Optimized medium for the efficient production ofBacillus intermedius glutamyl endopeptidase by the recombinantBacillus subtilis strain AJ73

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinantBacillus subtilis strain AJ73 bearing theBacillus intermedius 3–19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca²⁺, Mg²⁺, and Co²⁺.     

Effect of ethylene perception inhibitor on growth, water relations, and abscisic acid content in wheat plants under water deficit

The effects of treatment with 1-methylcyclopropene (1-MCP, the inhibitor of ethylene receptors) of 7-day-old wheat (Triticum durum Desf., cv. Bezenchukskaya 139) seedlings on growth characteristics, water relations, and the content of phytohormones during three days after watering cessation were studied. In treated seedlings, a decrease in the water content in the substrate resulted in a decrease in stomatal conductance in the leaves by one day earlier than in untreated seedlings. This could be related to the more rapid and substantial accumulation of ABA in treated plants. There was no clear relationship between changes in the content of cytokinins and water relations under the influence of 1-MCP under drought conditions. The role of ethylene and ABA in the regulation of growth and water relations in plants suffering from water deficit is discussed.     

Fast evolutionary genetic differentiation during experimental colonizations

Founder effects during colonization of a novel environment are expected to change the genetic composition of populations, leading to differentiation between the colonizer population and its source population. Another expected outcome is differentiation among populations derived from repeated independent colonizations starting from the same source. We have previously detected significant founder effects affecting rate of laboratory adaptation among Drosophila subobscura laboratory populations derived from the wild. We also showed that during the first generations in the laboratory, considerable genetic differentiation occurs between foundations. The present study deepens that analysis, taking into account the natural sampling hierarchy of six foundations, derived from different locations, different years and from two samples in one of the years. We show that striking stochastic effects occur in the first two generations of laboratory culture, effects that produce immediate differentiation between foundations, independent of the source of origin and despite similarity among all founders. This divergence is probably due to powerful genetic sampling effects during the first few generations of culture in the novel laboratory environment, as a result of a significant drop in N _e. Changes in demography as well as high variance in reproductive success in the novel environment may contribute to the low values of N _e. This study shows that estimates of genetic differentiation between natural populations may be accurate when based on the initial samples collected in the wild, though considerable genetic differentiation may occur in the very first generations of evolution in a new, confined environment. Rapid and significant evolutionary changes can thus occur during the early generations of a founding event, both in the wild and under domestication, effects of interest for both scientific and conservation purposes.     

Genetic mechanisms of bacilli adaptation

Adaptive strategies of bacilli involving genetic regulatory mechanisms are reviewed. The role of master regulators and signal transduction systems that coordinate the interaction of the extracellular signals and the genetic programs responsible for the metabolic state of bacteria are discussed. Most of the known regulatory pathways are directly or indirectly regulated by the DegU, Spo0A, AbrB, and CodY global regulators. The main factor affecting the development of cell phenotype is the concentration of the regulatory protein and its ability to bind with varying affinity to promoters of the genes and operons. The effect of the regulatory systems on the bistability of microbial populations is discussed.     

Isolation and study of polyadenylated RNA from Bacillus intermedius

A poly(A)+RNA fraction was isolated from the overall RNA of Bacillus intermedius using chromatography on poly(U) Sepharose and was shown to be electrophoretically heterogeneous. The presence of a polyadenylate segment was confirmed by hybridization with polyuridine. The biological activity of the poly(A)+RNA was proved by the translation in Xenopus laevis oocytes. The dynamics of poly(A)+RNA synthesis was studied in the course of B. intermedius growth and the content of poly(A)+RNA was assayed in the cells grown in different media.     

环境对沼泽红假单胞菌基因型的影响

<正>紫色光合细菌是不产氧光合细菌的一个重要分支,包括紫色非硫细菌和紫色硫细菌。其中紫色非硫细菌具有极其丰富的代谢模式,可进行光能异养、光能自养和化能异养生长,其代谢的多样性使得它们广泛存活于不同的生态系统中,如土壤、湖泊、海洋及底泥等[1-2]。沼泽红假单胞菌(Rhodopseudomonas palustris)是紫     

A novel secreted metzincin metalloproteinase from Bacillus intermedius

The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes.     

Growth conditions and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

Isolation and characteristics of Bacillus intermedius glutamyl endopeptidase secreted by a recombinant strain of Bacillus subtilis at various growth phases

The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid delta58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca+ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.