An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

The Biometric Analysis of Bacterial Cells in Soil

The biometric analysis of bacterial cells in soil by light, fluorescence, and scanning electron microscopy showed that their average size is 0.8 m in diameter, 1.4 m in length, and 0.7 m³ in volume. In soil loci with enhanced microbiological activity (the rhizoplane of plants and the intestinal tract of soil invertebrates), the average size of bacterial cells was found to be 40% smaller than that of cells occurring in other parts of soil. This is the first experimental evidence showing that the metabolic activity of soil bacteria, their concentration, and allometric parameters are related.     

Estimation of abundance dynamics of gram-negative bacteria in soil

Bacterial succession in soil was studied for two variants of initiation (moistening and moistening with addition of glucose). To determine the numbers of viable gram-negative bacteria, the modified nalidixic acid method was applied. The numbers of gram-negative bacteria revealed by this method were 2 to 3.5 times higher than those determined by the traditional method. In a developing community, the highest total bacterial numbers were observed on day 7; afterwards their numbers decreased and stabilized at a level exceeding four-to fivefold the initial one. In both experimental variants, the highest numbers of viable gram-negative bacteria were revealed on day 15 (75–85% of the total bacterial numbers). Morphology of these bacteria suggests their classification as cytophagas (chitinophagas) utilizing chitin from the dead fungal mycelium.     

Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6–8°C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the β-structural proteins (they have 59–96% total β structures and 0–17% α helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl β-D-glucoside for SDS: the content of β structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.     

Growth of epiphytic and soil yeasts on wheat seedlings

Colonization of wheat seedlings by epiphytic (Rhodotorula glutinis) and soil (Lipomyces starkeyi) yeasts was studied by scanning electron microscopy. Epiphytic yeast cells dominated on the plant surface. Soil yeast cells were randomly distributed among both the zones of a seedling and the particles of an inorganic substrate. It has been found that epiphytic yeast strains can readily grow on the surface of a plant.     

Primary Structures of Actinoporins from Sea Anemone <Emphasis Type="Italic">Oulactis orientalis</Emphasis>

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with primers specific to the N-terminal regions of the O. orientalis actinoporin sequences and to the C-terminal region, which is highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were elucidated by sequencing the cDNA clones obtained by the rapid amplification of 3′-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain the lower hemolytic activities of Or-A and Or-G compared to those of actinoporins from the tropical species.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 357–362.Original Russian Text Copyright © 2005 by Il’ina, Monastyrnaya, Isaeva, Guzev, Rasskazov, Kozlovskaya     

Properties of Soil Native and Ortho- and Pyrophosphate-Modified Ferro-Humic Complexes and Their Effect on Solubility and Toxicity of the Products of Exogenous Zn Binding

The properties of soil ferro-humic sorbent complexes proved to determine the degree of exogenous Zn binding and the toxicity of Zn-compounds for soil microbiota. The properties of ferro-humic complexes were profoundly altered under the action of phosphate anions as potential ligands. Distinct properties of ferro-humic complexes modified by ortho- and pyrophosphates were revealed.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 3, 2005, pp. 364–374.Original Russian Text Copyright © 2005 by Kudeyarova, Guzev.