Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3 kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4 kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific β-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.     

原核微生物的多样性

微生物是一群以分解代谢为主的重要生物类群,其生物学多样性十分丰富。但由于它们的微观性,尤其原核微生物简单的单细胞结构、以无性方式进行快速地繁殖而造成的无准确的基线难以对其进行种群数目和数量的统计,因而对微生物的多样性研究远没有宏观生物那样深入和受到重视。本文根据原核微生物的特性,从其物种、所代表的进化分支、生理代谢类群及遗传背景几个方面简述了它们的多样性及重要意义,意在引起科学界和全社会对这类生物资源的认识和保护的重视。     

Stability of Recombinant Plasmids in Transgenic Microorganisms under Different Environmental Conditions

The copy number of R plasmids weakly depends on the selective pressure of the respective antibiotic but does depend on the physiology of the host species and the type of plasmids and cloned genes, whose expression leads to a further load on the biosynthetic apparatus of cells. The last factor is critical in the maintenance of recombinant plasmids in transgenic microorganisms.     

Distribution of Microorganisms in the Al–Fe–Humus Podzols of Natural and Anthropogenically Impacted Boreal Spruce Forests

Natural and anthropogenically induced seasonal variations in the abundance and biomass of various groups of microorganisms in the Al–Fe–humus podzols of boreal spruce forests were analyzed. The fungal biomass in these soils was found to be considerably higher than the bacterial biomass. The microbial population was mainly concentrated in a thin surface layer (10–15 cm in thickness), which included the forest litter and the upper mineral root-inhabited soil horizon and greatly differed from other soil horizons in morphology and other properties. This layer was found to be optimal with respect to hydrothermal and nutritional conditions and is characterized by profound seasonal variations in the abundance and biomass of microbiota. The high acidity, typical of the Al–Fe–humus podzols, resulted from the metabolism of their microbial communities. In the polluted podzols, the population of prokaryotes increased and that of eukaryotes decreased.     

环境样品中DNA的分离纯化和文库构建

采用研磨 /冻融和SDS/蛋白酶K热处理等理化方法 ,直接从性质不同的环境样品中提取和纯化混合基因组DNA。所获得纯品DNA的产量为每克样品 2～ 1 6μg。对纯品DNA进行限制性内切酶处理后 ,构建了以pUC1 8为载体的DNA文库。建库效率为从每克环境样品获得约 1 0 3～ 1 0 4 个含 3～ 8kb外源随机插入片段的克隆。通过DNA序列测定和基因注释 ,对从文库中随机选取的克隆进行了分析 ,发现外源插入片段均含序列未见报道的新基因。本文所做的尝试对于保存、研究和开发未培养微生物基因资源具有意义     

生物合成谷胱甘肽种间耦合ATP再生系统的构建

利用重组大肠杆菌Ⅱ 1中的谷胱甘肽合成酶系和面包酵母WSH J7中的ATP生物合成酶系 ,构建了一个以葡萄糖为能源的种间耦合ATP再生系统。经过通透性处理的酵母细胞几乎不能消耗葡萄糖。在反应体系中添加 1mmol/LAMP和 0 0 5mmol/LNADH ,即可启动酵母的酵解途径。提高耦合系统中的葡萄糖浓度 ,可促进GSH的合成。当葡萄糖浓度为 40 0mmol/L时 ,系统内GSH浓度达到 1 0 4mmol/L(3 2 g/L)。Mg2 +缺乏时 ,耦合系统和外加ATP的非耦合系统均不能合成谷胱甘肽。耦合系统中Mg2 +与ATP形成螯合物 ,可能是导致耦合系统中GSH产量较低的原因。在耦合系统中补加Mg2 +,反应 6h时GSH浓度达到 1 4 3mmol/L(4 4g/L)。     

Influence of the Degree of Aeration on Halotolerance of Yeasts of the Genera Candida,Rhodotorula, and Malassezia

The biochemical mechanisms were studied that determine different reactions of yeasts of different genera to two simultaneously imposed stressors, hypoxia and osmotic shock. For Candida lipolytica, these two stressors were antagonistic, which resulted in stimulation (and not suppression) of the growth of this yeast by NaCl (in a wide range of concentrations) under microaerobic conditions. The reaction of Malasseziasp. was different: the degree of halotolerance of this microorganism was lower under microaerobic conditions. An intervening reaction pattern was characteristic of Rhodotorula aurantiaca.These differences were found to be determined, above all, by the induction of a salt-resistant respiratory system (oxidase) in C. lipolytica, which could not be induced in Malassezia sp. In addition, the synthesis of catalase was enhanced in C. lipolytica, which provided for neutralization of the active forms of oxygen accumulating as a result of inhibition of other protective enzymes by salt.     

The Growth-promoting Effect of Beijerinckia mobilis and Clostridium sp. Cultures on Some Agricultural Crops

New strains of Beijerinckia mobilis and Clostridium sp. isolated from the pea rhizosphere were studied with respect to their promoting effect on the growth and development of some agricultural crops. Seed soaking in bacterial suspensions followed by the soil application of the suspensions or their application by means of foliar spraying was found to be the most efficient method of bacterization. The application of B. mobilis andClostridium sp. cultures in combination with mineral fertilizers increased the crop production by 1.5–2.5 times. The study of the population dynamics of B. mobilis by the method of genetic marking showed that this bacterium quickly colonized the rhizoplane of plants and, therefore, had characteristics of an r-strategist. At the same time, Clostridiumsp. was closer to K-strategists, since this bacterium slowly colonized the econiches studied. The introduction of the bacteria into soil did not affect the indigenous soil bacterial complex. The presence of Clostridium sp. slowed down the colonization of roots by the fungal mycelium. The possible mechanisms of the plant growth–promoting activity of B. mobilisand Clostridiumsp. are discussed.     

Isolation ofTricholoma matsutake andT. bakamatsutake cultures from field-collected ectomycorrhizas

Tricholoma matsutake was isolated into pure cultures from field samples of ectomycorrhizas onPinus densiflora. The mycorrhizal tips were collected at different times of the year from a colony ofT. matsutake in aP. densiflora stand. The mycorrhizal tips were continuously washed with sterilized distilled water and diluted Tween 80 solution, surface-sterilized with calcium hypochlorite solution, and inoculated on several kinds of nutrient agar media. Most of the mycorrhizal tips collected in winter and spring produced colonies that were morphologically similar to cultures ofT. matsutake isolated from basidiocarps. The identity of isolates obtained from mycorrhizas was further confirmed to beT. matsutake based on fungal morphology and RFLP patterns of PCR amplified rDNA. The feasibility ofT. bakamatsutake isolation into pure culture from ectomycorrhizas onQuercus serrata was also confirmed. These results indicated that mycelium of matsutake mushrooms can be isolated into pure culture from ectomycorrhizas at different times of the year. Mycorrhizas of bothT. matsutake andT. bakamatsutake were not observed to have any specific association with soil fungi such asMortierella spp.