Fusarium strains, strain 5a1 was the most suppressive and enhanced enzyme activity more than did the other strains, whereas strain T was inactive and did not induce changes in activity. These results are in accordance with previous work showing that induced resistance is associated with enhanced activity of glycosidases and phenol oxidizing enzymes, and increased phenols content. It is suggested that induced resistance is part of the mechanism of the natural suppressiveness of soil and that this resistance is induced by non-pathogenic Fusarium strains.     

Altered Expression of Auxin-binding Protein 1 Affects Cell Expansion and Auxin Pool Size in Tobacco Cells

Auxin-binding protein 1 (ABP1) has an essential role in auxin-dependent cell expansion, but its mechanisms of action remain unknown. Our previous study showed that ABP1-mediated cell expansion is auxin concentration dependent. However, auxin distribution in plant tissue is heterogeneous, complicating the interpretation of ABP1 function. In this study, we used cells in culture that have altered expression of ABP1 to address the mechanism of ABP1 action at the cellular level, because cells in culture have homogeneous cell types and could potentially circumvent the heterogeneous auxin-distributions inherent in plant tissues. We found that cells overexpressing ABP1 had altered sensitivity to auxin and were larger, with nuclei that have undergone endoreduplication, a finding consistent with other data that support an auxin extracellular receptor role for ABP1. These cells also had a higher free auxin pool size, which cannot be explained by altered auxin transport. In cells lacking detectable ABP1, a higher rate of auxin metabolism was observed. The results suggest that ABP1 has, beyond its proposed role as an auxin extracellular receptor, a role in mediating auxin availability.     

Protein Turnover in Response to Transient Exposure to Exogenous Auxin is Necessary for Restoring Auxin Autotrophy in a Stressed Arachis hypogea Cell Culture

An auxin autotrophic Arachis hypogea cell culture was sensitive to stress treatments leading to water loss whereas the growth of its auxin-supplemented counterpart was unaffected under similar conditions. Here we show that an hour of transient auxin treatment in the post stress period was sufficient for restoring the auxin autotrophic growth potential of the stress driven quiescent Arachis cells. Qualitative proteome analysis revealed protein turnover to have a role in mediating auxin-originated signals in these cells. In consonance, MG132 a cell permeable inhibitor of the ubiquitin mediated protein turnover completely inhibited the auxin dependent growth restoration of the stressed Arachis cells. Thus protein turnover is a necessary downstream event in exogenous auxin mediated stress tolerance in Arachis cells.     

Susceptibilities of Yeasts to Yeast Cell Wall Lytic Enzyme of Arthrobacter luteus

The susceptibilities of various strains of yeast to a yeast cell wall lytic enzyme produced by Arthrobacter lutens were examined. Twenty six strains of yeasts, mainly in the genera Saccharomyces and Candida were tested. They were tested after growth attained to the logarithmic or to the resting stage in different media (malt extract medium or n-paraffin medium) and various culture conditions (shaking or stationary liquid cultures or agar slopes).

The effects of various treatments, such as heating, or treatment with 2-mercaptoethanol or sodium dodecylsulfate on their susceptibility were also examined.

These various conditions and treatments greatly influenced the susceptibilities of the yeast cells, suggesting that they affected the composition and/or structure of the yeast cell walls.     

Enzymatic Release of Invertase from Cell Ghosts of Candida utilis

1. The liberation of invertase (β-fructofuranosidase, EC 3.2.1.26) from Candida utilis at autolysis of the cells was found to begin after the autolysis was almost completed. The autolysis residue at this stage consisted mainly of cell walls (ghosts). A suspension of washed cell ghosts released invertase on further incubation and this liberation was stimulated by the addition of reducing agents such as mercaptoethanol, or proteolytic enzymes such as papain, as has been known in the release of the invertase of Saccharomyces cerevisiae.

2. The invertase activity of the cell ghosts was not lost when the suspension was heated at 60°C. However, the invertase of the heated cell ghosts was not liberated even if the above stimulative agents were added.

3. Several commercial enzymes were shown to stimulate the liberation of invertase from the heated cell ghosts and “Zymolyase,? one of the effective enzymes, was fractionated. One fraction isolated from the preparation showed a striking effect on the liberation of invertase but this fraction did not show lytic activity on brewer’s yeast cells.     

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell–cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg® led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg® of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg® crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg® crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.     

Additional Volatile Compounds Produced by Pyrolysis of Sulfur-containing Amino Acids

Bacillus circulans WL-12, a yeast and fungal cell wall lytic bacterium, secretes a variety of polysaccharide degrading enzymes into the culture medium. When β-1,3-glucanase was induced with pachyman, a β-1,3-glucose polymer obtained from the tree fungus Poria cocus Wolf, six distinct active molecules of the enzyme with different molecular weights were detected in the culture supernatant of this bacterium. Molecular cloning of one of the β,3-gIucanase genes into E. coli was achieved by transforming E. coli HB101 cells with recombinant plasmids composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pUC 19. A recombinant plasmid containing 4.4 kb of inserted DNA in the Pst I site of pUC 19, designated as pNT003, conferred the ability to degrade pachyman on E. coli cells. The presence of pNT003 was harmful for E. coli cells and caused cell lysis, especially at higher temperatures of cultivation. β,3-Glucanase activity detected in E. coli was mainly recovered in the periplasmic fraction when cell lysis did not occur. SDS-PAGE analysis revealed that the periplasmic fraction contained four active molecules of β-1,3-glucanase which corresponded to four of the six active molecules produced by B. circulans WL-12.     

Distribution and Solubilization of Particulate Gluconate Dehydrogenase and Particulate 2-Ketogluconate Dehydrogenase in Acetic Acid Bacteria

The distribution of two particulate enzymes, gluconate dehydrogenase (GDH) and 2-ketogluconate dehydrogenase (2KGDH), was investigated with cell free extract through 26 strains of genus Acetobacter and genus Gluconobacter. GDH activity was found in the cell free extracts from all strains of genus Gluconobacter and two species of genus Acetobacter, A. aceti and A. aurantium. High activity of 2KGDH was also found in the pigment-producing strains of genus Gluconobacter.

Best solubilization of particulate enzymes was attained with the highest recovery when 10 mg of Triton X–100 and 30 mg of protein of particulate fractions in 1 ml of 0.01 m phosphate buffer, pH 6.0, are incubated for 9 hr at 5°C with continuous stirring.

By comparison of the total enzyme activity of particulate enzymes with that of NAD(P)-linked enzymes in the cell free extract, it was obvious that the formation of ketogluconates by particulate enzymes was much more predominant, roughly over 100 times higher, as that of NAD(P)-linked enzymes.