Alain Asselin

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.

Antifungal activity of chitosan on post-harvest pathogens: induction of morphological and cytological alterations in Rhizopus stolonifer

The effect of chitosan preparations with different levels of deacetylation and other polyanions on the growth of post-harvest pathogens was investigated. Chitosan markedly reduced the radial growth of all the fungi tested, with a greater effect at higher concentration. Chitosan was more effective than N,O-carboxymethylchitosan, polygalacturonate or d -glucosamine, and its inhibitory activity appeared to increase with the level of deacetylation. In addition to inducing cellular leakage of amino acids and proteins in Botrytis cinerea and Rhizopus stolonifer, chitosan also caused morphological changes in R. stolonifer. The ultrastructural study showed that chitosan caused deep erosion of the cell wall as well as increasing the cell-wall thickness. Although chitosan treatment did not affect chitin distribution in R. stolonifer wall, it stimulated the activities of chitin deacetylase, an enzyme involved in the biosynthesis of chitosan. This may well upset the balance between biosynthesis turnover of chitin, thereby rendering the cell wall more viscoelastic.

Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli

SummarySeveral hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed inE. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.

Chitinase, chitosanase and β-1,3-glucanase activities in Allium and Pisum roots colonized by Glomus species

Abstract By using a two-dimensional polyacrylamide gel electrophoresis (PAGE) system, one major additional band with chitinase activity could be detected in Allium porrum (leek) root extracts after colonization by Glomus versiforme or Glomus intraradix when compared to control root extracts. After separation under native conditions in the first dimension, this band was observed both in the Davis system (designed to separate native acidic or neutral proteins) and in the Reisfeld system (designed to separate basic proteins). After the second dimension in sodium dodecyl sulfate (SDS)-PAGE under non-reducing conditions, its apparent molecular mass was determined to be about 30 kDa. In Glomus fasciculatum -colonized root extracts of Allium cepa (onion), four additional bands with chitinase activity were found. The same additional bands were obtained in onion roots colonized by the other Glomus species. Among the four bands, one of them was separated in the first dimension in both the Davis system and the Reisfeld system, two were only detected as acidic or neutral isoforms, while the last one was present as a basic isoform. Their apparent molecular masses were estimated at 33, 35 and 50 kDa. In vesicular-arbuscular mycorrhizae (VAM)-colonized pea root extracts, one acidic chitinase isoform was strongly stimulated while another acidic isoform was less highly induced in the various mycorrhizal interactions. Their apparent molecular masses were 30 and 47 kDA. Chitosanase activities were detected in leek and onion roots colonized by the different VAM fungi, while no chitosanase was observed in either non-mycorrhizal or VAM-colonized pea roots extracts. In VAM-colonized leek roots, the main chitosanase activity had an estimated apparent molecular mass of 20 kDa as determined by SDA-PAGE under non-reducing conditions. Endomycorrhizal onion roots exhibited three chitosanases with estimated apparent molecular masses of 14, 20 and 35 kDa, the two latter being induced by VAM fungal colonization. Finally, no induction of β-1,3-glucanase activity was detected in VAM-colonized roots of leek and onion. In pea roots, one β-1,3-glucanase activity was revealed, but it was not present in all VAM interactions.

Some pathogenesis-related proteins are chitosanases with lytic activity against fungal spores.

Chitosanases represent another type of hydrolase in IF extracts of stressed plant tissue with activity toward a specific fungal cell wall polysaccharide. This is the first report of extracellular stress-related plant chitosanases distinct from chitinases that can be lytic to fungal spores of some plant pathogens

Immunogold localization of beta-1,3-glucanases in two plants infected by vascular wilt fungi.

An antiserum raised against a purified tobacco beta-1,3-glucanase (PR-N) was used to study the subcellular localization of enzyme in fungus-infected plant tissues by means of post-embedding immunogold labeling. In susceptible tomato plants, the enzyme accumulation was found to occur as a result of successful tissue colonization, whereas it appeared to be an early event associated with limited spread of the fungus in resistant tissues. Although marked differences between susceptible and resistant tomato cultivars were observed in the rate of production of beta-1,3-glucanase, the pattern of enzyme distribution was similar. The enzyme was found to accumulate predominantly in host cell walls and secondary thickenings of xylem vessels. By contrast, a very low amount of enzyme was associated with compound middle lamellae. The occurrence of beta-1,3-glucanase at the cell surface of invading fungi was an indication of their possible antifungal activity. A low enzyme concentration was detected in vacuoles of both healthy and infected tissues. In infected eggplant tissue, the pattern of beta-1,3-glucanase distribution was similar to that observed with tomato. Whether these hydrolases accumulate first in vacuoles and are subsequently conveyed toward the outside to participate in fungal wall lysis remains to be determined.

