Role of prodigiosin and chitinases in antagonistic activity of the bacterium Serratia marcescens against the fungus Didymella applanata.

The molecular features of antagonism of the bacterium Serratia marcescens against the plant pathogenic fungus Didymella applanata have been studied. The chitinases and the red pigment prodigiosin (PG) of S. marcescens were isolated and characterized. Specific antifungal activity of the purified PG and chitinases against D. applanata was tested in vitro. The antagonistic properties of several S. marcescens strains exhibiting different levels of PG and chitinase production were analyzed in vitro with regard to D. applanata. It was found that the ability of S. marcescens to suppress the vital functions of D. applanata depends mainly on the level of PG production, whereas chitinase production does not provide the bacterium with any competitive advantage over the fungus.

Chitotriosidase activity in the blood serum and organs of mice of various strains under the influence of chitin.

Mouse chitotriosidase cleaving chitin belongs to the family of mammalian chitinases, whose biological functions are poorly understood. Chitotriosidase activity in mouse serum was shown to be much higher than in humans. The following interstrain differences were revealed in mouse chitotriosidase activity: GR>C57Bl/6>BALB/c>A/Sn>CBA. Chitotriosidase activity in CBA mice was lowest and practically did not differ from that in C3H/He and ICR mice. No sex-related differences were found in enzyme activity. Hybrids of opposite strains CBA and C57Bl/6 were characterized by dominant inheritance of this sign (elevated activity of chitotriosidase in the serum). Intragastric administration of chitin in a single dose of 100 mg/kg was followed by a decrease in chitotriosidase activity in the lungs, but not in the blood serum and homogenate of gastric cells from CBA mice. These data indicate that intragastric administration of chitin does not induce chitotriosidase in mice.

Extracellular chitinases of mutant superproducing strain Serratia marcescens M-1.

Four extracellular proteins with chitinase activity capable of binding chitin substrates have been revealed in the culture liquid of chitinase superproducing mutant strain M-1 of Serratia marcescens. Proteins were analyzed by SDS-PAGE and MALDI-TOF mass spectrometry. Based on the data obtained, the proteins were identified as typical chitinases of S. marcescens: ChiA, ChiB, ChiC, and CBP21.

[Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens]. / Obrazovanie vnekletochnykh khitinaz prirodnym (B-10) i mutantnym (M-1) shtammami Serratia marcescens.

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).

[Isolation and properties of extracellular lipase of native (B-10) and mutant (M-1) Serratia marcescens strains]. / Vydelenie i svoistva preparatov vnekletochnykh lipaz prirodnogo (B-10) i mutantnogo (M-1) shtammov Serratia marcescens.

The cultivation conditions of wild-type strain V-10 and mutant strain M-1 (overproducer of endonuclease and chitinase) of Serratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximal lipase yield (840 AU/ml) after 10-12 h of cultivation; the strain M-1 (33 AU/ml), after 25-30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acid medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed a higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and inactive in the presence of cetyltrimethylammonium bromide.

[Comparative analysis of the structure of double-stranded RNA from killer strains of Saccharomyces cerevisiae]. / Sravnitel'nyi analiz stroeniia dvuspiral'nykh RNK killernykh shtammov Saccharomyces cerevisiae.

Double-stranded RNAs (M and L molecules) of two strains of the killer system Saccharomyces cerevisiae M437 (wild type) and ski-5 (superkiller mutant) were studied by means of electron microscopy and high resolution thermal melting. The M molecules of the ski-5 mutant were by 100 b.p. shorter than those of M437. L molecules were of the same length for both strains. Analysis of the differential melting curves of L molecules showed that L molecules differ significantly in their nucleotide sequences, whereas M molecules were practically identical. It was found that M molecules contained a long AU region: that of M molecules of M 437 was 170-180 b.p. long and contained almost no GC pairs, whereas the AU region of M molecules of the ski-5 mutant was three times shorter and contained GC pairs.

[Structural organization of double-stranded RNA from killer yeasts Saccharomyces cerevisiae by the thermal denaturation method]. / Izuchenie strukturnoi organizatsii dvuspiral'noi RNK killernykh drozhzhei Sacharomyces cerevisiae metodom termicheskoi denaturatsii.

The thermal denaturation method for studying the structural organization of double-stranded RNA (dsRNA) from virus-like particles of killer yeasts Saccharomyces cerevisiae was used. High resolution derivative denaturation profiles of total dsRNA and its L- and M-types were obtained. Comparative analysis of these data with those on phage DNA denaturation demonstrated that the processes of denaturation of dsRNA and phage DNA were identical in quality. Increase of thermostability, interval of thermal denaturation and width of local helix-to-coil transitions in dsRNA as compared with phage DNA are caused by the differences of corresponding thermodynamic parameters. Derivative denaturation profiles of L- and M-types of yeasts dsRNA were shown to have certain identical local transitions. Low melting transition, consisting of three local thermalites, is due to the denaturation of AU-rich region (about 200 n.b.p.) in M-dsRNA.

[Natural interferon inducers: double-stranded RNA of killer plasmids of Saccharomyces cerevisiae]. / Prirodnye induktory interferona: dvuspiral'naia RNK killernykh plazmid Saccharomyces cerevisiae.

Methods for isolation and purification of yeast double spiral RNA (dsRNA) are described. The most simple method includes reprecipitation of dsRNA in solutions of lithium chloride and its interphase distribution in the system of chloroform: isoamilic alcohol: water. Preparations with the content (w/w) of dsRNA from 30 to 90 per cent were obtained. They possess a high interferon-inducing activity and an antiviral effect on the experimental infection caused by the virus of mouse encephalomyocarditis.