Diversity of cucumber chitinase isoforms and characterization of one seed basic chitinase with lysozyme activity

Abstract Up to six bands with chitinase activity could be detected in seeds or young seedlings after native polyacrylamide gel electrophoretic (PAGE) separation of proteins at pH 8.9 in the Davis system. Two root-specific acidic chitinases were found between 2 and 11 days after germination. Up to eight bands with chitinase activity were observed after native PAGE separation of proteins at pH 4.3 in the Reisfeld system. Since one chitinase activity migrated with the lowest electrophoretic mobility in both polyacrylamide gel systems, a total of 13 chitinases could be found. Seeds (0, 1 and 2 days after germination) were the best source of basic chitinase activities. The strongest activity had a relative mobility ( R m ) value of 0.80 when compared to the mobility of purified hen egg white lysozyme set at 1.00. This activity ( R m 0.80) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography and preparative PAGE. The enzyme had an estimated molecular weight of 27 000 after sodium dodecyl sulfate (SDS)-PAGE. It exhibited lysozyme activity as determined by lysis of Micrococcus cells. Its lysozyme activity was optimal at pH 4.5 and at low ionic strength. Its chitinase activity was typical of an endochitinase type as estimated by the relative efficiency of hydrolysis of 4-methylumbelliferyl-chitotriose versus 4-methylumbelliferyl-chitobiose Cucumber seeds thus contain a highly basic endochitinase/lysozyme which disappears almost completely 3 days after germination. The role of this seed hydrolase has yet to be determined.

Attempted localization of a substrate for chitinases in plant cells reveals abundant n acetyl d glucosamine residues in secondary walls

Ultrathin sections of healthy and fungus‐infected plant tissue were treated with either wheat‐germ agglutinin (WGA) ovomucoid‐gold complex or microbial chitinase‐gold complexes for localizing putative chitin‐like macromolecules. Fungal cell walls, known to contain chitin, were labeled with both probes and were considered as positive controls. Plant secondary cell walls of both healthy and infected tissues were also intensely labeled whereas compound middle lamella‐primary walls and cell cytoplasm were free of labeling. Enzymatic digestion of plant tissues with chitinase from Streptomyces griseus abolished the fungal cell wall labeling but did not interfere with that of plant secondary cell walls. This suggests that polymers analogous to fungal chitin are absent in plant cell walls. Tissue digestions with either proteinase K or lipase led to surprising results as far as the possible nature of N‐acetylglucosamine‐containing molecules is concerned. The loss of labeling over plant secondary walls following lipase digestion suggests that N‐acetylglucosamine residues may be linked to lipids to form glycolipids. However, these results have to be viewed with caution since the possibility that peptides may be present but inacessible to proteinase K should be considered. The role of the detected N‐acetylglucosamine containing molecules as possible substrates for plant chitinases is discussed.

Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis

Mucor racemosus and Rhizopus nigricans were used as sources of chitin deacetylases. Crude protein extracts were subjected to polyacrylamide gel electrophoresis at pH 8.9 (Davis system) or 4.3 (Reisfeld system) under native conditions. After electrophoresis, an overlay gel containing 0.1% (w/v) glycol chitin as substrate was incubated in contact with the separation gel. Chitin deacetylase activity was revealed by uv illumination with a transilluminator after staining for 5 min in 0.01% (w/v) Calcofluor white M2R. Chitosan (deacetylated chitin) generated by chitin deacetylases appeared more fluorescent than the intact chitin embedded in the overlay gel. Chitosan in a separate overlay gel was also subjected to a nitrous acid treatment which specifically depolymerizes chitosan while leaving chitin intact. Hydrolysis of chitosan by nitrous acid followed by Calcofluor staining yielded dark (nonfluorescent) bands (chitin deacetylase activities) in the fluorescent chitin-containing gel. Both assays revealed the presence of several chitin deacetylases from Zygomycetes. The same assays were performed after denaturing electrophoresis in 12% (w/v) polyacrylamide gels containing 0.1% (w/v) glycol chitin. Enzymes were renatured in buffered 1% (v/v) purified Triton X-100. Chitin deacetylases with estimated molecular weights between 26,000 and 64,000 were detected after Calcofluor staining. The assays were also performed in two-dimensional gel electrophoretic systems. Chitin deacetylases can be rapidly revealed by using the assay involving the nitrous acid treatment. However, both assays (with and without nitrous acid treatment) should be run to conclusively demonstrate chitin deacetylase activity after polyacrylamide gel electrophoresis.

Chitinase isoforms in roots of various pea genotypes infected with arbuscular mycorrhizal fungi

Chitinase activity has been investigated in mycorrhiza-resistant (myc−), non-nodulating (nod−) isogenic pea (Pisum sativum L.) mutants in an attempt to understand the reasons for such resistance to symbiotic fungi. Activities from control and Glomus mossease-inoculated roots of myc− mutants were compared to the corresponding mycorrhizal (myc+), nodulating (nod+) wildtype pea genotype cv. Frisson. A pea mutant only deficient for the nod− character [myc+, nod−] was also studied. Two acidic and two basic chitinase isoforms were detected in control roots of all peas tested after native polyacrylamide gel electrophoretic (PAGE) separation of proteins at pH 8.9 or pH 4.3. No difference in basic chitinase isoforms patterns occurred in the various pea genotypes in the presence of G. mosseae. However, as soon as 1 week after infection, an additional acidic chitinase isoform was observed in extracts of G. mosseae-colonized roots from the wildtype pea genotype and the [myc+, nod−] mutant. The isozyme had a molecular mass of approximately 27 kDa, estimated by sodium dodecyl sulfate (SDS)-PAGE under non-reducing conditions, and exhibited a faint lysozyme activity by lysis of Micrococcus luteus cells. It was not detected in the [myc−, nod−] pea mutant, unless plants were grown under conditions which increased the number of appressoria. The host origin of the 27 kDa chitinase isoform was indicated by its presence after infection with another Glomus species, and from the fact that it was not detected in a mixture of germinated spores and mycelia of G. mosseae.